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Abstract

Oncolytic viruses (OVs) are engineered and/or evolved to propagate selectively in cancerous tissues. They have a dual mechanism of action; direct killing of infected cancer cells crossprimes anticancer immunity to boost the killing of uninfected cancer cells. The goal of the field is to develop OVs that are easily manufactured, efficiently delivered to disseminated sites of cancer growth, undergo rapid intratumoral spread, selectively kill tumor cells, cause no collateral damage and pose no risk of transmission in the population. Here we discuss the many virus engineering strategies that are being pursued to optimize delivery, intratumoral spread and safety of OVs derived from different virus families. With continued progress, OVs have the potential to transform the paradigm of cancer care.

viruses as anticancer drugs

The key desirable characteristics of any OV are specificity, potency and safety; specificity for the targeted cancer, potency to kill infected cells and cross-prime antitumor immunity, and safety to avoid adverse reactions and pathogenic reversion.

• • Limiting pathogenicity

Although it will be interesting to determine whether OVs encoding modified polymerases with higher fidelity will exhibit an improved safety profile [176] , it should be noted that OV reversion to a more pathogenic state has not yet been observed clinically. This is also true of many time-honored viral vaccines. For example, in 50 years of widespread measles vaccination using attenuated Edmonston lineage substrains, there has never been a documented reversion back to a wild-type phenotype, even after IV doses as high as 10 11 TCID 50 were administered to immunosuppressed, measles antibody negative myeloma patients in an oncolytic virotherapy study [177] .
Limiting the pathogenic potential of OVs can also be accomplished by enhancing the host innate immune response. VSV has been engineered to express IFN-β, which induces an antiviral state and reduces proliferation of cells. Cells capable of responding to interferon signaling will limit viral replication in response to the virally produced interferon, further restricting viral replication to tumors with defective innate responses [188] . The expression of IFN-β from infected tumor cells can also prevent off-target infection of nearby tissue through paracrine signaling. Another way to achieve a similar effect is mutating the VSV matrix protein, which is responsible for limiting cellular production of type I interferons [189] . This, and similar approaches, to inactivate viral genes that combat innate immunity has been extensively utilized in the OV field, across a broad range of viral families. Sensitization of OVs to innate immune responses can limit off-target infections and enhance tumor-specific tropism while increasing the therapeutic index.

engineering viruses: a diversity of platforms

In nature, viruses are continuously evolving and adapting to occupy almost every imaginable biological niche. Viruses infect bacterial, archaea, protist, fungal, plant and animal cells. Their genomes are composed of DNA or RNA, which may be single or double stranded, positive or negative sense, ranging in size from 2 to 300 kb, in complexity from 1 to 300 genes and in capacity for foreign genetic material from a few hundred bases to several kilobases. The particles of viruses from different families also vary enormously in size and structure, ranging from 20 to 1000 nm, from icosahedral to helical symmetry, with or without lipid envelope, integument or matrix and with variable susceptibility to physical disruption. Naturally occurring viruses also offer a vast diversity of virus life cycles, cell entry and replication mechanisms, cell and species tropisms, cycle times, burst sizes, innate immune evasion, apoptosis, antiviral state prevention and immune combat strategies, modes of transmission and pathogenic mechanisms. The diversity of viruses that have been investigated as oncolytic platforms and the different strategies used to improve their efficacy continue to be expanded (Table 1) .

Delivery of Ovs

Viral infections are well understood but for OV applications must be viewed as drugs obeying pharmacological principles. A drug is typically administered to a patient in an extremely controlled way to produce a reliable, consistent and predictable pharmacokinetic profile (absorption, biodistribution, metabolism and excretion) and bioavailability. Natural viral infections do not obey these rules since the inoculum is of variable size; host resistance varies from person to person; and the kinetics of the adaptive immune response differ greatly between individuals. Thus, the outcomes of natural infections with a given pathogenic virus range from asymptomatic seroconversion to full blown disease. The primary virus inoculum must contain sufficient virus particles to overcome initial host defenses at the site of entry. Some viruses enter through mucous membranes in the GI or respiratory tracts, while others enter via direct inoculation into the blood stream following a needle stick or arthropod blood meal [58] . OVs are delivered in the same way as traditional drugs, by introducing a highly concentrated virus inoculum into the body via oral, intravenous (IV), intranasal, transdermal, subcutaneous or intramuscular routes whereupon the dispersion of the inoculated virus, or its progeny, takes it to the targeted cancerous tissues. A major factor distinguishing OVs from traditional drugs is that they self-amplify and spread after delivery so their peak concentration may not be reached until sometime after the treatment is administered. Biological amplification by viral replication is the most important difference between viral therapies and traditional drugs. The concentration of a drug diminishes over time at a very well described rate depending on the clearance and elimination from the body. Through the course of a viral infection the viral load is initially small and then increases and finally decreases rather than just following a specific rate law of elimination. Limited amounts of virus particles replicate at the site of inoculation [59] . Input or progeny viruses then can either drain with the lymphatic fluid to the nearest lymph node, go directly into circulation or spread locally before spreading systemically to eventually arrive at a specific target organ. Pathology is induced by high levels of viral replication in the target organ directly killing infected cells or recruiting the immune system to kill them, at the same time provoking a local inflammatory response.

• • Limiting pathogenicity

Polymerase infidelity is not the sole driver of virus genome diversification and evolution. For many viruses, recombination between homologous viral genomes can occur when two related viruses infect the same cell [178] . This type of recombination is not a feature of negative strand RNA virus evolution because the nascent progeny viral genomes are cotranscriptionally incorporated into a helical ribonucleocapsid structure which prevents recombination. However, for positive sense RNA picornaviruses, and DNA viruses such as adenovirus, HSV and vaccinia, it is important to consider the possibility that the input virus may encounter and recombine with a homologous 'wild-type' virus in a treated patient. While such recombination has never been documented in OV trials, there are well described examples in the field of vaccinology, such as the re-emergence of pathogenic polioviruses through the recombination of vaccine genomes and naturally circulating picornavirus genomes [179] . In view of these risks, it is generally considered inadvisable to arm viruses with therapeutic transgenes that have theoretical potential to increase pathogenicity if transferred to a related naturally circulating virus. Genes encoding immunosuppressive, antiapoptotic or directly cytotoxic proteins are on this list.
Certain viruses with segmented genomes, most notably the orthomyxoviruses (e.g., influenza), are capable of rapid evolution by genome fragment reassortment in multiple infected cells. Reassortment of genome fragments in virus-infected birds and pigs is considered to be a key driver of the antigenic shifts that occur during evolution of new pathogenic influenza virus strains [180] . This important safety concern may explain why oncolytic influenza viruses have not yet been advanced to human clinical trials [181] . Because of the toxicity risks associated with the proliferative and evolutionary capacity of OVs, there is considerable interest in contingency plans to terminate the spread and/or transmission of an OV infection. Reliable antiviral medications are available for TK-expressing herpes viruses, but this is not the case for the majority of viruses being developed as OV platforms [182, 183] . Attention has therefore been directed to the development of safety switches that can be engineered into the OV genome and triggered on demand to terminate in vivo replication.

• • Sodium iodide symporter (radioconcentrator)

Iodide is a critical component of thyroxine and is concentrated in thyroid follicular cells by the thyroidal sodium iodine symporter (NIS), a cell surface glycoprotein with 13 transmembrane domains [146] . Radioiodine is therefore used routinely in the clinic for thyroid imaging and for ablation of overactive thyroid tissue, including metastatic thyroid cancer. This is facilitated by the ready availability of several iodine radioisotopes, most notably 123 Ifor γ camera and single-photon emission computed tomography imaging, 124- for PET imaging and 131 I -, a β-emitting isotope, for thyroid ablation. Besides radioiodine, NIS can concentrate several related anions, of equal or greater value for single-photon emission computed tomography ( 99m TcO 4 -, pertechnetate), PET (B 18 F 4 -, tetrafluoroborate) and tissue ablation ( 211 At -, astatide, 188 ReO 4 -, perrhenate) [147] . The β emissions of 131 Ihave an average path length of approximately 1.8 mm in tissue and can therefore inflict significant damage on cells adjacent to an 131 I --loaded NIS expressing cell. For 188 ReO 4 the β emission path length is longer such that NIS negative tumor cells are even more likely to be damaged in the β particle crossfire when this radioisotope is used. Unsurprisingly, therefore, the NIS gene has been engineered into several OVs whose IT spread has been elegantly mapped and monitored in tumor-bearing mice by serial radiotracer imaging, and whose potency has been substantially boosted by appropriately timed administration of 131 I - [148] . At least two NIS-expressing OVs, a prostate-targeted oncolytic adenovirus and a CD46-targeted measles virus, have been advanced to human clinical trials with positive imaging data reported [11, 149] .

viruses as anticancer drugs

Oncolytic viruses (OVs), evolved and engineered for cancer specificity, are gaining momentum as a new drug class in the fight against cancer. Besides, causing the death of virus-infected cancer cells, the spreading intratumoral (IT) infection can also boost the anticancer immune response, leading to immune destruction of uninfected cancer cells. The paradigm of OVs has been reviewed extensively [1] [2] [3] [4] [5] [6] [7] .
It is well established in several rodent cancer models that a single dose of an effective OV can completely cure disease [8, 9] . This has been shown for DNA and RNA viruses in diverse tumor models. However, while the single shot cure is an exciting prospect for cancer therapy, to date clinical outcomes have typically fallen short of this, and repeat IT virus administration has proven to be a more reliable approach [6, 10] . But there are a number of anecdotal case reports that give credence to the idea that a single shot OV cure may be achievable in clinical practice [4, 11] suggesting that OVs have the potential to transform the practice of oncology.
Based on these clinical advances, development of OVs is rapidly accelerating. The purpose of this review is to discuss the virus engineering approaches and OV performance optimization strategies that are being pursued in the field, and to point out some of the challenges that remain. We believe this perspective will be particularly valuable to virologists entering the field. We apologize to the numerous investigators whose work has not been acknowledged; we are well aware of these omissions but due to space constraints our approach has been choosing illustrative examples that demonstrate the key principles. The review is divided into five main sections: (i) an introduction to viruses and virus engineering, (ii) delivery, (iii) spread, (iv) arming and (v) safety. All five topics are highly interdependent.

engineering viruses: a diversity of platforms

Aside from their differences, viruses share fundamental similarities including their dependence on the host cell to provide a suitable environment for genome amplification, gene expression and progeny virus production and their adaptation to a specific set of host cell conditions and factors such that propagation is precluded in cells that fail to provide the necessary environment. These characteristics allow viruses to be targeted to cancer cells as a self-replicating antineoplastic therapy.

Delivery of Ovs

OV therapy begins with the administration of the virus to the patient just as with any other drug. Several approaches can be taken, each with to optimize oncolytic virotherapy indicated that maximizing delivery, so more tumor cells are infected, has a profound impact on the degree to which the virus must propagate from each infected cell for curative therapy [60] . The higher the number and the more random the dispersion of infectious centers established in a tumor after delivery of virus, the more effective the virus will be, although these parameters must be optimized for each virus and model [61] . IV and IT administrations of OVs are common delivery methods that introduce large amounts of virus into one compartment in a very short time. This rapid delivery of a massive virus inoculum does not parallel what typically occurs in a natural infection and potentially allows the IT spread of the OV infection to outpace host defenses. Successful delivery in the context of an OV infection requires that a critical viral concentration is achieved within the tumor to allow for sufficient oncolysis and systemic spread of the virus to all sites of disease in a safe and reproducible pattern.
• • iv delivery IV delivery of an OV seems advantageous in the setting of metastatic disease, allowing the virus to potentially access all sites of disease via the circulation soon after infusion. In a clinical setting, systemic delivery may be preferred since it is more broadly applicable than IT inoculation, regardless of tumor location and total tumor burden. Yet, attempts at systemic delivery have shown limited success; the administered virus is immediately diluted in the circulating blood volume (∼5 l), so extremely high doses are needed to achieve meaningful circulating titers. A clinical trial using an oncolytic pox virus, JX-594, demonstrated that virus could only be detected in tumors of patients that received at least 10 9 p.f.u. per IV dose without any virus being detected in tumors of the lower dose cohorts of patients [62] . Because of this important dilution effect, doses required for IV therapy may have to be up to 1000-fold higher than for IT administration, which results in significant manufacturing challenges as well as a unique set of toxicities [2] . Administration of high titers of virus intravenously can lead to hepatotoxicity, thrombocytopenia and lymphopenia [33, 63] .
Following the path of an OV from the IV line to the tumor will quickly illustrate some of the challenges for IV delivery. First, the virus is rapidly diluted in the circulation, where its infectivity is neutralized by serum proteins including antibodies and complement, and a large proportion is sequestered by reticuloendothelial phagocytes in the spleen and liver. Any virus particles that reach the tumor vasculature without first being neutralized must then extravasate through fenestrations, and pores between the vascular endothelial cells of the tumor capillaries, and once in the interstitial space of the tumor, must negotiate the extracellular matrix (ECM) to reach and infect the resident cancer cell [64] .
Tumor vasculature can both aid and limit viral access. Tumors typically have an unorganized growth pattern, producing vasculature that is tumultuous and insufficient resulting in heterogeneous perfusion throughout the parenchyma [65] . Decreased perfusion limits the formation of infectious centers evenly throughout a tumor. Manipulating physiological parameters such as blood pressure and systemic vascular resistance can preferentially increase tumor perfusion and promote better delivery of viral vectors [66] . Once in a tumor vessel, the virus must move from the intravascular compartment into the interstitium by crossing the vascular endothelium to continue its journey through the interstitial space until finally reaching a permissive tumor cell.
At least in theory, viruses can cross the endothelial lining of tumor neovessels by diffusion, active infection, or by trafficking inside of or on the surface of marrow-derived cells that are capable of diapedesis. Tumors generally have poor lymphatic drainage and can have leaky blood vessels with a wide range of pore sizes which helps to explain the phenomenon of enhanced permeability and retention of nanoparticles such as OVs in the tumor parenchyma [67] . In some tumors, the pores and fenestrations in tumor capillaries may be large enough to permit passive diffusion of viruses (depending on their size) but in many tumors review Maroun, Muñoz-Alía, Ammayappan, Schulze, Peng & Russell future science group the endothelium is sufficiently organized that this type of extravasation is simply not possible.
Another approach to help OVs' extravasate is by temporally creating pores throughout the tumor microvasculature to enhance transport of the virus into tumor interstitium. This can be done through application of focused ultrasound in the presence of a contrast agent or a polymer. The microbubbles or polymers are injected intravenously and can freely circulate without harming tissues, but when they are exposed to ultrasound waves they can oscillate and induce pore formation or cavitation in the surrounding tissue. The ultrasound can be focused on a tumor just after the infusion of the virus to increase viral extravasation. This has been shown to enhance vaccinia virus delivery to tumor xenografts and can increase viral transgene expression by more than a 1000-fold in the tumor [71, 72] . Many strategies are being developed to enhance OV IV delivery.
• • iT delivery IT administration directly delivers a high concentration of the OV into the parenchyma of the injected tumor but may not result in spread of the virus to distant sites of metastasis. T-Vec is administered by IT injection to accessible skin tumors of patients with metastatic malignant melanoma. The injection is repeated every 2 weeks until lesions have resolved or failed to respond [12, 73] . Interestingly, viremia was not documented in patients, suggesting that the regression of noninjected lesions was likely immune-mediated and not due to direct oncolysis [74] .
Immediately following IT delivery, small levels of virus can be detected in the blood from leakage through injured tumor vasculature [75] , but that small amount of virus is quickly cleared from the system. Also, virus that has been injected into a tumor is frequently extruded immediately from the injection track once the syringe is removed, especially in smaller higher pressure tumors. However, systemic viral spread can occur following IT administration if the initially exposed tumor cells are capable of amplifying the input virus and releasing progeny into the bloodstream. Such secondary viremia may not peak until several days after IT virus delivery and, with currently used OVs, is often not detected. Secondary viremia, when it occurs, more closely mimics the natural propagation of a viral infection from the site of inoculation to more distant target organs.

• Other routes of delivery

Intraperitoneal virus administration is often pursued in virotherapy studies aiming to impact ovarian cancer and other disseminated intraperitoneal malignancies, while intrapleural administration is pursued for mesothelioma therapy. Both of these approaches are similar to IT administration in that the virus comes into direct contact with tumor cells in the injected cavity, although there is a greater risk that input virus will be immediately neutralized by antibodies and other proteins in ascites or pleural fluid. Intravesical instillation of virus is the favored route of administration for treatment of early-stage bladder cancer, requiring only that future science group www.futuremedicine.com the input virus be stable in urine. Also, immediately after brain cancer surgery, in an attempt to control residual disease, virus is often instilled directly into the resection cavity.

• Limiting viral neutralization & clearance

Perhaps the most significant barrier to widespread OV delivery is rapid neutralization and clearance of circulating virus particles. Antibodies and complement proteins can coat the virus blocking its ability to interact with its cellular receptor and accelerate Fc receptor-mediated clearance by splenic macrophages and hepatic Kupffer cells.
For high seroprevalence viruses, one approach to circumvent viral neutralization by preformed antibodies is to engineer or switch the viral coat proteins. Some OVs (e.g., adenovirus) offer a menu of different serotypes, providing a basis for serotype switching between successive doses of the therapy to avoid antibody neutralization, although this does greatly complicate the product development pathway since each serotype is considered to be a distinct pharmaceutical product [82] . For monotypic viruses such as measles, serotype switching is not an option, but antimeasles antibodies can be circumvented by substituting the surface glycoproteins of measles with those of a related but noncross-reactive morbillivirus such as canine distemper virus [83] . Alternatively, the immunodominant epitopes of the measles surface glycoproteins can be modified by mutating key surface residues to eliminate them, or by introducing glycosylation signals so they are shielded by N-linked glycans [84] . It is worth noting that all of these virus engineering strategies have the potential to alter viral receptor usage and hence tumor cell tropism which may limit their utility [83, 85] .
Mengovirus, VSV and Newcastle disease virus are examples of animal pathogens being developed as oncolytic agents. Their appeal as oncolytic platforms is due to the coupling of their ability to selectively propagate in human tumors with their low seroprevalence in the human population [77] . Unlike viruses such as measles which have almost exclusively human cell tropism, these zoonotic viruses are capable of infecting both rodent and human tumor cells allowing preclinical testing in more informative immunocompetent mouse cancer models.
Virus-infected cell carriers can also be used to transport viruses via the bloodstream to sites of tumor growth. In part, this is possible because there is an 'eclipse period' after the cell carriers have been infected during which they do not display viral proteins on their surface and are therefore not bound by virus neutralizing antibody, but are still able to extravasate from tumor neovessels and release infectious progeny into the tumor parenchyma. Several cell carrier/OV combinations have been used including mesenchymal stem cells, dendritic cells, T cells and endothelial progenitor cells [89] [90] [91] [92] . Some carrier cells are believed to home more efficiently to tumors responding to chemotactic signals arising from hypoxia or IT inflammation.

Ov spread

Replication and amplification in the tumor is the major feature that sets OVs apart from other anticancer drugs. No traditional chemotherapy, immunotherapy or small molecule inhibitor can target tumor cells and then amplify at the site of action and spread to other sites of tumor growth. Specificity as well as speed and extent of IT virus replication are the key determinants of therapeutic index (efficacy/toxicity ratio) for an OV therapeutic, and each of these parameters can be modified by virus engineering. Additionally, the kinetics of spread can be impacted by combining the virus with immunomodulatory drugs. The idea of a virus with selective tropism for cancerous tissue has obvious appeal. Even for naturally occurring viruses that have not been tropism modified, a tumor offers a favorable environment to support a productive infection, and this is borne out by case reports of temporary remission or regression of different cancers concurrent with viral infections [4, 93] . There are several reasons why tumors are generally more susceptible than normal tissues to virus attack; poorly developed lymphatics, high nonsuppressible metabolic activity, resistance to apoptosis, poor responsiveness to interferon and intrinsic suppression of immune effector cells. But as we move in the direction of intentionally using virus infections to mediate tumor destruction, it is apparent that a targeted virus with exquisite tumor specificity will be superior to its non targeted counterpart, allowing for the administration of higher tumor destructive doses without toxicity to normal tissues.
Virus tropism is determined by many factors, most of which, if understood sufficiently, can be manipulated to enhance tumor specificity. The receptor tropisms of naturally occurring viruses are rarely of interest for tumor targeting, but this is not true for tissue-culture-adapted vaccine lineage viruses, some of which have evolved in the laboratory to use receptors more abundantly expressed on cancer cells. Examples include the CD46 receptor tropism of vaccine lineage measles virus and the heparan sulfate tropism of laboratory-adapted Sindbis virus [94, 95] . Where greater specificity is desired, it may be possible to engineer new receptor tropisms by modifying the structure of a viral attachment protein, for example, by displaying polypeptide ligands at the extreme C-terminus of the measles H glycoprotein [96, 97] . However, for many viruses, displaying a polypeptide ligand on the surface does no more than redirect attachment and does not confer a new receptor tropism [98] [99] [100] . This remains an area of active investigation.
In addition to their dependence on specific entry receptors, the surface glycoproteins of many enveloped viruses (and sometimes other critical viral proteins) must be proteolytically activated before they are competent to mediate virus entry [101] . By engineering the protease target sequences in these viral glycoproteins it is possible to generate viruses whose propagation is now dependent on exposure to a specific protease with high IT abundance, such as urokinase, matrix metalloproteinase 1 or cathepsin D [102] [103] [104] .
Beyond the step of cell entry, viruses are exquisite sensors of intracellular processes and can therefore be adapted or engineered in several ways for intracellular targeting of cancer cells. This is best understood by considering some fundamental aspects of the interplay between a virus and an infected cell. The incoming virus aims to usurp the cellular synthetic machinery for generation of progeny viruses. The cell resists this takeover bid by rapidly detecting virus invasion, then triggering a signaling cascade that leads to establishment of an antiviral state and release of interferon which induces an anti viral state in adjacent cells [105, 106] . The antiviral state is very complex but suppression of protein translation is a key component. Apoptosis is also triggered by the virus detection machinery so that the infected cell dies before it is able to manufacture virus progeny [107] .
For a virus to be 'successful' it must combat these host cell responses, avoiding detection, as long as possible, for suppressing the establishment of an antiviral state and preventing apoptosis. Virtually all naturally occurring viruses therefore encode proteins that inhibit apop tosis and the antiviral state [108] . Removing these accessory functions from the viral genome leads to virus attenuation in normal tissues, but to a much lesser degree in cancerous tissues. This is because cancer cells are intrinsically resistant to apoptosis and to the establishment of an antiviral state, making them highly susceptible to attenuated viruses that are no longer able to control those processes. This has provided a mechanistic basis for physiologic targeting of several viruses; VSV by mutating the matrix gene whose encoded protein blocks the interferon response [109] ; HSV by mutating both copies of the γ-34.5 gene which interferes with interferonmediated shutoff of host protein synthesis and enhances neurovirulence [110, 111] ; adenovirus by mutating the E1B protein, one of whose actions is to inhibit the apoptotic activity of p53 [112] .
Viruses can be further engineered to exclusively replicate in tumor cells by combining a virus' needs with physiologic peculiarities intrinsic in tumorigenesis. For example, a virally encoded thymidine kinase (TK) is required for HSV and vaccinia virus infection to increase the supply of deoxynucleotide triphosphates required for synthesis of progeny virus genomes [113, 114] . Elimination of the TK future science group www.futuremedicine.com gene from the viral genome restricts viral replication to cancer cells where there is an upregulation of human TK [115, 116] . Another example of a virus that exploits the high replication rate of tumor cells is Toca-511, a replication competent C-type retrovirus encoding the drug activating enzyme cytosine deaminase (CD) [117] . Since the integration of C-type retroviruses is S-phase dependent [118] , this virus is selectively amplified in rapidly proliferating tumor tissue.
Engineering tumor specificity can also be achieved by detargeting viruses to ablate unwanted tropisms that can cause off target pathology in normal tissues. MircroRNA targeting is the best example of this approach. For example, miRNA targeting was used to control the tropism of an oncolytic coxsackievirus A21 virus which caused rapid tumor regression followed by fatal myositis in murine models of myeloma and melanoma [119] . Insertion of muscle-specific miRNA targets into the viral genome eliminated muscle toxicity but left the antitumor potency of the virus intact. This was shown to be due to miRNA-mediated recognition and rapid destruction of the viral genome in muscle cells. miRNA targeting has since been applied to many OVs from diverse virus families and provides a convenient and economical strategy (using sequence insertions of only ∼100 bases) to control unwanted virus tropisms [25] .

• viral amplification & spread

After an appropriately targeted virus has infected a tumor cell, it is the extent of its subsequent propagation that becomes the key driver of potency. Data-driven mathematical models of systemic oncolytic virotherapy indicate that tumor eradication is dependent on two major parameters: the initial density and distribution of infectious foci in the tumor; and the ultimate size of the infectious centers arising from each individual infected cell (i.e., virus spread) [120] . Viral spread may occur by various mechanisms. Local spread may occur by intercellular fusion, by direct transfer of virus from infected to adjacent cells, or by release and local migration of progeny virions through the interstitial space. Systemic spread as free virus particles or as virusinfected migratory cells occurs via lymphatic channels or via the bloodstream.
Direct cell-to-cell transfer of viruses has the advantage of stealth as the virus cannot be neutralized by antiviral antibodies in the interstitial fluid [121] . Nonfusogenic viruses can be armed with fusogenic membrane glycoproteins (FMGs) to enable stealthy spread through intercellular fusion leading to the formation of large, nonviable multinucleated syncytia which may also serve as excellent antigen presenting cells for amplification of the antitumor immune response [122] . By way of example, VSV encoding measles fusogenic glycoproteins and HSVs encoding the fusogenic gibbon ape leukemia virus glycoproteins have shown superior efficacy when compared with their corresponding parental viruses [123, 124] .
In the case of free virus particles, which are susceptible to antibody neutralization, the stroma of the tumor also has the potential to limit IT diffusion and block systemic release. Fibroblasts, endothelial cells, immune cells and the ECM make up the tumor stroma, and the exact composition varies widely depending on tumor type [125] . The ECM is a collagenous matrix of protein fibrils, adhesive proteins and proteoglycans that create a web with pore sizes similar or slightly smaller than virions [126] . Destruction of ECM components can facilitate viral spread throughout the tumor and can be achieved using conventional chemoradiotherapy or by delivering matrix-degrading enzymes such as collagenase and hyaluronidase [127] . An alternative approach is to encode a matrix-degrading enzyme or inducer of matrixdegrading enzymes in the viral genome. For example, hyaluronidase and relaxin encoding oncolytic adenoviruses have each been shown to spread more efficiently in experimental tumors [128] [129] [130] .
Another factor that significantly impacts the kinetics of virus spread is the burst size, or the number of progeny viruses released by a productively infected cell, which varies widely between viral families. Picornaviruses, VSV and vaccinia virus can release up to 10,000 progeny from a single infected cell after a delay of only 6-18 h [131] [132] [133] . In addition to innate antiviral immunity, adaptive cell-mediated immune responses are typically required for the complete elimination of a viral infection and act by eliminating infected cells before progeny can be released. Oncolytic virotherapy can therefore be viewed as a race between the spreading virus and the responding immune system. For this reason, faster moving viral infections are often considered capable of inflicting greater damage to an infected tumor before they can be contained by the immune system [134] . However, in defense of the viruses with smaller burst sizes, or which release progeny by budding, they tend to be less review Maroun, Muñoz-Alía, Ammayappan, Schulze, Peng & Russell future science group rapidly controlled both by the innate and adaptive host immune responses [135] . Therefore, as with the classic race between the hare and the tortoise, it is very difficult to predict whether a fast or slow replicating virus will show superior efficacy in a given preclinical cancer model.

• • Secreted toxins

As a general rule, if a virus is to be armed with a gene encoding a secreted toxin, that toxin should be targeted so that it can kill only cancer cells. In the absence of such targeting, there would be little prospect of avoiding off-target toxicities. Immunotoxins are bifunctional proteins in which plant or bacterial toxins (typically ribosomal inhibitors) are fused to a single chain antibody or other polypeptide domain to target endocytosis in cancer cells [136] . In theory, such molecules that have been extensively investigated and advanced to human clinical trials particularly for the treatment of B-cell and T-cell malignancies [137] , could be expressed from an engineered OV genome.

• • Fusogenic membrane glycoproteins

Fusion of the lipid envelope of an incoming virus with the limiting membrane of its target cell is a necessary step in the life cycle of an enveloped virus, to deliver the encapsidated viral genome into the target cell cytoplasm. This virus-to-cell fusion reaction is mediated by FMGs embedded in the envelope of the virus, and may occur at the cell surface at neutral pH, or in the endo-somal compartment at acidic pH [150] . Neutral pH fusion is triggered by receptor attachment. Cells expressing certain virally encoded, neutral pH-active FMGs on their surface may fuse with neighboring receptor positive cells (cell-to-cell fusion), giving rise to multinucleated syncytia, the hallmark cytopathic signature of a fusogenic virus (e.g., measles) [151] . But more often the FMG is activated only after its incorporation into a budding virus particle, so cannot cause cell-tocell fusion, and must be modified, for example, by cytoplasmic tail truncation, to render it constitutively fusogenic. Either way, FMG-driven fusion of OV-infected cells with uninfected neighboring cells leads to increased bystander killing because multinucleated syncytia are nonviable as well as being highly immunogenic [152] . Based on these observations, nonfusogenic OVs have been armed with FMGs thereby conferring superior oncolytic potency [123, 153] . In one example, an oncolytic herpes virus was rendered highly fusogenic when engineered to encode a cytoplasmically truncated gibbon ape leukemia virus envelope glycoprotein [154] [155] [156] while in another study, an oncolytic VSV was rendered highly fusogenic by replacing its surface glycoprotein (G) with the hemagglutinin and fusion glycoproteins of measles virus.

• • Arming Ovs to amplify antitumor immunity

Each step of the above process can be impacted by virus engineering. Not only can the kinetics and mode of cell killing be manipulated (see the previous section on virus spread), but also the OVs can be armed with a wide variety of immunoregulatory genes whose products will be secreted into the interstitial fluid space. Thus, OVs have been engineered to express the following: high levels of type I interferons to better drive the early innate/inflammatory response; GM-CSF to stimulate the phagocytic activity and lymph node trafficking of professional antigen presenting cells; chemokines to enhance the IT recruitment of immune and inflammatory effector cells, especially cytotoxic T cells; cytokines to drive the activation and proliferation of tumor-resident cytotoxic T cells; bispecific T-cell engagers to enable tumor cell killing by T cells not recognizing tumor antigens; checkpoint inhibitor antibodies to block the protective PD-L1/B7.1 shield that protects tumor cells from T-cell attack; cytokines to increase MHC-peptide neoantigen expression on uninfected tumor cells; or cloned tumor antigens to further amplify tumor-specific immunity [165] .
While each of these approaches has proven beneficial in selected preclinical animal models where immune destruction of the tumor is the dominant OV effect, they are equally likely to decrease potency by speeding virus elimination in situations where the spread of the virus is dominant.

Safety

Engineering viruses for increased stealth, greater specificity, faster spread and enhanced potency is not without risk. As OVs gain new properties, checks and balances must be applied to assure that they do not readily evolve into pathogens, let alone transmissible pathogens that could pose a risk not only to immediate contacts of the treated patient, but also to the greater population.

• • Limiting pathogenicity

Viral cytotoxicity is the basis of tumor cell death necessary for oncolytic activity. Off-target infection and killing of normal cells by poorly targeted OVs or by OVs that have evolved new tropisms in a treated patient can cause unwanted normal tissue pathology. OVs are therefore delicately balanced between retaining enough virulence to substantially decrease tumor burden versus being sufficiently targeted (or attenuated) to not cause a new disease in the patient. Cancer-specific targeting is the most critical safety feature, but viruses evolve and viral populations are dynamic [172] . Evolution is a constant accompaniment of a spreading virus infection, whether or not the virus is oncolytic; in vivo progeny of the therapeutic OV differs on average by a single -point mutation per genome from the input virus [173] . Hence, as the input virus is amplified, it generates a swarm of quasispecies viruses, each one a slightly imperfect replica of the input virus. This swarm of progeny viruses is subjected to selective pressure as it encounters new biological niches in the treated cancer patient. Thus, if it so happens that a member of the swarm is capable of infecting a normal host tissue, the virus may have gained a new foothold from which to further evolve. Gaining new cell tropisms or losing restriction factors is therefore a significant theoretical concern in oncolytic infections, but has not yet been documented in human trials, nor in preclinical models. However, given the importance of this particular scenario, a great deal of attention is paid to the problem not just by investigators, but also by regulatory agencies.

• Limiting transmissibility

For obvious reasons OV transmission from a treated patient to a caregiver, family member, coworker, pet or other species is highly undesir-able. Infectious virus particles may be present in the blood of a treated patient, and may be shed into the environment in urine, feces, saliva and other bodily secretions. Contact with the body fluids of an OV-treated patient therefore may have the potential to spread an infection to new hosts [190] .
Because the risks of OV transmission are typically unknown when first-in-human Phase I trials are initiated, it is usual to implement standard infection containment measures throughout these studies and to carefully monitor body fluids for the appearance and disappearance of viral genomes and infectious virus progeny [191] . For certain viruses, containment may be considered unnecessary, particularly when there is already widespread population immunity to the OV in question (e.g., measles) or when there has been extensive human experience of exposure to a related virus in the form of a live viral vaccine (e.g., vaccinia). Contingency plans may also be required calling for quarantine of OV-treated patients if treatment toxicity is associated with a longer period of shedding. In reality, to date there has been no instance in which transmission of an OV from a patient to a caregiver or other contact has been demonstrated, and there are no examples of long-term virus persistence or shedding in a treated patient.
In light of the inconvenience and undesirability of OV shedding, there is interest in engineering strategies that may selectively interfere with the process. As one example, wild-type measles virus is a highly transmissible airborne virus that uses the nectin-4 receptor to enter into airway epithelial cells from whence its progeny are shed into respiratory secretions [192, 193] . Eliminating the nectin-4 tropism by strategically mutating key surface residues in the hemagglutinin attachment protein results in a virus that still causes measles in nonhuman primates, but which is no longer shed into respiratory secretions or urine [194] . Measles virus RNA (but not infectious virus) was detected in the blood, urine and saliva of myeloma patients up to 3 weeks after IV administration of an oncolytic measles virus, especially at higher dose levels, and it is possible that this shedding might be eliminated by using a nectin-4 blind version of the virus [195] . Virus shedding has also been detected in human clinical trials of HSV, reovirus, vaccinia, reovirus and adenovirus oncolytics, but was less readily detected following IT therapy with oncolytic VSV and polioviruses [167] . However, future science group www.futuremedicine.com it should be noted that while genome sequences have been detected in shed material, infectious virus particles have not been recovered.
In an OV-treated patient pre-existing antiviral immunity can be a formidable barrier to efficacy, particularly if the virus is administered intravenously. However, pre-existing antiviral immunity in caregivers and patient contacts provides reassuring protection against virus transmission. Conversely, when this particular efficacy barrier is circumvented by using OVs engineered for antibody evasion or selected for low seroprevalence, the risk of epidemic spread in the human population, unchecked by pre-existing herd immunity, looms larger.
An alternative strategy often used to side step antibody neutralization, at least of the first dose of virus administered, is to use OVs derived from zoonotic animal viruses such as New Castle disease virus (chicken), VSV (cattle), myxomavirus (rabbit), Seneca Valley virus (pig) or mengovirus (e.g., mouse and monkey). Additional regulatory executive summary viruses as anticancer drugs • Viruses naturally possess many properties that favor infection of cancer cells. Enhancing these natural properties and adding new properties through directed evolution and genetic engineering are used to create oncolytic viruses (OVs), which are emerging as a new anticancer drug class.
• The diversity of virus families and engineering techniques allows for the creation of OVs with a wide range of properties that can be tailored for each type of cancer.

Delivery of Ovs

• Intravenous delivery allows a virus to reach distant sites of metastasis via the circulation, but extravasation into the tumor parenchyma is inefficient.
• Intratumoral injection can concentrate virus at a site of tumor growth, but regression of distant tumors requires that the virus spread systemically or induce a systemic antitumor immune response.
• Neutralizing antibodies, hepatosplenic sequestration of the virus by macrophages and dilution of the virus in blood or tissue may limit the effectiveness of treatment.

viral spread

• Targeting viral spread to tumor cells can be accomplished by transductional targeting (modifying receptor tropism), transcriptional targeting (controlling virus gene expression with tumor-specific promoters), physiologic targeting (disrupting viral immune combat proteins), apoptosis targeting (disrupting viral antiapoptotic proteins) or miRNA targeting.

Safety

• Ideally, OVs should be nontransmissible. scrutiny is generally required for these viruses (e.g., from the US Department of Agriculture) to address the additional risks of environmental release and epidemic spread in domestic animals, particularly as they relate to agricultural livestock.

Conclusion & future perspective

Viruses are at last being harnessed for the benefit of cancer patients. The OV field has moved well beyond proof of principle in human studies, and virus engineering will be the key to its continued advancement in the coming years. Virtually every component of every naturally occurring or laboratory-adapted virus can be engineered and/or evolved to enhance its suitability for cancer therapy and we are currently witnessing unstoppable creative activity in this area. Safety is obviously of paramount importance in this relatively new field, and is therefore closely regulated from the design stage to clinical implementation. Considering the current trajectory of OV research, there can be little doubt that viruses are on their way to becoming one of the foundational modalities of future cancer treatment regimens.

viruses as anticancer drugs

In light of recent clinical progress, interest in the approach is burgeoning. One critical milestone was the 2015 marketing approval granted in Europe and the USA for talimogene laherparepvec (T-Vec, Imlygic™), an engineered HSV encoding GM-CSF. This virus, administered intratumorally every 2 weeks for malignant melanoma, led to complete resolution in 47% of injected tumors and boosted systemic antitumor immunity leading to resolution of 9% of distant un infected visceral tumors [12] . Subsequent clinical studies have shown that responses are more frequent and more durable when T-Vec (and other OVs such as coxsackievirus A21) is combined with immune checkpoint For reprint orders, please contact: reprints@futuremedicine.com review Maroun, Muñoz-Alía, Ammayappan, Schulze, Peng & Russell future science group inhibitor antibody therapy [13] . Another significant milestone was the demonstration that a systemically administered oncolytic measles virus can target and destroy disseminated cancer in a human subject [11] .

engineering viruses: a diversity of platforms

As might be expected, proponents of a given OV are typically able to advance strong arguments to support their choice of platform, emphasizing unique features such as replication kinetics, genome plasticity, targetability, seroprevalence and stability that may lead to superior oncolysis. However, it is too early to determine which unique viral characteristics will be the critical drivers of clinical success for a given cancer type. Reverse genetics systems are available for virtually all virus families, and the rules of engagement for new virus creation are well established. In general, the most effective strategy is to combine rational design with evolution, allowing each engineered virus to mutate and fully adapt to its intended target cells after it has been rescued. Biosafety oversight is in place at all academic centers responsibly engaging in virus engineering activities, and it is now a relatively straightforward matter to generate and test new virus configurations using what now amounts to the world's best lego set. Viral gene and noncoding sequences can be modified in a variety of ways to add or eliminate functions and nonviral genes or noncoding regulatory elements, whether synthetic or naturally occurring, can be added into viral genomes to confer additional desirable properties. The overarching engineering goal for the oncolytic virotherapy field is to generate viruses that can be efficiently delivered to disseminated tumors in the body where they will spread and selectively kill both infected and uninfected tumor cells, without causing collateral damage and posing no risk of transmission to the population.

Delivery of Ovs

Clearly, the ability to infect the activated endothelial cells of tumor neovessels would be an attractive targeting property for OVs to enhance entry into the tumor parenchyma by releasing viral progeny on the ablumenal side of the blood vessel. Intravascular coagulation in the capillary could also be provoked by virusinfected endothelial cells reacting with clotting and inflammatory factors. Virus engineering strategies have been pursued to achieve this goal, for example, by displaying polypeptide ligands echistatin and urokinase plasminogen activator on the surface of measles virus to target integrin αVβ3 and UPaR endothelial cell surface receptors, respectively [68, 69] . Also, at least one OV (vesicular stomatitis virus VSV) has been shown naturally capable of infecting neovessel endothelium in implanted mouse tumors, but the study did highlight the potential toxicity of the approach, namely intravascular coagulation requiring heparin therapy for its prevention [70] .
Mouse models with implanted flank tumors provide a convenient model for IT injection as the tumor is superficial, but in human patients with spontaneous malignancies tumors often develop internally in visceral organs. Melanoma, head and neck cancer, and lower gastrointestinal malignancies may be better suited for IT injections due to anatomical position, although with advanced interventional radiology using computed tomography or MRI image guidance to place the needle many more tumors may be accessible for IT virotherapy. One additional advantage of IT injection over the IV route is that the threshold concentration of input virus required to initiate a spreading infection in the tumor tissue can be more easily achieved.

• Limiting viral neutralization & clearance

Antibody neutralization has been shown to reduce the efficacy of systemically administered OVs such as measles, VSV and vaccinia in preclinical models [76] [77] [78] . When previous exposure has occurred, preformed antibody reduces the effectiveness of the initial treatment. However, the situation is typically far more problematic for second and subsequent doses, even for low seroprevalence viruses, because the first dose induces a powerful primary antiviral antibody response or boosts the pre-existing response [79, 80] . These troublesome responses can be constrained by coadministering immunosuppressive drugs such as cyclophosphamide. Cyclophosphamide administered concurrently with an OV has been shown to suppress or delay the development of humoral and cytotoxic antiviral T-cell responses [81] .
Another strategy for avoiding neutralization is to block the reticuloendothelial system with polyinosinic acid or with clodronate-loaded liposomes which poison or deplete splenic macrophages and Kupffer cells. This approach has been shown to slow the clearance of circulating virus particles that have been coated with antibodies or complement [86] [87] [88] .

Arming Ovs: extending the range

No matter how extensively an OV infection spreads through a tumor, a sizable percentage of the cancer cells will escape infection [61] . Killing of these uninfected (bystander) cancer cells is therefore critical if oncolytic virotherapy is to become a curative, as opposed to just a tumor debulking strategy. Bystander killing can be achieved, both locally at the site of a spreading infection and systemically at uninfected tumor sites, by genetically arming the virus using one of several possible approaches. For example, an OV can be engineered to encode a secreted protein that selectively mediates the destruction of neighboring and/or distant cancer cells. Alternatively, it can be armed with a 'suicide gene' or prodrug convertase whose encoded protein converts a harmless prodrug to a diffusible anticancer drug. In a third approach, the OV can be armed with a 'radioconcentrator gene' so that infected tumor cells are able to concentrate a β-emitting radioisotope whose emitted electrons damage adjacent uninfected tumor cells. Fourth, it can be engineered to fuse infected tumor cells with uninfected neighboring cells. And last, but by no means least, it can be engineered to express one or more genes capable of amplifying immune-mediated killing of uninfected tumor cells. Each of these approaches is discussed below using specific examples to illustrate the concepts.

• • Prodrug convertases

CD converts 5-flurocytosine (5-FC), an inert small molecule that is administered intravenously, to 5-flurouracil (5-FU), an antimetabolite that irreversibly inhibits thymidylate synthase and is an approved chemotherapeutic agent for a variety of cancers (anal, breast, colorectal, esophageal, stomach, pancreatic and skin) [29] . The argument supporting the CD/5-FC system is that local production of 5-FU in the OV-infected tumor will create a 5-FU concentration gradient that will expose tumor cells to a higher concentration of the drug compared with distant tissues, thereby ameliorating toxicity and enhancing the therapeutic index of the drug. However, 5-FU is freely diffusible so local production of the drug in a CD-positive tumor exposed to high concentrations of 5-FC can lead to systemic toxicity [140] . The dose of 5-FC must therefore be adjusted accordingly. CD has been incorporated into several OVs including adenoviruses, paramyxoviruses and poxviruses, but the one that has advanced ahead of all others in clinical testing is a C-type retrovirus, Toca-511, which is currently being evaluated in a Phase III clinical trial for the treatment of patients with malignant glioma [141] [142] [143] . Besides the TK and CD prodrug convertase systems, there are reports of OVs engineered to express the cyclophosphamide-activating protein CYP2B1, future science group www.futuremedicine.com the CPT11-activating secreted human intestinal carboxylesterase (shiCE) and the fludarabine phosphate activating purine nucleotide phosphorylase [144, 145] .

Safety

So far OVs have an exceptional safety record in the clinic with few serious adverse events and minimal mortality reported from human trials [166, 167] . However, since efficacy has also been limited, it remains possible that toxicity profiles may appear less favorable as doses are increased and as newer OVs or virus-drug combinations are advanced to clinical testing. Currently, there are over 40 active clinical trials ongoing using OVs alone or in combination with other therapies [168] . As the number of patients participating in trials grows so will the insight into rare adverse effects. The most common adverse effects reported in recent OV trials are fever and flu-like symptoms [169] . However, the potential for severe adverse reactions was much more clearly demonstrated in the earliest virotherapy trials undertaken in the 1950s and 1960s when deaths and severe adverse reactions were not infrequently recorded due to presumed OV replication in normal tissues, notably the brain, especially in immunocompromised cancer patients [4, 170, 171] .

• • Limiting pathogenicity

Understanding the selective pressures that operate within the tumor and the host, as well as the role of viral quasispecies in treatment outcomes is an active area of research. RNA and DNA viruses exist as a population of quasispecies or collections of related viral genomes undergoing variation and selective pressure [174] . Generation of quasispecies occurs when viral genomes are copied during the replication cycle. Viral polymerases typically have an error rate that introduces an average of one or more base mutations per progeny genome [175] . In general, the larger the virus genome, the lower the polymerase error rate. Picornaviruses are among the smallest viruses being developed for oncolytic therapy with positive sense RNA genomes ranging from 7 to 9 kb in length, and their RNA polymerases have a correspondingly high intrinsic error rate. Production of a virus for clinical application is a highly regulated process and involves multiple rounds of replication to achieve enough virus for patients. The viral product is therefore already a swarm of quasispecies at the time it is administered to the patient and mutates further as it undergoes additional rounds of replication in vivo. Studies of the mutation rates of viral polymerases, the generation of quasi species, the evolution of viral populations and the evolution between dominant subspecies within a virus population are therefore of great interest and relevance to the OV field.
Two so-called suicide genes, TK and inducible caspase 9, are of interest in this regard. Both of these genes have been used as a safety switches to eliminate genetically modified T cells that were causing graft versus host disease in human clinical trials [184] [185] [186] . Viruses engineered to encode TK have also been controlled with ganciclovir therapy [187] , but this cannot be considered a reliable safety switch because of the ever-present risk of the emergence of viral quasispecies with TK inactivating mutations. The development of a universal and highly reliable safety switch remains one of the significant research challenges facing the field of oncolytic virotherapy.

• • Prodrug convertases

Unlike secreted toxins, prodrug convertases do not pose a risk of increasing OV virulence because their toxic potential is manifest only in the presence of an exogenously added prodrug. For this reason, the approach has been extensively studied. Perhaps the most well-known convertase-prodrug combination is HSV TK, used with ganciclovir. TK converts ganciclovir to ganciclovir monophosphate which is further processed intracellularly to ganciclovir triphosphate, a DNA synthesis chain terminator that kills dividing cells as they enter S phase [138] . A major weakness of the TK-ganciclovir system, aside from its inability to kill nondividing tumor cells, is that it has very limited bystander killing potential. This is because ganciclovir monophosphate is not released from the cell in which it is generated, so does not impact uninfected tumor cells unless they are connected to the infected cell via gap junctions through which it can pass [139] . Encoding connexin, a gap junction protein, in the OV genome can enhance the bystander killing effect of TK, but attention is shifting to other convertases, most notably CD.

Arming viruses with transgenes

• Secreted toxins, prodrug convertases and immunostimulatory proteins have been incorporated into OVs to increase treatment efficacy.
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Abstract

Viruses replicate inside the cells of an organism and continuously evolve to contend with an ever-changing environment. Many life-threatening diseases, such as AIDS, SARS, hepatitis and some cancers, are caused by viruses. Because viruses have small genome sizes and high mutability, there is currently a lack of and an urgent need for effective treatment for many viral pathogens. One approach that has recently received much attention is aptamer-based therapeutics. Aptamer technology has high target specificity and versatility, i.e., any viral proteins could potentially be targeted. Consequently, new aptamer-based therapeutics have the potential to lead a revolution in the development of anti-infective drugs. Additionally, aptamers can potentially bind any targets and any pathogen that is theoretically amenable to rapid targeting, making aptamers invaluable tools for treating a wide range of diseases. This review will provide a broad, comprehensive overview of viral therapies that use aptamers. The aptamer selection process will be described, followed by an explanation of the potential for treating virus infection by aptamers. Recent progress and prospective use of aptamers against a large variety of human viruses, such as HIV-1, HCV, HBV, SCoV, Rabies virus, HPV, HSV and influenza virus, with particular focus on clinical development of aptamers will also be described. Finally, we will discuss the challenges of advancing antiviral aptamer therapeutics and prospects for future success.

Introduction

Vaccination is the most effective means to prevent individuals from being infected with pathogenic viruses [1] . However, some viruses, such as HIV-1 and hepatitis C virus, can evade the immune system, and thus impede the effectiveness of vaccines for those viruses [2] [3] [4] . Therefore, antiviral small molecule inhibitors that inhibit critical steps in the virus lifecycle in infected individuals are critically needed in the battle against virus infections. These inhibitors could curb the virus number in the body by interfering with viral entry into host cells, the function and assembly of viral replication machinery or the release of viruses to infect other cells [5] . Ideally, these antiviral agents should completely eradicate viruses from the body without affecting normal cellular metabolism. However, these features have not yet been achieved because of two main problems associated with use of these drugs: (1) the emergence of resistant viral strains and (2) cytotoxicity to host cells [6] [7] [8] . Some viruses, such as HIV-1, replicate its genome with high error rate [9] . These mutations in the viral genes that code for surface antigens and enzymes in the replication components often confer drug resistance capabilities to viruses [10] . Also, cytotoxicity often arises because antiviral drugs are usually designed to target and inhibit certain functional motifs of a viral protein. These motifs share a high degree of amino acid sequence similarity across different species and associated with conserved functions. One example demonstrating the motif similarities between viral and human proteins is helicases in which their DEAD-box domain is largely conserved in HCV helicase and human DDX3 RNA helicase [11] . Although off-target cross reactivity often leads to mild side effects, sometimes they are serious and can have a major effect on health. For example, the HIV-1 reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (zidovudine) is a nucleoside analogue that competes with natural deoxynucleotides (dNTPs) and is incorporated into the growing DNA chain by viral reverse transcriptases. Treatment with zidovudine delays the progression of AIDS, but does not clear the virus because drug resistant mutants usually arise [12, 13] . Moreover, long-term, high-dose treatment with zidovudine can cause serious complications, such as anemia, neutropenia, hepatotoxicity, cardiomyopathy and myopathy [12, [14] [15] [16] .

Aptamers as Antiviral Therapeutics

In summary, aptamer technology is well-suited for treating viral infections, and there are now many examples, illustrating that a wide range of viruses can be inhibited by aptamers and have potential for clinical applications.

HIV-1 Integrase

HIV-1 integrase (IN), a retrovirus encoded enzyme, catalyzes the insertion of proviral DNA into the host-cell genome [124] . Because HIV-1 IN is essential for retrovirus replication, it is a promising target for the development of antiretroviral drugs.

HIV-1 Gag Protein

Gag polyprotein is a major HIV-1 structural protein that is synthesized in the cytoplasm of infected cells, orchestrates the assembly and release of HIV-1 particles, and is necessary and sufficient for the formation of noninfectious virus-like particles [130, 131] . It is involved in both assembly and virion maturation after particle release, as well as early post-entry steps in virus replication. During virion maturation, Gag is cleaved into several component proteins: matrix protein (MA), capsid protein (CA), nucleocapsid protein (NC), the late domain (p6), and two small, spacer peptides, SP1 and SP2 [132] . These proteins play specific roles in the life cycle of HIV-1, and mutations in any domain of Gag lead to defects in particle assembly and loss of infectivity.

HIV-1 TAR Element

Virus-resistant transgenic T cells and macrophages that express HIV-1 TAR aptamer either alone or in combination with others nucleic acid therapeutics have been produced by lentiviral gene transduction of CD34 + progenitor cells. When the differentiated T lymphocytes and macrophages were challenged with HIV-1, marked resistances against HIV-1 infection was seen [58] . For example, a triple combination lentiviral construct composed of a U6-driven TAR RNA decoy, a U6-promoted HIV-1 tat/rev shRNA, and a VA1-promoted anti-CCR5 trans-cleaving hammerhead ribozyme efficiently transduced human progenitor CD34 + cells [155] . These transduced cells had increased suppression of HIV-1 over 42 days when compared to cells that received a single anti-tat/rev shRNA or double combinations of shRNA/ribozyme or decoy [155] . Recently, this triple construct has been used for ex vivo gene delivery to hematopoietic stem cells in a human clinical trial [156] . The lentivirally transduced cells successfully engrafted in all four infused patients by day 11 and no unexpected infusion-related toxicities were seen [156] . Long-term (up to 24 months) expression of an ectopically expressed shRNA and ribozyme in multiple peripheral blood cell lineages of two of the transplanted patients was observed [156] .

Inhibition of Hepatitis B Virus

Infection with hepatitis B virus (HBV) is one of the most prevalent chronic infections associated with serious clinical outcomes, including hepatitis, cirrhosis and liver cancer. It is estimated that HBV infection affects approximately 350 million people worldwide, but available therapies against HBV infection are limited in term of their efficacy and safety. Licensed interferon-α and nucleoside analogs, such as adefovir and lamivudine, are routinely used to treat chronically infected patients. However, although these drugs have therapeutic effects, their use is limited by the development of resistant HBV strains and unwanted side effects [169, 170] .

Inhibition of Severe Acute Respiratory Syndrome Coronavirus

Severe acute respiratory syndrome (SARS) is a life-threatening form of pneumonia that caused almost 800 deaths between 2002 and 2003 [176] . The causative agent of SARS is the SARS coronavirus (SCoV), which is an enveloped, single-stranded RNA virus with a genome of ~30 kb [176] . Within weeks of the start of the outbreak, the SCoV genome structure had been determined; it encodes two large replicative polyproteins, pp1a and pp1ab, and structural proteins including spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins [177] . The four structural proteins generate the virus particle. Although infection control measures and surveillance successfully contained the spread of SCoV in humans, there is still no effective therapeutic regimen against the virus.
The SCoV helicase (nsp13) is necessary for viral replication and proliferation, and thus offers an attractive target for development of anti-SCoV aptamers [178] . Two studies independently isolated potent nucleic acid aptamers, both DNA and RNA, against the protein. These aptamers demonstrated sub-nanomolar IC 50 values against the helicase [103, 104] . Interestingly, the SCoV helicase contains a functional domain with double-stranded nucleic acid unwinding and ATPase activities. However, only the nucleic acid unwinding activity of the protein was inhibited by aptamers, while the ATPase activity was not affected, suggesting that the aptamer may bind to the nucleic acid binding site of the helicase and block the unwinding activity [103, 104] . Intriguingly, this phenomenon is observed in HBV and HCV helicase inhibition mediated by aptamers. Although both DNA and RNA aptamers against SCoV helicase were selected, no similarity was seen in their sequences. Recently, a newly discovered coronavirus, namely Middle East respiratory syndrome coronavirus (MERS-CoV), was identified in Saudi Arabia that produced clinical symptoms resembling SARS [179] . Both MERS-CoV and SCoV belong to the genus beta-coronavirus [180] ; however, they are genetically distinct and their replicase domains only have less than 50% amino acid identity [181] . So, it is unlikely that the SCoV helicase aptamers will be used for the treatment of MERS-CoV infection.

Inhibition of Influenza Virus

Influenza is considered the most prevalent infectious disease in humans. Over the past century, this virus has accounted for three major pandemics, which cumulatively killed tens of millions of individuals [182] . In particular, H1N1 influenza, also known as Spanish flu, killed more than 20 million people around the globe [183] . Recently, the emergence of H7N9 and H5N1 in China has raised concerns [184, 185] . Fortunately, no sustained person-to-person spread of these viruses has been confirmed to date.
There are three major genetically distinct influenza viruses (types A, B and C). Influenza A, which can infect various animal hosts, has a high evolutionary rate and has been responsible for many of the major flu pandemics. On the other hand, influenza B and C, which usually cause relatively mild illnesses, are of less concerns [182] . Generally, influenza A viruses are enveloped, single-stranded RNA viruses that contain eight negative strand RNA segments and two membrane glycoprotein components, hemagglutinin (HA) and neuraminidase (NA) [186] . Mutations (antigenic drift) and re-assortment of genome segments (antigenic shift) have led to the evolution of various strains of influenza. These strains are classified according to the H number (for HA) and the N number (for NA). For example, an "H5N1 virus" designates an influenza A virus subtype that has an HA 5 protein and an NA 1 protein. The HA and NA proteins have served as prime targets for several of FDA-approved small molecules and vaccines [187] [188] [189] ; however, because influenza A is highly adaptive and these drugs are widely used, there are now strains that are resistant to these treatments.
The HA antigen is ubiquitously expressed (~900 copies per virus) on the surface of each viral particle and is required for binding and membrane fusion with host cells to mediate the early stage of viral infection [190] . So far, eight aptamers against influenza viruses have been developed for therapeutic purposes, and all of these aptamers target HA (Table 1 ). In an early study, Jeon et al. evolved DNA aptamers against a HA peptide (position: 91-261) that had a conserved receptor binding pocket among all H3 subtypes [105] . Based on a hemagglutination assay and enzyme-linked immunosorbent assay (ELISA), the A22 aptamer was found to be the most efficient at blocking hemagglutination of various H3N2 strains (A/Texas/1/77, A/Japan/57 and A/Port Chalmers/1/73) when used at picomolar ranges [105] . Moreover, the A22 aptamers not only inhibited the hemagglutinin capacity of the virus, but also prevented the viral binding and entry to cells [105] . Importantly, intranasal administration of the A22 aptamer into influenza-infected mice reduced the virus burden by 95%, which is comparable with the amount of inhibition seen in mice after treatment with NA-based drugs currently available in the clinic [105] .
In a similar study, Gopinath et al. devised a novel selection strategy that used the whole virus, instead of purified proteins, as a selection target to isolate aptamers [107] . In this approach, an H3N2 subtype (A/Panama/2007/1999) was initially incubated with the RNA library, followed by counter-selection with another subtype of H3N2 A/Aichi, which is closely related to A/Panama/2007/1999 [107] . In this way, the aptamers (P-30-10-16) were identified as blocking viral-cell interactions by binding to the HA of the target strains and failing to recognize other influenza A subtypes [107] . Although there are many parallels between aptamer and antibody technology, this study clearly demonstrated that the isolated aptamers displayed different molecular recognition mechanisms and had >15-fold higher binding affinity to the HA in comparison with the commercially available anti-HA monoclonal antibody [107] . These aptamers not only have the potential to be developed as therapeutic agents, but could be used as genotyping reagents for identifying different influenza subtypes. Recently, Gopinath et al. isolated 2'-fluoro modified aptamers that specifically targeted the HA protein of H1N1 viruses (A/California/07/2009). One aptamer, D26, was further optimized and displayed strong binding affinities, as determined by surface plasmon resonance. Moreover, this aptamer could distinguish the HA of H1N1 from other subtypes and interfere with HA-glycan interactions.

Inhibition of Rabies Virus

Rabies is a viral disease that causes nerve damage and death in many mammals. The causative agent of rabies is the rabies virus (RABV), which can be transmitted between species, often through saliva transferred during a bite from a rabid animal. Currently, there is no effective treatment for rabies once symptoms develop, and death occurs within weeks. Although post-exposure rabies treatments are available to treat RABV-infected patents before the virus invades the central nervous system, the virus still causes about ~60,000 deaths annually worldwide [192] .

Inhibition of Human Papillomavirus

Human papillomavirus (HPV) infects keratinocytes of the skin or mucous membranes, and is considered one of the most common sexually transmitted diseases. HPV causes 440 million infections and 288,000 deaths worldwide [194] . There are more than 180 genotypes of HPVs. Although the majority do not cause severe clinical outcomes, some types of HPV are linked to cervical and throat cancers [195, 196] . For example, infection with HPV16 (50% of cases) or HPV45 (30% of cases) is strongly connected to the cause of cervical cancer [197, 198] , which is the second most common cancer in women, while HPV18 is commonly found in patients with head and neck cancer [199] . Recent progresses in HPV vaccines, such as Gardasil ® (Merck) and Cervarix ® (GSK, London, UK), are effective means to prevent infection with certain types of HPV, but are ineffective at treating those who are already infected [200] . In fact, because the HPV family is so large, it is challenging to design effective and universal prophylactic and therapeutic approaches against this disease [200] .

Inhibition of Herpes Simplex Virus

Herpes simplex virus (HSV) often infects primary epithelial tissues before invading the nervous system, where it becomes latent. There are two forms of HSV, HSV1 and HSV2, which share 70% genome identity [206] . Both HSV-1 and HSV-2 are ubiquitous and highly contagious, but there is no effective treatment that can eradicate the virus totally from the body [207] . Akin to other antiviral drugs that can block viral entry, anti-HSV aptamers were evolved against a glycoprotein D (gd) of HSV-1 and HSV-2 [119] . The gD protein is an essential viral surface protein that is required for binding to the host cell receptors for viral entry and fusion [208] . In HSV-1 aptamer selection, an aptamer was evolved that can specifically bind to the HSV-1 gD protein using protein-based SELEX. Although the gD proteins of HSV-1 and HSV-2 are highly homologous (86% sequence identity) [209] , the aptamer can discriminate between the two. Chemical modifications of the HSV-1 aptamers with 2' fluoro increased the stability of the RNA against nuclease degradation, but did not greatly affect their binding capacity [119] . Furthermore, the aptamer effectively inhibited HSV-1 entry to the cell by blocking the interaction between the gD protein and the HSV-1 target cell receptor [119] . Similarly, the HSV-2 aptamers were selected against the gD protein and counter-selected against IgG to remove non-specific binders [120] . A panel of aptamers was shown to neutralize HSV-2 infection dependent on two entry receptors, Nectin 1 and HVEM. One effective aptamer, G7a, inhibited HSV-2 infection in Vero cells with an IC 50 of 20 nM and its inhibition required the ACCCA motif for binding and function [120] . Given the importance of HSV-2 infection in enhancing the risk of HIV-1 transmission, this aptamer is proposed to be incorporated into a microbicide to reduce the spread of HSV-1 as well as HIV-1.

Conclusions

Furthermore, as typical nucleic acid entities, naked nucleic acid aptamers are relatively small and are sensitive to nuclease degradation. Their average diameter is usually less than 10 nm, and therefore they are rapidly removed from the blood by renal clearance [44] . Thus, the intrinsic physicochemical features of aptamers pose serious challenges for their transport to infected organs or cells, such as the liver and central nervous system, following systemic administration into the blood stream. Typically, respiratory viruses, such as influenza viruses and SCoV, are well-suited for targeting with aptamer therapeutics because the upper airways and lungs are relatively easy to access as target organs [1] . Therefore, it may be possible to block respiratory virus infections by using an aptamer-containing aerosol [211] . Similarly, sexually transmitted viruses, such as HIV-1 and HPV, might be targeted by intravaginal application of a microbicide or cream that contains the neutralizing aptamers [211] . Although a topical microbicide might protect women against the viruses before the intercourse, the female genital tract is abundant in various nucleases that can degrade nucleic acid aptamers, even the 2' F modified ones, in minutes [212] . One way to improve the aptamer stability is to chemically introduce the 2'-O-Me modifications on the purine nucleotides or phosphorothioate linkages [212] . Moreover, zinc ions can be incorporated into the formulation because nucleases are sensitive to inhibition by zinc ions [212] . Recently, Wheeler et al. developed a topical microbicide containing chemically modified CD4 aptamer-siRNA chimeras that target the HIV co-receptor CCR5, gag and vif for the protection from sexual transmission of HIV-1. The chimeras were stabilized and formulated in a hydroxyethyl cellulose gel, which is a FDA-approved polymer already used in HIV-1 clinical trials, to achieve durable gene knockdown and inhibit HIV-1 transmission in mice [213] .

Introduction

Aptamers are in vitro evolved nucleic acids that are capable of performing a specific function [17, 18] . The process to identify a functional ligand from a vast population of random sequences is called Systematic Evolution of Ligands by Exponential enrichment (SELEX). Typically, an initial combinatorial library contains a central random region with 30 to 70 nucleotides flanked by a fixed sequence at both ends. The fixed sequence is used for PCR amplification during each SELEX round. Random sequences with at least 10 12 entities represent extraordinary molecular diversity and structural complexity to screen high affinity and bioactive aptamers to the target. To date, a dozen of SELEX methodologies have been developed in isolating aptamers against purified proteins or even whole cells (or whole viruses) [19] [20] [21] . The use of purified proteins as selection targets has the advantage of easy control to achieve optimal sequence enrichment during the SELEX. But whole cell or virus selection is preferred, when the biomarker is unknown. Moreover, since the target protein may be present in a modified form or exist as a protein complex that may be masked and therefore inaccessible to the aptamers, it reflects a more physiological condition when the protein is displayed on the cell surface rather than isolated as purified proteins.

Aptamers as Antiviral Therapeutics

Aptamers are single-stranded non-coding nucleic acids screened by SELEX to perform a defined function by forming a complex structure complementary to their targets. Because of their versatility in structure and function, aptamers have many advantages over small molecules and biologics for therapeutic applications, particularly as antiviral therapeutics [26, 39] . First, traditional antiviral small molecules, such as HCV protease inhibitors (boceprevir, Victrelis ® , Merck, Whitehouse station, NJ, USA) and HIV integrase inhibitor (elvitegravir, GS-9137, Gilead, Foster City, CA, USA), fit into crevices on protein surfaces, especially into the active site of enzymes to inhibit their catalytic activity, while aptamers can also form clefts that bind protruding parts of protein [26, 40] . Thus, aptamers can bind more specifically and tightly because they extend surface contact with their targets, and thereby disrupt protein-protein or protein-nucleic acid interactions more effectively than small molecules [41] . For example, one HIV reverse transcriptase (RT) aptamer is able to mask ~2600 Å 2 of the enzyme's surface, which is likely to slow down the evolution of resistant viruses [41] [42] [43] .
A third advantage is that potent aptamers can be identified through the iterative SELEX process and evolved in a test tube within a month. Canonical SELEX requires a purified soluble form of the target proteins; however, if the purified protein does not fold into a stable conformation or the native protein exists as a protein complex that may be inaccessible for aptamer screening, then a cell-SELEX strategy that uses whole living cells or inactivated viruses as targets may be useful for aptamer identification [36, [53] [54] [55] . Many aptamers were developed against viral proteins (e.g., HIV reverse transcriptase and HCV NS3), nucleic acid elements (e.g., HIV TAR, HCV IRES), as well as the entire viruses (e.g., influenza A virus) ( Table 1 ). In addition, since the cell-SELEX approach relies on the differences between two populations of cells (infected cells versus healthy cells), it can be performed when the target is unknown and without going through the tedious process of protein expression and purification [53] . Moreover, large-scale manufacture of high quality current good manufacturing practice (cGMP)-grade nucleic acids is possible through solid-phase chemical synthesis, and aptamers, composed entirely of nucleic acids, are regarded as chemical drugs by FDA instead of biologics. These features all speed up the process of aptamer research and development.

HIV-1 Reverse Transcriptase

Currently, RT is one of the main drug targets for HIV/AIDS, and many nucleoside inhibitors (e.g., AZT, 3TC, ddI, ddC, d4T) and non-nucleoside inhibitors (e.g., snevirapine, delavirdine, efavirenz) are currently used in patients [61, 62] . In 1992, Tuerk and Gold identified RNA aptamers against HIV-1 RT from an RNA pool that spanned a 32-nt random region by using in vitro SELEX [42] . The selected anti-RT aptamer contained a consensus sequence that resulted in the formation of an RNA pseudoknot. This RNA pseudoknot was reported to bind to HIV-1 RT in the picomolar range [63] and had an inhibitory effect on HIV-1 replication [64, 121, 122] . Since this discovery, numerous RNA or DNA aptamers of various lengths have been raised against HIV-1 RT and their uses for inhibiting the virus replication have been explored. For example, single stranded DNA aptamers (ODN112) with high affinity for the RNase H domain of HIV-1 RT were isolated by using recombinant RTs [66] . The selected DNA ligands could greatly diminish the infectivity of HIV-1 in human cells [66] . Similarly, double-stranded DNA thioaptamers (R12-2) that contained thiophosphate backbones were selected against the RNase H domain of HIV-1 RT by using in vitro combinatorial selection methods [67] . One lead thioaptamer could specifically bind to HIV-1 RT (dissociation constant (K D ) of 70 nM) and significantly inhibited HIV-1 infection in a dose-dependent manner [67] . In addition to aptamers against wild-type (WT) HIV-1 RT, several RNA aptamers targeting a drug-resistant HIV-1 RT, mutant 3 (M3) have been isolated [65] . One of these aptamers, M302, bound M3, but did not have significant affinity for WT HIV-1 RT, which would likely allow specific detection of HIV-1 RT variants [65] .
Anti-RT aptamers have been intracellularly expressed together with flanking, self-cleaving ribozymes to generate aptamer RNA transcripts that have minimal flanking sequences [122] . The expression of aptamers in the HIV-1 infected cell led to encapsidation of the aptamer in the virion particles, which subsequently blocked the HIV-1 replication [122] . These aptamers also effectively suppressed drug-resistant variants and other HIV subtypes (e.g., subtypes A, B, D, E, and F) [122] . More recently, Lange et al. further optimized this expression cassette, in which an extended, tertiary-stabilized hammerhead ribozyme was replaced to enhance its self-cleavage activity [123] . Stable clonal cell lines that expressed aptamers from these optimized constructs strongly suppressed infectious virus when used at a high multiplicity of infection (MOI) [123] .

Inhibition of Hepatitis C Virus

Hepatitis C infection is a serious public health problem that chronically affects 170 million people worldwide [157] . Infection with hepatitis C virus (HCV) places individuals at high risk for scarring of the liver and ultimately leads to cirrhosis and liver cancer. HCV has limited therapeutic options, including a combination therapy of PEGylated interferon-α and ribavirin, which is not effective against certain HCV genotypes [158] . Thus, there is a need for new HCV therapies. The HCV is a 9.6 kb enveloped virus with a positive linear, single stranded sense RNA genome that contains a 5' non-translated region (NTR), protein coding region and 3' NTR for viral replication [159] . The 5' coding region consists of a single polyprotein of 3,010 amino acids, which is further processed by viral and cellular proteases into structural (C, E1, E2 and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins [159] . Because some of these proteins are considered important for viral replication and proliferation, they are prime targets for the development of antiviral therapies.

HCV Non-Structural Protein (NS) 3

Because the G6-16 aptamers had low efficacy, a new set of aptamers against the NS3 proteins was re-evolved, using a slightly modified selection strategy [91] . A truncated protease domain of NS3 was used as a selection target instead of the entire NS3 protein, and a 30-nucleotide randomized core region was used for SELEX instead of a 120-nucleotide randomized core region [91] . Consequently, three classes of conserved aptamers were identified after 9 rounds of selection. The dissociation constant of these aptamers was about 10 nM, which could inhibit 90% of the protease activity of both truncated NS3 and entire NS3 in a non-competitive manner [91] . Mutational analysis of the aptamer G9-1 revealed that the sequence required for protease inhibition was in stem I, stem III and loop III of the aptamers [91] . Use of these aptamers in vivo required that they may be expressed intracellularly. Therefore, an in vivo aptamer expression system was designed that combined the G9-1 aptamer with cis-acting genomic human hepatitis delta virus (HDV) ribozymes to construct a chimeric HDV-ribozyme-G9-aptamer [162] . The G9-1 aptamer was inserted into the non-functional stem IV region of the HDV ribozyme so that the aptamer would be protected from exonuclease degradation in the presence of the tightly packed structure of the HDV ribozyme and hence remains stable and functional in cells [162] . Furthermore, the chimera was attached with a nuclear export signal (CTE-M45) and arrayed as a tandem repeat in the expression plasmid in order to increase its cytoplasmic level in cells. Evaluation of this expression system in cultured cells showed efficient protease inhibition activities similar to the aptamer G9-1 alone [162] .

HCV Non-Structural Protein 5B

Other HCV NS5B aptamers include 2' F modified aptamers evolved by Lee et al. Transfection of this aptamer into cells significantly suppressed replication of both HCV genotype 1b and 2a, but did not generate escape mutant viruses or cause cellular toxicity [95] . This potent aptamer was further modified through conjugation of cholesterol or galactose-polyethylene glycol ligands to increase its stability and specificity for the liver.

Inhibition of Influenza Virus

Although the influenza A and B have different biological mechanisms, as well as evolutionary characteristics and genetic lineages, they are largely similar in the symptoms they cause during infection [191] . Therefore, aptamers that can specifically recognize influenza B virus would be very useful for both therapeutic and diagnostic perspectives. Similarly, RNA aptamers specific for influenza B were isolated against the influenza virus B/Johannesburg/05/1999. The isolated aptamers bound specifically to the HA of influenza B virus, but not to that of influenza A virus [112] . Interestingly, HA recognition by these aptamers required magnesium ions in order to inhibit HA-mediated membrane fusion [112] .

Inhibition of Rabies Virus

Liang et al. twice used a cell-SELEX approach to attempt to isolate DNA aptamers against RABV. In the first study, aptamers were isolated through 35 iterative rounds of selection [113] . The library was first incubated with rabies virus (CVS-11)-infected baby hamster kidney (BHK)-21 cells, followed by counter-selection of uninfected BHK-21 cells. This process identified five aptamers that inhibited replication of RABV [113] . Although the aptamers bound very tightly to the virus, their inhibitory capacity was limited and they did not cross-react with other rabies strains. This led the group to use the same cell-SELEX approach to select a new batch of aptamers. In their second attempt, 16 DNA aptamers were isolated, but only the aptamer, FO21, inhibited significantly the replication of RABV but not other related viruses [114] . The FO21 aptamers were further modified with polyethylene glycol (PEG). In vivo testing showed that the aptamers effectively protected infected mice, enabling a 87.5% survival rate when mice were treated with aptamers for a day prior to challenge with RABV [114] . However, almost all mice died when they were challenged with RABV prior to treatment with aptamers because RABV replicates rapidly and produces a large amount of infectious particles once it enters the brain, making the aptamer ineffective [193] .

Conclusions

Since the first publication of SELEX over two decades ago, the development of aptamer technology has advanced rapidly from the laboratory to early or mid-stage clinical development [210] . Aptamers, also described as chemical versions of antibodies, can inhibit their targets through specific and strong interactions that are superior to those of biologics and small molecule therapeutics, and yet avoid the toxicity and immunogenicity concerns of these traditional agents derived from their nucleic acid compositions [26] . The latest advances in SELEX technology and chemical conjugation methods have given aptamers remarkable potential to be used as "smart bombs" that delivers secondary therapeutic cargos to diseased cells. Several examples (e.g., aptamer-siRNA chimeras, aptamer-ribozyme chimeras and aptamer-aptamer chimeras) discussed in this review demonstrate complementary and versatile approaches for combining the strength of aptamers with other nucleic acid-based therapeutics, offering a polyvalent platform for treating various diseases [23, 38, 143] . These chimeras offer a huge potential to provide enhanced therapeutic potency and reduced cellular toxicity of the drug. However, despite the substantial advances described above, no aptamers have yet reached clinical development pipeline for antiviral therapy. Aptamers whose targets are expressed intracellularly are unlikely to be used in the clinic because aptamers are hydrophilic and therefore cannot pass through epithelia and the hydrophobic plasma membrane [211] . Consequently, only aptamers that target extracellular viral proteins or capsid proteins of virions, such as HIV-1 gp120 or influenza A HA, are likely to be suitable for clinical therapeutic development [211] . In addition to the aptamer chimera approach, another potential approach to solve this problem would be to use a viral vector that will transiently express the aptamer intracellularly. For example, Bai et al. designed lentiviral vectors that encode anti-HIV ribozymes together with anti-Tat aptamers [51] . The construct was tested in HIV-infected humanized mice and was able to inhibit virus replication [51] . However, it was not conclusive whether the aptamers contributed any inhibitory effect.

Introduction

Generally, SELEX comprises of cycles of four sequential steps: (1) binding to the target; (2) partition of target-bound aptamers; (3) recovery of target-bound aptamers; and (4) amplification of recovered sequences [22] [23] [24] . The selection cycle is complete when a functional aptamer sequence is enriched among the random sequence library. Since the inception of SELEX technology two decades ago, the extraordinary diversity of molecules screened in this manner has led to the discovery of aptamers that bind with exquisite specificity and extraordinary strength [25, 26] . Macugen (Pfizer), which is used to treat age-related macular degeneration, was the first aptamer therapeutic approved by United States Food and Drug Administration (FDA) and has proven to be a milestone in the aptamer history [27, 28] . Many novel aptamers are currently being evaluated in clinical trials for treating life-threatening diseases, such as acute myeloid leukemia, renal cell carcinoma, acute coronary syndrome, and choroidal neovascularization [29] [30] [31] [32] [33] . In addition, because aptamers can easily be conjugated to chemicals and manufactured, the use of aptamer chimeras for targeted delivery and enhanced potency of secondary agents has progressed rapidly [23, [34] [35] [36] [37] [38] . In this review, we will focus on the recent progress and prospective use of aptamers against a variety of human viral pathogens; representative examples of aptamer chimeras will be highlighted. Finally, we will discuss the challenges of advancing antiviral aptamer therapeutics and the prospects for future success.

Aptamers as Antiviral Therapeutics

Second, because the folding of nucleic acid aptamers is mainly governed by Watson and Crick base-pairing, aptamers can intrinsically form various loops and diverse thermodynamically stable structures in a highly programmable way [44] . Thus, aptamers can serve as building blocks for bottom-up fabrication of an aptamer chimera system [45, 46] . For example, aptamers and other nucleic acid therapeutics can link with a nanovector that has polyvalent functionalities [46] [47] [48] [49] . Several novel aptamer-based chimeras, such as aptamer-aptamer chimeras, aptamer-ribozyme chimeras and aptamer-siRNA chimeras, have been designed [50] [51] [52] . An innovative use of such chimeras is as drug delivery carriers for cell-or tissue-specific targeted delivery [34] . These cell-internalizing aptamers act as "smart bombs" that only target a particular cell population and deliver their therapeutic cargos specifically into the cell, thereby offering enhanced therapeutic efficacy and reduced cellular toxicity [37] .
20 section matches

Abstract

Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 mg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 mg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFkB, JNK, and p38, redoxsensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.

Introduction

Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .

Cytopathogenic effect (CPE) reduction assay

The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.).

Influence of glycyrrhizin on monocyte recruitment of H5N1-infected A549 cells

Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).

Influence of glycyrrhizin on H5N1-induced caspase activation and nuclear export of ribonucleoprotein (RNP) complexes

Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ).

Discussion

Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity [53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs.

Introduction

H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .

Discussion

In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease.

Virus strains

Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates.

Influence of glycyrrhizin on replication of H5N1 virus in A549 cells

The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .
To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection.

Influence of glycyrrhizin on H5N1-induced caspase activation and nuclear export of ribonucleoprotein (RNP) complexes

Influence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ).
In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ).

Discussion

Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations.
Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations.
Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells.
Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] .

Introduction

The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] .

Detection of influenza A nucleoprotein

To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).

Cytopathogenic effect (CPE) reduction assay

Unless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus.
27 section matches

Abstract

Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer PLOS Neglected Tropical Diseases | The infection of cells by enveloped viruses involves the fusion of membranes between viruses and cells. This process is mediated by viral fusion proteins that have been grouped into at least three structural classes. Membrane-enveloped hantaviruses are worldwide spread pathogens that can cause human disease with mortality rates reaching up to 50%, however, neither a therapeutic drug nor preventive measures are currently available. Here we show that the entrance of Andes hantavirus into target cells can be blocked by fragments derived from the Gc fusion protein that are analogous to inhibitory fragments of class II fusion proteins. The Gc fragments acted directly over the viral fusion process, preventing its late stages. Together, our data demonstrate that the hantavirus Gc protein shares not only structural, but also mechanistic similarity with class II fusion proteins, suggesting its evolution from a common or related ancestral fusion protein. Furthermore, the results outline novel approaches for therapeutic intervention.

Introduction

The genus Hantavirus of the family Bunyaviridae comprises diverse viruses that are highly pathogenic to humans. In Asia and Europe the Hantaan, Seoul and PUUV viruses cause hemorrhagic fever with renal syndrome, while in America ANDV and Sin Nombre virus can lead to hantavirus pulmonary syndrome with mortality rates above 30% [1] [2] [3] [4] [5] . Hantaviruses are currently the most lethal human pathogenic viruses known to occur in America and, due to the lack of Food and Drug Administration (FDA)-approved preventive or therapeutic measures [6, 7] , they have been classified as category A pathogens. Like other members of the Bunyaviridae family, hantaviruses have a tri-segmented single strand RNA genome of negative polarity packaged by the nucleoprotein into viral ribonucleocapsids, which are also associated to the viral RNA-dependent RNA polymerase [8] . A lipid membrane, which further envelopes the viral ribonucleocapsids, anchors the viral Gn and Gc glycoproteins. This viral envelope is derived from a host cell membrane during the budding process of the virus [9, 10] . To infect new cells, hantaviruses bind to cell surface receptors [11] [12] [13] [14] , and are subsequently taken up by endocytosis [15, 16] . A crucial step in viral cell entry is the fusion of the virus with an endosomal membrane of the host, escaping from its degradation in lysosomes. Yet, little is known about the fusion process of hantaviruses; however, our recent data show that the low pH of endosomes triggers a non-reversible fusion process, in which the Gc protein inserts into target membranes and forms a highly stable post-fusion homotrimer [17] .

ANDV infectivity titration

Production of simian immunodeficiency virus (SIV) vectors pseudotyped with vesicular stomatitis virus (VSV) G protein or ANDV glycoproteins and transduction SIV vectors bearing the VSV glycoprotein were prepared as indicated elsewhere [59] . Briefly, 293FT cells were transfected with 8 μg of plasmid coding for SIV Gag-Pol (pSIV3+), 8 μg of plasmid encoding GFP as an RNA minigenome (pGAE1.0) [60] , and 4 μg of plasmid coding for the envelope protein G of VSV (pVSV-G, Clontech). Alternatively, the plasmid pI.18/GPC coding for ANDV glycoproteins was used to generate SIV vectors pseudotyped with ANDV glycoproteins. At 48 h post-transfection, supernatants containing pseudotyped particles were concentrated by centrifugation at 100,000 g for 75 min. Different dilutions of VSV-G pseudotyped SIV vectors were incubated for 1 h with Vero E6 cells in the presence and absence of protein or peptide inhibitor candidates. Three days later, the expression of GFP in transduced cells was analyzed by flow cytometry (FACScan, Becton Dickinson). !10.000 cells were counted for each experimental condition.

stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.

Introduction

In general, virus-cell membrane fusion is thought to be accomplished by multiple steps [reviewed in 18, 19] . After the activation, viral fusion proteins insert a fusion peptide or fusion loop into a target membrane. At this intermediate stage, the fusion peptide is located at one end of the fusion protein while the opposite end is anchored to the viral envelope membrane by a transmembrane region, thereby bridging the viral and cellular membranes. By undergoing additional conformational changes, the fusion protein is thought to pull both anchors together, until it reaches a hairpin-like conformation in which both membrane-inserted domains are located at the same end of the protein. Once the opposed membranes have been brought into a close distance by the introduction of local membrane curvature, the fusion of the outer leaflets of the membranes produces a hemifusion intermediate, followed by the full fusion of the membranes. The fusion culminates in the formation of a fusion pore through which the virus can deliver its ribonucleocapsids into the cell cytosol to initiate replication. Viral fusion proteins are currently grouped into at least three different classes based on their molecular structures: class I fusion proteins have a high alpha helical content, class II proteins consist principally of beta sheets, while class III fusion proteins include characteristics from the first two classes [reviewed in [19] [20] [21] . Early in silico and in vitro analyses suggested that the Gc glycoprotein of hantaviruses shares structural similarity with class II fusion proteins [22, 23] . This notion has also been proposed for other members of the Bunyaviridae [24] [25] [26] [27] [28] , and the crystal structure of Gc from Rift Valley Fever virus (RVFV) ultimately confirmed this notion for phleboviruses [29] .
Class II fusion proteins are composed of three domains (I-III) and a stem region that connects the ectodomain to the transmembrane anchor [30] [31] [32] [33] [34] . To adopt a hairpin-like structure, DIII moves towards the fusion loop [35, 36] , while the stem region, which is connected to the transmembrane anchor, is thought to follow the movement of DIII by folding against the trimeric core formed by the fusion protein [37] [38] [39] . The extensive conformational changes that occur during the fusion process offer opportunities to disrupt the fusion cascade, thereby blocking viral infection. Ligands that bind selectively to an intermediate form of the fusion protein preceding its post-fusion conformation can delay or inhibit viral entry. In the case of human immunodeficiency virus 1, which has a fusion protein with a class I fold, there is a licensed drug based on a 20-residue peptide [reviewed in 40, 41] . This peptide comprises a partial sequence of the outer layer of the trimeric post-fusion hairpin conformation of gp41 and binds to the trimeric core of the fusion protein in its extended intermediate conformation, preventing the foldback reaction [reviewed in 42, 43] . Among class II proteins, DIII and the stem region form the outer layer of the trimeric post-fusion conformation [35] [36] [37] . Liao & Kielian (2005) showed that the addition of soluble DIII with or without the stem region of Semliki Forest virus E1 protein and soluble DIII of Dengue virus type 2 (DV2) E protein inhibit the entry of the respective virus into cells and confirmed a common inhibitory mechanism of class I and class II fusion proteins [44] . Other studies have shown that peptides derived from the stem region of the fusion protein of flavi-and phleboviruses also inhibit viral cell entry [45, 46] . The binding of stem peptides to fusion proteins is thought to prevent the post-fusion conformation as in the case of DIII; however, their amphipathic characteristics seem to allow binding to the virus before attachment to the cell, and are hence thought to be carried on the virus into endosomes [47, 48] . This characteristic provides an advantage for the delivery of the inhibitor to the site of virus-cell membrane fusion when this process occurs in a closed endosomal compartment. Here, we hypothesized that if hantavirus Gc shares mechanistic similarity with class II fusion proteins, then it should be inhibited with strategies used for other class II fusion proteins. To test this hypothesis, we predicted and produced DIII and the stem region of ANDV Gc and assessed them for ANDV inhibition. Our results show that indeed both, recombinant DIII and synthetic stem peptides, interfered with the ANDV infection, acting at late stages of the ANDV fusion process.

ANDV infection mediated by virus-plasma membrane fusion

Vero E6 cells were pre-chilled on ice for 10 min with 20 mM NH 4 Cl. The adsorption of ANDV (MOI = 0.2) was performed at 4°C for 1h. Next, cells were washed, and the fusion of the virus with the plasma membrane was triggered by incubation in low pH media (E-MEM, 20 mM sodium succinate, pH 5.5) for 5 min at 37°C in the presence and absence of inhibitor candidates. Next, cells were washed, and infection was followed by incubation for 16 h at 37°C in the presence of 20 mM NH 4 Cl. Subsequently, viral infection was assessed as described above.

Prediction of DIII and stem fragments with inhibitory potential

For the hantavirus Gc protein, we predicted DIII and the stem region by sequence alignments with known class II fusion proteins and subsequent model derivation. These proteins included among others the more recently crystallized RVFV Gc [29] . None of the new sequence alignments achieved a higher sequence identity, greater cysteine match or model validation scores compared to the alignment used for the original Gc model structure [23] , which is based on the pre-fusion structure of the tick-borne encephalitis virus (TBEV) E protein (PDBid: 1SVB) [31] . For this reason, we used the alignment from the original ANDV Gc model [23] to identify a putative DIII in ANDV Gc (Fig 1A and 1B) . The sequence that was derived from this model (residues Asp315-Leu414) was termed ANDV DIII, and served as template to predict a putative DIII in Gc of other hantaviruses such as PUUV. We subsequently defined the putative stem region in ANDV Gc as the sequence encompassing residues Leu414-Asn456, which corresponded to the region between the C-terminal end of the predicted DIII and the predicted Gc transmembrane region obtained by the TMpred server [64] (Fig 1C) .

Production and characterization of ANDV DIII and stem fragments

The production of DIII from different class II fusion proteins, including those of flaviviruses and alphaviruses, has been previously established in E. coli [52, [67] [68] [69] [70] [71] . The feasibility of preparing soluble DIII from flavi-and alphaviruses in a prokaryotic expression system retaining the native structure may be related to its globular IgG-like fold and lack of glycosylation [44] . The purification of DIII is generally achieved from inclusion bodies followed by refolding [44, 71] , or from the supernatant of the cell lysate [52, [67] [68] [69] [70] . Here, we prepared three recombinant DIII proteins; DIII derived from ANDV Gc with or without N-terminal His-tag (ANDV hDIII, ANDV DIII), and DIII from PUUV Gc with N-terminal His-tag (PUUV hDIII). The proteins were obtained from the supernatant of E. coli BL21 or Origami 2(DE3) cells, and eluted after their purification as a single peak detected by absorbance at 280 nm. Fig 2A shows an elution profile example of purified ANDV DIII from the last size exclusion chromatography column together with the homogeneity of the preparation assessed by SDS-PAGE. Because the predicted ANDV and PUUV DIII sequences contain eight highly conserved cysteine residues, the DIII proteins were next characterized for the presence of disulfide bridges by reduction and subsequent alkylation. An increase in the electrophoretic mobility could be detected for reduced ANDV DIII, ANDV hDIII and PUUV hDIII compared to its unreduced control (Fig 2B) , indicating that the cysteines seemed to be arranged in disulfide bridges. We next explored the presence of secondary structure elements in DIII from hantaviruses by circular dichroism. The spectra showed a unique negative maximum at 209 nm (Fig 2C) , confirming the presence of secondary structure. Deconvolution of the circular dichroism spectra into four components by different servers [54] [55] [56] indicated that DIII contained 40-41% β-sheets,~60% random coils and turns with an α-helical content close to zero. This composition coincides with the high content of β-sheets and turns observed in DIII of class II fusion proteins [31] . Taking these data together, the monomeric form of recombinant ANDV DIII in solution, the presence of disulfide bridges, the secondary structure content, and the solubility of the recombinant protein (>20 mg/ml) indicate that DIII was folded.

Inhibition of ANDV cell entry by exogenous DIII and stem fragments

Once the predicted DIII and stem fragments were synthesized, purified, and characterized, we measured their inhibitory activity against ANDV during virus cell entry via the native, endosomal infection route. For this purpose, ANDV was incubated with Vero E6 cells in the presence of the Gc fragments. After 1 h incubation, the cells were washed and the infection monitored after 16 h based on an earlier established protocol [57] . ANDV DIII and ANDV hDIII reduced ANDV infection up to 60%, at 3-4 μM (Fig 3A) . The N-terminal His-tag did not further improve inhibition of viral infection, as observed for the alpha-and flavivirus DIII proteins [44] . Interestingly, PUUV hDIII did not show any cross-inhibition of ANDV. This result was unexpected since cross-reactivity by DIII has been reported within the alphavirus and flavivirus genera [44, 72] . It is likely that the absence of cross-inhibition was due to the presence of the N-terminal His-tag in PUUV DIII (see results below; Fig 4C) .
To further explore the specificity of the Gc fragments on viral inhibition, we used as a model the unrelated vesicular stomatitis virus (VSV). While VSV enters cells by endocytosis and low pH-triggered fusion, this virus achieves fusion by a different class of fusion protein (class III). To analyze VSV-mediated entry, SIV vectors [24] were pseudotyped with the envelope glycoprotein G of VSV and the transduction of cells by this vector was evaluated by the expression of the GFP reporter gene. Neither ANDV DIII nor the ANDV stem peptides altered cell transduction at any tested concentrations up to 6 μM and 60 μM, respectively (Fig 3C) . In contrast, when the pH of the endocytic route was neutralized with the weak base ammonium chloride, VSV-G mediated transduction was blocked up to 80% (Fig 3C) . On the other hand, when SIV vectors were pseudotyped with ANDV glycoproteins, the DIII and R2 fragments produced~40% and~30% of inhibition of these vectors at 6 μM and 20 μM, respectively ( Fig 3D) , corroborating that the inhibitory fragments were active in this system.
It was previously reported that flavivirus stem peptides bind to the virus before it enters the cell, helping its delivery into endosomal compartments [47] . Based on this observation, we compared the inhibition of ANDV by (a) pre-incubation of the virus with the R2 peptide, or (b) co-incubating the R2 peptide with the virus during its adsorption to cells. Comparing the results of both experimental designs, a similar dose-dependent inhibition could be observed (Fig 3F) , coinciding with the results obtained for flaviviruses (47) . Based on this result, it seems plausible that binding of the R2 peptide to ANDV may occur very fast, making longer incubation times unnecessary.

Cross-inhibition of hantaviruses by exogenous DIII and stem fragments

The ANDV-derived Gc fragments were likewise tested for their potential to cross-inhibit other hantaviruses such as PUUV. Therefore, we performed a cell-cell fusion assay driven by the PUUV glycoproteins. In this assay, PUUV hDIII blocked the PUUV glycoprotein-mediated fusion process, reducing fusion up to 65% at 25 μM (Fig 4C) . This result coincides with the concentration range in which the ANDV DIII proteins block the ANDV glycoprotein fusion activity and confirms the activity of PUUV hDIII. When we tested the ANDV DIII proteins for cross-inhibition, we found that ANDV DIII, but not ANDV hDIII, blocked PUUV glycoprotein-mediated fusion up to 50% (Fig 4C) . These results, together with the data on absent crossreaction of PUUV hDIII with ANDV, suggest that the hantavirus DIII proteins without the Nterminal His-tag have cross-inhibiting activity; however the N-terminal His-tag of these domains seems to interfere in the inhibition. Thus so far, this notion remains to be corroborated with PUUV DIII lacking the N-terminal His-tag.

Exogenous DIII and stem fragments block the fusion process

ANDV cell entry can be blocked at different steps such as receptor binding and membrane fusion. For hantaviruses, the envelope glycoprotein or the specific domain involved in binding to receptors has not yet been identified. To discard a possible effect of the ANDV inhibitors in steps preceding virus-cell membrane fusion, we incubated the cells with ANDV DIII or the R2 peptide for 1 h before the addition of the virus. Unbound fragments were subsequently washed out, and cells were then infected with ANDV. The addition of neither DIII nor R2 at 10 and 20 μM, respectively, before the cells were incubated with ANDV led to a significant decrease in virus infection (Fig 5A) . These data emphasize that the pre-incubation of DIII and stem fragments did not abrogate early steps in virus infection such as receptor binding or cellular signaling pathways. Furthermore, the results coincide with data obtained with stem peptides derived from DV2, where the pre-incubation of cells with these peptides also did not affect infection by DV2 [47] . Next, we asked whether the DIII and stem fragments interfered directly in the virus-cell fusion process. To test this, we assessed inhibition in a fusion infection assay, fusing ANDV with the plasma membrane. Therefore, ANDV was pre-bound to cells at 4°C and then ANDV DIII or R2 were added during the 5 min low pH pulse that triggers fusion. The blockade of this viral entry pathway was highly efficient in the case of the R2 peptide, reaching over 95% of inhibition at 20 μM (Fig 5B) . Recombinant DIII led to a lower inhibition efficiency of 70% at 20 μM, result that in comparison to that obtained with R2 could be explained by the short incubation time in this experimental design. Compared to inhibition of the normal entry route of the virus, higher concentrations of ANDV DIII might have been necessary for inhibition since more input virus was used to reach similar levels of infection (MOI = 0.2). Taking these data together, our results confirm that the exogenous DIII and stem fragments function specifically during the viral fusion process.

Discussion

The fusion of the viral membrane with a host cell membrane is a crucial step in the entry of enveloped viruses into cells. In the present study we demonstrated that predicted DIII and stem fragments blocked acid-induced fusion of ANDV within the endosomal entry pathway and with the cell surface. Fusion was allowed to proceed until Gc trimerization, but prevented membrane hemifusion and fusion pore formation. These results not only provide novel information about inhibitory strategies against ANDV and other hantaviruses, but also provide a proof of concept that Gc shares structural similarity with the overall fold of class II fusion proteins.
Comparing the inhibition of hantaviruses by exogenous DIII with that of other class II fusion proteins, ANDV and PUUV hantaviruses were blocked by ANDV DIII without additional N-terminal His-tag or C-terminal residues, although containing seven N-terminal residues derived from the GST-tag. While the addition of an N-terminal His-tag achieved a 100-fold improvement in blocking fusion of Semliki Forest virus [44] , ANDV DIII -with or without N-terminal His-tag -achieved similar inhibitory results. For SFV DIII it has been proposed that this N-terminal tag may mimic the domain I-DIII linker region, thereby stabilizing the interaction with the fusion protein core [44] . On the other hand, it has been reported that the presence of C-terminal residues derived from the stem region is necessary for inhibition by exogenous DIII of DV2 and chikungunya virus E proteins [44, 72] . More specifically, for chikungunya virus it has been shown that nine residues from the E stem region are required for DIII to bind to the fusion protein domain I-domain II core. Hence, the potency of inhibiting the ANDV fusion process through ANDV and PUUV DIII may be further improved in future studies by adding N-or C-terminal residues.
The ANDV DIII and stem peptides blocked not only fusion mediated by ANDV glycoproteins, but also the fusion activity of the glycoproteins of another hantavirus (PUUV). This result is in accordance with the high sequence identity of 72% between DIII of these viruses and also with reports on the cross-inhibition activity of DIII within the genus Alphavirus [72] , where DIII conservation is as high as 50%. However, the N-terminal His-tag of ANDV hDIII seemed to prevent the cross-inhibition of PUUV fusion activity, indicating that this tag may interfere in specific binding to Gc from heterologous species. It is likely that the histidines of the tag become positively charged in the low pH environment, which in turn may induce repulsion with positively charged residues in the Gc of hantaviruses. Such repulsion may be overcome by a higher binding affinity of DIII to Gc from the same hantavirus, but not to heterologous viruses. In addition to the DIII of ANDV, peptides derived from the stem region of ANDV also cross-inhibited the PUUV fusion activity, which further corroborates the presence of conserved residues among hantavirus Gc proteins that are involved in the likely binding of this peptide. Cross-inhibition of fusion proteins by stem peptides has been previously reported for viruses from the genus Flavivirus; Dengue virus stem peptides blocked different Dengue virus serotypes but not other flaviviruses. [48] . The absence of cross-inhibition in that case was related not to a poor interaction with the respective E protein, but rather to a poor interaction with the viral membrane [48] . Finally, stem peptides derived from the RVFV Gc protein have been reported to block the three different classes of viral fusion proteins [46] , acting as a broad-spectrum fusion inhibitor [80] . For the exogenous stem peptides from ANDV we did not observe cross-inhibition of other fusion proteins such as that of VSV at concentrations up to 60 μM. Therefore, it is more likely that the ANDV stem fragments may be applied to inhibit similar viruses within the same genus, but not other viral fusion machineries.
Taken as a whole, our results demonstrate that strategies employed against class II fusion proteins allow for the inhibition of hantaviruses such as ANDV and PUUV. Although targeting the endosomal site of virus fusion has not yet been optimized, it was possible to block fusion and infection under physiological virus entry conditions. Hopefully, the novel inhibitory strategy based on ANDV DIII and stem peptides will help in the future development of therapeutic strategies against different hantaviruses.

Preparation of DIII expression plasmids

For the PCR amplification of the predicted DIII and DIIIS sequences we used the cloned cDNA of the M segment from ANDV isolate CHI-7913 [49] and from PUUV strain K27 cloned into pWRG/PUUV-M(s2) expression plasmid [50] (kindly provided by Jay Hooper, USAMRIID, USA), GenBank accession numbers AAO86638 and L08754, respectively. The PCR products were cloned into pET28a, which gave rise to fusion proteins with an N-terminal tag of 34 residues including polyhistidine (His-tag). For the preparation of DIII without the His-tag, the PCR product of ANDV DIII was cloned into pGST-Parallel-1 [51] . The expression product of this plasmid contained an N-terminal tag of 314 residues including a Glutathione S transferase (GST) affinity tag followed by a cleavage site for the tobacco etch virus (TEV) protease. After cleavage with TEV protease, 7 residues from the GST-fusion protein remained fused to the N-terminal of DIII, corresponding to the sequence GAMDPEF.

Virus and cells

ANDV isolate CHI-7913 (kindly provided by Héctor Galeno, Instituto de Salud Pública, Chile) was propagated in Vero E6 cells (ATTC) as described before [57] . All work involving the infectious virus was performed under biosafety level 3 conditions (Centro de Investigaciones Médicas, Pontificia Universidad Católica de Chile, Chile). 293FT cells (Invitrogen) were propagated in DMEM supplemented with 10% fetal calf serum (FCS). CHO-K1 cells (ATTC) were grown in F12-K medium containing 10% FCS.

ANDV infectivity titration

The infection of Vero E6 cells by ANDV (multiplicity of infection (MOI) = 0.1) was quantified by flow cytometry as formerly established [57] . Briefly, cells were incubated with ANDV for 1 h at 37°C in the presence and absence of protein or peptide inhibitor candidates. Subsequently, cells were washed and infection was allowed to proceed for 16 h. Cells were next fixed with 2% (w/v) paraformaldehyde for virus inactivation, and permeabilized with 0.1% Triton X-100. For immunofluorescence staining, cells were incubated for 45 min with anti-ANDV N monoclonal antibody (mAb) 7B3/F6 [58] and then the primary antibody was detected with goat antimouse immunoglobulin conjugated to Alexa 555 (Life Technologies). !10.000 cells of each condition were analyzed using a flow cytometer (FACS CAN II, Becton Dickinson). The standard deviation of at least n = 3 experiments is indicated as the error bar of each value.

Acid-induced Gc homotrimerization

The multimerization state of Gc was assessed by sucrose gradient centrifugation as previously established [17] . Briefly, ANDV was treated at pH 5.5 to allow for the rearrangement of glycoproteins on the viral envelope. Where indicated, DIII or R2 were added to the virus during its low pH incubation during 30 min at 37°C. Subsequently, viral glycoproteins were extracted by 1% Triton X-100 and separated in a gradient of 7-15% (w/v) sucrose by centrifugation at 150,000 g for 16 h. Fractions were collected, and the presence of Gc in each fraction was assessed by western blot analysis using anti-Gc mAb 2H4/F6 [61] . The molecular mass of each fraction was assessed by the Coomassie staining of a molecular marker (Gel filtration standard, Bio-Rad) that was applied to the same sedimentation gradient. The experimental molecular mass of the marker was next plotted against the log of its theoretical molecular mass in the panel above the western blots of the gradient. The stability of the Gc homotrimer was further tested by trypsin digestion as indicated before [17] . Briefly, well-characterized VLPs projecting ANDV glycoproteins were prepared as described elsewhere [62] and were incubated for 30 min at pH 5.5. After the acidification, VLPs were incubated with TCPK trypsin (Sigma) for the indicated times. Finally, digestion was stopped by adding sample buffer and heating to 95°C for 10 min. The digestion of Gc was tested by western blot analysis, using anti-Gc mAb as described above.

Cross-inhibition of hantaviruses by exogenous DIII and stem fragments

When we tested the ANDV stem peptides to block PUUV-mediated fusion, we found that they also had a cross-inhibition function (Fig 4D) . Among them, the R2.1 peptide reached the highest cross-reduction result of~70% at 20 μM, in line with the observation that this peptide also achieved the highest inhibition value of ANDV-mediated cell-cell fusion. Collectively, these results on hantavirus cross-inhibition suggest that residues in DIII and stem fragments are involved in intramolecular interactions. Some of these residues seem to be conserved between ANDV and PUUV (Fig 1D) , allowing for the cross-interaction with exogenous DIII and stem fragments from a different hantavirus.

Exogenous DIII and stem fragments do not affect Gc homotrimer formation

Since the fusion process involves multiple steps, we next assessed at which specific stage the Gc fragments interfere in this process. To this end, we started analyzing the trimerization of Gc using an earlier protocol [17] . ANDV was therefore incubated at neutral or low pH with or without DIII. Subsequently, the viral glycoproteins were extracted from the virus and applied to a sucrose gradient (7-15%) to evaluate their molecular mass. After ultracentrifugation, each fraction was examined by western blot analysis for the presence of Gc. Gradient sedimentation at pH 7.4 led to the detection of Gc in fractions corresponding to the molecular mass of Gc monomers of~50 kDa (fractions 5-7), in the presence or absence of ANDV DIII (Fig 6A) . Two Gc migration bands could be observed in the reducing electrophoretic system, which may correspond to different oxidation forms of Gc as described earlier [73] . Only the lower molecular mass band was previously found to shift from Gc monomers at neutral pH to Gc trimers at low pH [17] . When we incubated ANDV at pH 5.5, in the presence or absence of the DIII inhibitor, the lower molecular mass band of Gc shifted from the fractions corresponding to monomers and was found in fractions corresponding to Gc homotrimers (fractions 11-12; Fig 6A) . This result indicates that ANDV DIII abrogated neither Gc fusion activation, nor Gc trimerization.

Exogenous DIII and stem fragments prevent the formation of a stable Gc post-fusion trimer

The post-fusion conformation of ANDV Gc corresponds to a highly stable homotrimer [17] . In this context, we next explored the stability of the Gc homotrimer formed in the presence of exogenous DIII and stem fragments by assessing its resistance to protease digestion. For this experimental approach we used ANDV-like particles (VLPs) [62] , since they can be purified to higher concentrations than the virus. These VLPs resemble the viral envelope by exposing the Gn and Gc glycoproteins. To test their trypsin resistance, the VLPs were first incubated at neutral or low pH in the presence or absence of the inhibitors, and subsequently subjected to digestion with trypsin for different incubation times. As expected, the neutral pH form of Gc was fully degraded within 30 min, while the low pH form of Gc was largely resistant to digestion (Fig 6B) . In contrast, Gc lost its trypsin resistance and digestion could be observed when ANDV DIII or the R2 peptide was co-incubated with VLPs during the low pH incubation step, indicating that they interfered in the formation of a stable Gc homotrimer (Fig 6B) . Some residual Gc could be detected, most likely because the DIII or stem fragments did not block all Gc molecules, which coincides with the inhibitory results (Figs 3A and 3B and 5B) . The formation of a stable homotrimer was however not prevented when the control peptide NN was added before acidification, nor when either the NN peptide, DIII or the R2 peptide was added after the low pH incubation, confirming the specificity of the assay. Together, these data suggest that the exogenous DIII and stem fragments prevented the formation of a stable post-fusion hairpin structure, presumably by direct binding to Gc in its extended intermediate conformation.

Exogenous DIII arrests the fusion process in a step preceding the hemifusion intermediate

The presence of exogenous Gc fragments interfered neither with the activation of ANDV Gc nor with its homotrimerization; however it did not allow the formation of a stable post-fusion structure. The hemifusion intermediate is a stage that occurs between these fusion steps, in which the outer membranes of the virus and cell have fused, while the inner leaflets still remain apart. In order to test if the inhibition of ANDV by exogenous DIII occurs before or after the hemifusion intermediate state, we developed a hemifusion assay for the ANDV glycoproteins. For this purpose, we took advantage of a previously developed cell-cell fusion assay between 293FT cells expressing the ANDV glycoproteins (effector cells) and CHO-K1 cells (target cells) [17] . In this assay, the low pH-triggered transfer of GM1 from effector cells to target cells was analyzed by confocal microscopy, which allows detection of GM1 at the cell surface in a single focal plane, with minimal sample intervention (Fig 7A) . To allow unambiguous identification of GM1 transfer from effector cells to target cells, CHO-K1 cells were only defined as GM1 + when the label was detected on their full circumference (Fig 7A, red arrows) . Conversely, target cells in contact with effector cells, but showing only partial or no GM1 stain, were defined as GM1 - (Fig 7A, yellow arrows) . Although this assay allows for the detection of lipid mixing between cells, it does not discriminate between full fusion and hemifusion of membranes. To obtain a quantitative measure of GM1 transfer from effector to target cells, we quantified the percentage of transfer in each condition. At pH 7, a background level of transfer around 20% was observed in all conditions (Fig 7B) . When glycoproteins were activated at pH 5.5, GM1-transfer from effector cells to target cells was detected in~70% of Mock control treatment ( Fig 7B) . However, when ANDV DIII was incorporated in the low pH incubation, the transfer of GM1 appeared to be less frequent, decreasing to below 40% (Fig 7B) . This value is still higher than the background level observed for GM1 transfer at neutral pH, indicating that this domain reduced lipid mixing in an incomplete manner. Most probably, exogenous ANDV DIII did not make contact with all fusion proteins during the short low pH incubation, which coincides with the results for blocking cell-cell fusion (see Fig 4A) . The impairment of GM1-transfer was highly specific, since the incorporation of PUUV hDIII, which has no crossinhibition activity against ANDV (see Figs 3 and 4A), did not abrogate the acid-induced GM1 transfer of~70% (Fig 7A and 7B ). In summary, our results show that ANDV DIII arrests the fusion process after Gc trimerization, but before reaching a hemifusion intermediate. In this context, it seems likely that Gc fragments prevent the movement of the endogenous DIII or the stem region towards the core of the Gc trimer as described for other class II fusion proteins [39, 44] .

Discussion

The ANDV DIII, including the stem region, was largely insoluble and therefore this larger Gc fragment could not be tested in the ANDV entry assays. The R1 peptide spanning the first 17 N-terminal residues of the 44-residue stem region did not affect ANDV entry, while the R2 peptide spanning the last 20 C-terminal residues blocked ANDV infection to a similar extent as DIII. When we tested the two peptides R2.1 and R2.2, comprising either the N-or C-terminal half of the ANDV R2 stem peptide, the inhibitory activity against ANDV was largely retained by both peptides. Hence, the R2 peptide seems to contain residues in these two regions that participate similarly during inhibition. Similar to the C-terminal region of the ANDV stem region, the C-terminal region, but not the N-terminal, of DV2 E protein binds and blocks the fusion protein [47] . Interestingly, inhibitory peptides derived from membrane proximal regions (or stem regions) of diverse fusion proteins such as those of RVFV, DV2, SARS-coronavirus, Influenza virus, and Hepatitis C virus have been found likely to interact with membrane interfaces by a hydropathy segment [74] , which is predicted by the Wimley-White interfacial hydrophobicity scale (WWIHS) for the transfer of a peptide from an aqueous environment to a palmitoyl-oleoyl-phosphatidyl choline interface [75, 76] . A need for such a membrane-binding property for inhibitory activity coincides with studies on peptides derived from the stem region of alphavirus fusion proteins; these peptides generate WWIHS below values of 1 (S1 Table) , indicative of weak membrane binding [74] , and fail to block alphavirus fusion activity [44, 72] . In the case of the DV2 E protein, such a membrane-binding sequence is found at the C-terminal end of the peptide [48] with a high WWIHS value (S1 Table) . In fact, the stem peptides of the DV2 E protein have been shown to interfere with the fusion of the virus in the endosomal compartment by a two-step mechanism: first by binding to the viral membrane outside the cell, and next by binding against the E core trimer once fusion has been triggered in the endocytic compartment (47) . Using the Wimley-White scale [77] , an interfacial hydropathy segment with a WWIHS vale >5 was predicted for the ANDV Gc stem region that coincides with the sequence of the R2 peptide (S1 Table) , indicative for moderate membrane partitioning [74] . If the ANDV R2 peptide would interact with membranes by a hydropathy segment, then such an interaction did not favor ANDV inhibition sufficiently in the endosomal route, since fusion impairment was most efficient when directly present in the fusion compartment; when applied at the same concentration, the R2 stem peptide blocked 45% of ANDV infection when entry occurred via the endocytic pathway, while R2 reached over 95% inhibition of ANDV infection when fusion occurred at the plasma membrane. In this sense, modifications to the R2 peptide that favor membrane interaction, such as those introduced to peptides derived from the West Nile virus E stem region [48] or the membrane proximal region of Influenza virus hemagglutinin and HIV gp41 [78, 79] , may help in the future to improve its inhibitory activity and to direct it towards the closed environment of endosomes.
26 section matches

Abstract

The most recent Ebola virus outbreak in West Africa -unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected -highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae 1 . No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we describe the discovery of a novel anti-EBOV small molecule antiviral, GS-5734, a monophosphoramidate prodrug of an adenosine analog. GS-5734 exhibits antiviral activity against multiple variants of EBOV in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternate substrate and RNAchain terminator in primer-extension assays utilizing a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com

NTP levels in peripheral blood mononuclear cells (half-life = 14 h) and distribution to sanctuary sites for viral replication including testes, eye, and brain. In a rhesus monkey model of EVD, once daily intravenous administration of 10 mg/kg GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two of six treated animals. These results provide the first substantive, post-exposure protection by a smallmolecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses -including filoviruses, arenaviruses, and coronaviruses -suggests the potential for expanded indications. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.
The most recent outbreak of Ebola virus disease (EVD) in West Africa, was the far largest and most complex Ebola virus (EBOV) outbreak in the recorded history of the disease with >28,000 EVD cases and >11,000 reported deaths 1 . Medical infrastructures in Guinea, Sierra Leone, and Liberia were seriously impacted by a loss of >500 healthcare workers 1 . Additionally, EVD-related sequelae (joint and muscle pain, as well as neurological, ophthalmic, and other symptoms) together with viral persistence and recrudescence in individuals who survived the acute disease have been documented [2] [3] [4] [5] .
EBOV is a single-stranded, negative-sense, non-segmented RNA virus from the Filoviridae family. In addition to EBOV, other related viruses -namely Marburg, Sudan, and Bundibugyo -have caused outbreaks with high fatality rates 6 . Although the efficacy of various experimental small molecules and biologics have been assessed in multiple clinical trials during the West African outbreak [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] , there are no therapeutics for which clinical efficacy and safety have been established for treatment of acute EVD or its sequelae. The availability of broadly effective antiviral(s) with a favorable benefit/risk profile would address a serious unmet medical need for the treatment of EBOV infection.
A 1′-cyano substituted adenine C-nucleoside ribose analogue (Nuc) exhibits antiviral activity against a number of RNA viruses 19 . The mechanism of action of Nuc requires intracellular anabolism to the active triphosphate metabolite (NTP), which is expected to interfere with the activity of viral RNA-dependent RNA-polymerases (RdRp). Structurally, the 1′-cyano group provides potency and selectivity towards viral RNA polymerases, but because of slow first phosphorylation kinetics, modification of parent nucleosides with monophosphate promoieties have potential to greatly enhance intracellular NTP concentrations 20 . GS-5734, the single Sp isomer of the 2-ethylbutyl L-alaninate phosphosphoramidate prodrug (Supplementary Information), effectively bypasses the ratelimiting first phosphorylation step of the Nuc (Fig. 1a) . In human monocyte-derived macrophages, incubation with GS-5734 rapidly loads cells with high levels of NTP that persist with T 1/2 = 24 h following removal of GS-5734 (Extended Data Fig. 1a) , resulting in up to 30-fold higher levels compared to incubation with Nuc (Fig. 1b) . In cell-based assays, GS-5734 is active against a broad range of filoviruses including Marburg virus and several variants of EBOV (Fig. 1c) . GS-5734 inhibits EBOV replication in multiple relevant human cell types including primary macrophages and human endothelial cells with EC 50 values of 0.06 to 0.14 μM (Table 1) . As expected, the parent Nuc was less active with EC 50 values of 0.77 to >20 μM. Treatment with GS-5734 of liver Huh-7 cells infected with the EBOV-Makona variant isolated during the West African outbreak resulted in profound dosedependent reductions in viral RNA production and infectious virus yield (Extended Data Fig. 2 ). GS-5734 and the Nuc inhibited replication of other human RNA viral pathogens including respiratory syncytial virus, Junin virus, Lassa fever virus, and Middle East respiratory syndrome virus, but was inactive against alphaviruses or retroviruses (Table 1) . Prior studies have reported activity of the Nuc against flaviviruses, parainfluenza type 3, and severe acute respiratory syndrome associated coronavirus but little or no activity against West Nile virus, influenza A, or Coxsackie A 19, 21 . The antiviral activity of GS-5734 was selective as demonstrated by low cytotoxicity in a wide range of human primary cells and cell lines (Extended Data Table 1 ).

EBOV Huh-7 and HMVEC antiviral assay

Antiviral assays were conducted in biosafety level-4 containment (BSL-4) at the CDC. EBOV antiviral assays were conducted in primary HMVEC-TERT and in Huh-7 cells. Huh-7 cells were not authenticated and were not tested for mycoplasma. Ten concentrations of compound were diluted in 4-fold serial dilution increments in media, and 100 μL per well of each dilution was transferred in duplicate (Huh-7) or quadruplicate (HMVEC-TERT) onto 96-well assay plates containing cell monolayers. The plates were transferred to BSL-4 containment, and the appropriate dilution of virus stock was added to test plates containing cells and serially diluted compounds. Each plate included four wells of infected untreated cells and four wells of uninfected cells that served as 0% and 100% virus inhibition controls, respectively. After the infection, assay plates were incubated for 3 days (Huh-7) or 5 days (HMVEC-TERT) in a tissue culture incubator. Virus replication was measured by direct fluorescence using a Biotek HTSynergy plate reader. For virus yield assays, Huh-7 cells were infected with wild-type EBOV for 1 h at 0.1 pfu per cell. The virus inoculum was removed and replaced with 100 μL per well of media containing the appropriate dilution of compound. At 3 days post-infection, supernatants were collected, and the amount of virus was quantified by endpoint dilution assay. The endpoint dilution assay was conducted by preparing serial dilutions of the assay media and adding these dilutions to fresh Vero cell monolayers in 96-well plates to determine the tissue culture infectious dose that caused 50% cytopathic effects (TCID 50 ). To measure levels of viral RNA from infected cells, total RNA was extracted using the MagMAX ™ -96 Total RNA Isolation Kit and quantified using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay with primers and probes specific for the EBOV nucleoprotein gene.

High content imaging assay detecting viral infection

Antiviral assays were conducted in 384-or 96-well plates in BSL-4 at USAMRIID using a high-content imaging system to quantify virus antigen production as a measure of virus infection. A "no virus" control and a "1% DMSO" control were included to determine the 0% and 100% virus infection, respectively. The primary and secondary antibodies and dyes used for nuclear and cytoplasmic staining are listed in Supplementary Table 1 . The primary antibody specific for a particular viral protein was diluted 1,000-fold in blocking buffer (1× PBS with 3% BSA) and added to each well of the assay plate. The assay plates were incubated for 60 minutes at room temperature. The primary antibody was removed, and the cells were washed 3 times with 1× PBS. The secondary detection antibody was an antimouse (or rabbit) IgG conjugated with Dylight488 (Thermo Fisher Scientific, Waltham, MA, Cat. No. 405310). The secondary antibody was diluted 1,000-fold in blocking buffer and was added to each well in the assay plate. Assay plates were incubated for 60 minutes at room temperature. Nuclei were stained using Draq5 (Biostatus, Shepshed Leicestershire, UK) or 33342 Hoechst (ThermoFisher Scientific) for Vero and HFF-1 cell lines. Both dyes were diluted in 1× PBS. The cytoplasm of HFF-1 (EBOV assay) and Vero E6 (MERS assay) cells were counter-stained with CellMask ™ Deep Red (Thermo Fisher Scientific, Waltham, MA). Cell images were acquired using a Perkin Elmer Opera confocal plate reader (Perkin Elmer, Waltham, MA) using 10× air objective to collect five images per well. Virus-specific antigen was quantified by measuring fluorescence emission at a 488 nm wavelength and the stained nuclei were quantified by measuring fluorescence emission at a 640 nm wavelength. Acquired images were analyzed using Harmony and Acapella PE software. The Draq5 signal was used to generate a nuclei mask to define each nuclei in the image for quantification of cell number. The CellMask Deep Red dye was used to demarcate the Vero and HFF-1 cell borders for cell-number quantitation. The viral-antigen signal was compartmentalized within the cell mask. Cells that exhibited antigen signal higher than the selected threshold were counted as positive for viral infection. The ratio of virus positive cells to total number of analyzed cells was used to determine the percent infection for each well on the assay plates. Effect of compounds on the viral infection was assessed as percent inhibition of infection in comparison to control wells. The resultant cell number and percent infection were normalized for each assay plate. The Z′ values for all antiviral assays were >0.3. Analysis of dose response curve was performed using GeneData Screener software applying Levenberg-Marquardt algorithm (LMA) for curve fitting strategy. The curve-fitting process, including individual data point exclusion were pre-specified by default software settings. R 2 -value quantified goodness of fit and fitting strategy was considered acceptable at R 2 > 0.8.

In vivo efficacy

Rhesus monkeys (Macaca mulatta) were challenged on day 0 by intramuscular injection with a target dose of 1000 pfu of EBOV-Kikwit (Ebola virus H.sapiens-tc/COD/1995/ Kikwit), which was derived from a clinical specimen obtained during an outbreak occurring in The Democratic Republic of the Congo (formerly Zaire) in 1995. Challenge virus was propagated from the clinical specimen using cultured cells (Vero or Vero E6) for a total of four passages. Animals (3-6 years) were randomly assigned to experimental treatment groups, stratified by sex (with equal number of males and females per group) and balanced by body weight, using SAS ® statistical software (Cary, North Carolina, USA). Study personnel responsible for assessing animal health (including euthanasia assessment) and administering treatments were experimentally blinded to group assignment of animals. The primary endpoint for efficacy studies was survival to day 28 following virus challenge. GS-5734 was formulated at Gilead Sciences in water with 12% sulfobutylether-βcyclodextrin (SBE-β-CD), pH adjusted to 3.0 using HCl. Formulations were administered to anesthetized animals by bolus intravenous injection at a rate of approximately 1 min/dose in the right or left saphenous vein. The volume of all vehicle or GS-5734 injections was 2.0 mL/kg body weight. Animals were anesthetized using IM injection of a solution containing ketamine (100 mg/mL) and acepromazine (10 mg/mL) at 0.1 mL/kg body weight.

Extended Data Figure 2. Virus yield assay

Huh-7 cells seeded in 96-well plates were infected with wild-type EBOV (Makona) for 1 h at 0.1 plaque forming unit (pfu) per cell. The virus inoculum was removed and replaced with 100 μL per well of media containing the appropriate dilution of compound. At 3 days postinfection, supernatants were collected, and the amount of virus was quantified by endpoint dilution assay. The endpoint dilution assay was conducted by preparing serial dilutions of the assay media and adding these dilutions to fresh Vero cell monolayers in 96-well plates to determine the tissue culture infectious dose that caused 50% infection (TCID50). To measure levels of viral RNA from infected cells, total RNA was extracted using MagMAX ™ -96 Total RNA Isolation Kit (Invitrogen, Carlsbad, CA) and quantified using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay with primers and probes specific for the EBOV nucleoprotein gene. Values represent mean ± SD of log 10transformed values, n=4 replicates.
Clinical signs of disease in individual rhesus monkeys exposed to Ebola virus. Animals were observed multiple times each day and were subjectively assigned a clinical disease score ranging from 0 to 5 based on responsiveness, posture, and activity. Maximum daily scores were converted to color code, with darker colors indicative of more severe disease signs. The schematic was truncated to emphasize clinical scores during the acute disease phase, and none of the animals exhibited clinical disease signs outside of the times that are shown. Extended Data Table 1 In vitro cytotoxicity of GS-5734 and Nuc in human cell lines and primary cells.

GS-5734 pharmacokinetics, metabolism, and distribution were examined in NHPs. Upon intravenous administration of a 10 mg/kg dose in rhesus monkeys, GS-5734 exhibits a short plasma half-life (t 1/2 = 0.39 h) with fast systemic elimination followed by the sequential appearance of transient systemic levels of the key intracellular intermediate alanine metabolite (Ala-Met) and more persistent levels of Nuc (Fig. 2a) . GS-5734 is rapidly distributed into peripheral blood mononuclear cells (PBMCs), and efficient conversion to NTP is apparent within 2 h of dose administration. In PBMCs, NTP represents the predominant metabolite and is persistent with a t 1/2 = 14 h and levels required for >50% virus inhibition for 24 h ( Fig. 2a; Extended Data Fig. 1c) . In cynomolgus monkeys, intravenous administration of a 10 mg/kg dose of [ 14 C]GS-5734 demonstrated that the drugderived material distributed to testes, epididymis, eyes, and brain within 4 h of administration (Fig. 2b) . In brain, levels at 4 h were low relative to other tissues but remained detectable above the drug plasma levels 168 h post dose. Taken together, the pharmacokinetic analysis indicates that once-daily dosing of GS-5734 provides sustained intracellular NTP levels and efficiently delivers drug metabolites to sanctuary sites where virus may persist.
To evaluate the in vivo efficacy of GS-5734 we conducted a sequential two-part, adaptivedesign study in EBOV-infected rhesus monkeys (Fig. 2c) . In part 1, animals intramuscularly inoculated with EBOV were administered a 12-day regimen of vehicle (n = 3) or 3 mg/kg GS-5734 beginning on day 0 (D0; 30-90 min following virus challenge) or Day 2 (D2) (n = 6/treatment group). Regardless of the time of initiation, GS-5734 treatment conferred improved survival, 33% (2/6) in the 3 mg/kg D0 group and 66% (4/6) in the 3 mg/kg D2 group, and an antiviral effect by reducing systemic viremia relative to vehicle (Fig. 2d, Tables 2,3) ; however, mortalities observed in both treatment groups suggested that drug exposure at 3 mg/kg was sub-optimal. In part 2 of the efficacy study, GS-5734 was administered once at a loading dose of 10 mg/kg followed by once-daily 3 mg/kg doses beginning either 2 days (10/3 mg/kg D2) or 3 days (10/3 mg/kg D3) after virus exposure, or 10 mg/kg doses were administered beginning 3 days after virus exposure (10 mg/kg D3) with n = 6/group. All animals (12/12) in which GS-5734 treatments were initiated 3 days after virus exposure survived to the end of the in-life phase (Fig. 2d) . However, the antiviral effects were consistently greater in animals administered repeated 10 mg/kg GS-5734 doses ( Tables 2,3) . On Day 4, plasma viral RNA was significantly decreased (P<0.05), with geometric means reduced ≥1.7 log 10 in all GS-5734-treated groups compared with combined vehicle-treated groups ( Fig. 2e-f ; Extended Data Table 3 ), and on Days 5 and 7, when geometric mean viral RNA concentration of the vehicle group exceeded 10 9 copies/mL, viral RNA was detected at levels less than the lower limit of quantitation (8 × 10 4 RNA copies/mL) in 4 of 6 animals in the 10 mg/kg D3 group. Deep sequencing analysis of the EBOV RdRp (L) gene from all viral RNA-positive plasma samples showed no evidence of genotypic change(s) potentially associated with the emergence of GS-5734-resistant EBOV variants (Extended Data Table 5 ). The 10 mg/kg D3 GS-5734 regimen was associated with amelioration of EVD-related clinical disease signs ( Table 4 and Extended Data Fig. 5 ). Although greater survival was observed in the 10/3 mg/kg D3 group than the 10/3 mg/kg D2 group, survival and viral RNA load were not statistically distinguishable (Fig. 2e ) and likely represent natural endpoint variation associated with suboptimal therapeutic effect.
In summary, GS-5734 is a potent and selective inhibitor of EBOV in multiple relevant permissive cell types. In an NHP model of clinical EVD, intravenous administration of GS-5734 results in rapid accumulation and persistence of intracellular NTP. Pronounced antiviral effects, amelioration of EVD signs, and significant survival benefit was achieved in EBOV-infected NHPs despite treatment initiation on day 3, a time when systemic viral RNA was detectable. These results represent the first case of substantive postexposure protection against EVD by a small-molecule antiviral compound in NHPs. Intravenous GS-5734 is currently being evaluated in multiple dose studies in healthy human volunteers to assess clinical safety and pharmacokinetics, and to help determine whether GS-5734 may provide therapeutic benefit in acute or recrudescent cases of EVD, or in survivors with prolonged virus shedding and/or chronic clinical sequelae. The broad-spectrum antiviral activity of GS-5734 and its amenability to large-scale production warrants further assessment of its therapeutic potential against other significant human viral pathogens for which no treatment is available.

EBOV human macrophage infection assay

Antiviral assays were conducted in BSL-4 at USAMRIID. Primary human macrophage cells were seeded in a 96-well plate at 40,000 cells per well. Eight to ten serial dilutions of compound in triplicate were added directly to the cell cultures using an HP D300 digital dispenser in 3-fold dilution increments 2 h prior to infection. The concentration of DMSO was normalized to 1% in all wells. The plates were transferred into the BSL-4 suite, and the cells were infected with 1 pfu per cell of EBOV in 100 μL of media and incubated for 1 h. The inoculum was removed, and the media was replaced with fresh media containing diluted compounds. At 48 h post-infection, virus replication was quantified by immuno-staining as described in Supplementary Table 1 .

RSV A2 antiviral assay

For antiviral tests, compounds were 3-fold serially diluted in source plates from which 100 nL of diluted compound was transferred to a 384-well cell culture plate using an Echo acoustic transfer apparatus. HEp-2 cells at a density of 5 × 10 5 cells/mL were then infected by adding RSV A2 at a titer of 1 × 10 4.5 tissue culture infectious doses (TCID 50 )/mL. Immediately following virus addition, 20 μL of the virus/cell mixture was added to the 384well cell culture plates using a μFlow liquid dispenser and cultured for 4 days at 37°C. After incubation, the cells were allowed to equilibrate to 25°C for 30 minutes. The RSV-induced cytopathic effect was determined by adding 20 μL of CellTiter-Glo ™ Viability Reagent. After a 10-minute incubation at 25°C, cell viability was determined by measuring luminescence using an Envision plate reader.

Marburg virus assay

HeLa cells were seeded at 2,000 cells/well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 1 pfu per cell MARV, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period. Virus infection was quantified by immuno-staining using antibodies that recognized the viral glycoproteins as described in Supplementary Table 1 .

Sudan virus assay

HeLa cells were seeded at 2,000 cells/well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.08 pfu SUDV per cell, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period. Virus infection was quantified by immuno-staining using antibodies that recognized the viral glycoproteins as described in Supplementary Table 1 .

Junin virus assay

HeLa cells were seeded at 2,000 cells/well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.3 pfu per cell JUNV, which resulted in ~50% of the cells expressing virus antigen in a 48-h period. Virus infection was quantified by immuno-staining using antibodies that recognized the viral glycoproteins as described in Supplementary Table 1 .

Lassa fever virus assay

HeLa cells were seeded at 2,000 cells/well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.1 pfu per cell LASV, which resulted in >60% of the cells expressing virus antigen in a 48-h period. Virus infection was quantified by immuno-staining using antibodies that recognized the viral glycoproteins as described in Supplementary Table 1 .

Middle East respiratory syndrome virus assay

African Green Monkey (Chlorocebus sp) kidney epithelial cells (Vero E6) were seeded at 4,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.5 pfu per cell of MERS virus, which resulted in >70% of the cells expressing virus antigen in a 48-h period. Virus infection was quantified by immuno-staining using antibodies that recognized the viral glycoproteins as described in Supplementary Table 1 .

Chikungunya virus assay

U2OS cells were seeded at 3,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.5 pfu per cell of CHIK, which resulted in >80% of the cells expressing virus antigen in a 48-h period. Virus infection was quantified by immuno-staining using antibodies that recognized the viral glycoproteins as described in Supplementary Table 1 .

Venezuelan equine encephalitis virus assay

HeLa cells were seeded at 4,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.1 pfu per cell VEEV, which resulted in >60% of the cells expressing virus antigen in a 20h period. Virus infection was quantified by immuno-staining using antibodies that recognized the viral glycoproteins as described in Supplementary Table 1 .

Quantitative real-time PCR for in vivo studies

For quantitative assessment of viral RNA nonhuman primate plasma samples, whole blood was collected using a K3 EDTA Greiner Vacuette tube (or equivalent) and sample centrifuged at 2500 (± 200) RCF for 10 ± 2 min. To inactivate virus, plasma was treated with 3 parts (300 μL) TriReagent LS and samples were transferred to frozen storage (−60°C to −90°C), until removal for RNA extraction. Carrier RNA and QuantiFast High Concentration Internal Control (Qiagen) were spiked into the sample prior to extraction, conducted according to manufacturer's instructions. The viral RNA was eluted in AVE Buffer. Each extracted RNA sample was tested with the QuantiFast Internal Control RT-PCR RNA Assay (Qiagen) to evaluate the yield of the spiked-in QuantiFast High Concentration Internal Control. If the internal control amplified within manufacturer-designated ranges, further quantitative analysis of the viral target was performed. RT-PCR was conducted using an ABI 7500 Fast Dx using primers specific to EBOV glycoprotein. Samples were run in triplicate using a 5 μL template volume. For quantitative assessments, the average of the triplicate genomic equivalents (ge) per reaction were determined and multiplied by 800 to obtain ge/mL of plasma. Standard curves were generated using synthetic RNA. The limits of quantification for this assay are 8.0 × 10 4 -8.0 × 10 10 ge/mL of plasma. Acceptance criteria for positive template control (PTC), negative template control (NTC), negative extraction control (NEC), and positive extraction control (PEC) are SOP-specified. For qualitative assessments, the limit of detection (LOD) was defined as Ct 38.07, based on method validation testing. An animal was considered to have tested positive for detection of EBOV RNA when a minimum of 2 of 3 replicates were designated as "positive" and PTC, NTC, and NEC controls meet specified method-acceptance criteria. A sample was designated as "positive" when the Ct value was

Viral Genomic Sequence Analysis

Analysis of viral genomic sequence was conducted with the purpose of evaluating genomic sequence change patterns consistent with development of resistance against GS-5734. Attempts were made to obtain sequence of RNA-dependent RNA polymerase gene (L) of Ebola virus from all EBOV-RNA positive rhesus monkey plasma samples obtained during the efficacy studies. Deep sequencing screening of the L gene was completed using previously described methods 36, 37 . Mutations and large sub clonal events (≥10% of population) were reviewed for: non-synonymous substitutions in treated animals that succumbed to infection, any substitution enriched in treated populations regardless of survival status and clusters of substitutions in any treated animal.
cDNA synthesis was performed using Invitrogen's Superscript III First-Strand Synthesis System. cDNA was amplified with Phusion Hot Start Flex DNA Polymerase (New England Biolabs) using overlapping 1,500-kb amplicons (primer information available upon request). After pooling and purification with AMPure XP Reagent (Beckman Coulter), PCR products were fragmented using the Covaris S2 instrument (Covaris). Libraries were prepared with the Illumina TruSeq DNA Sample Preparation kit (Illumina) on the Caliper ScicloneG3 Liquid Handling Station (PerkinElmer). After measurement by real-time PCR with the KAPA qPCR Kit (Kapa Biosystems), libraries were diluted to 4 or 10 nM. Cluster amplification was performed on the Illumina cBot, and libraries were sequenced on the Illumina Nextseq or Illumina HiSeq 2500 using the 150 or 100 bp paired-end format. Viral assemblies were completed in DNAStar Lasergene nGen. Amplification primer removal, quality trimming, and trim-to-mer were performed on reads with a minimum similarity to the reference of 93% (four-base mismatch). A target depth of 1200 is sought and SNPs at positions with fewer than 200 read depth were removed from the analysis. A consensus change was defined as a change relative to the deposited EBOV sequence (GenBank #AY354458) present in ≥50% of the population. Below that threshold, SNPs were considered sub-clonal substitutions and part of a minority subpopulation of the virus. Consensus sequence for the region covered in this screening is available in Genbank and the accession numbers are provided in Extended Data Table 5 . . The corresponding EBOV EC 50 values for the prodrug diastereomeric mixture were 100, 184, and 121 nM in human macrophages, HeLa, and HMVEC, respectively, suggesting that an average intracellular NTP concentration of approximately 5 μM is required for 50% inhibition of EBOV in vitro.

Intracellular metabolism studies

The intracellular metabolism of GS-5734 was assessed in different cell types (HMVEC, HeLa, and primary human and rhesus PBMC, monocytes and monocyte derived macrophages) following 2-h pulse or 72-h continuous incubations with 10 μM GS-5734. For comparison, intracellular metabolism during a 72-h incubation with 10 μM of Nuc was completed in human monocyte derived macrophages. For pulse incubations, monocyte derived macrophages isolated from rhesus or human were incubated for 2 h in compound containing media followed by removal, washing with 37°C drug-free media, and incubated for an additional 22 h in media not containing GS-5734. Human monocyte derived macrophages, HeLa and HMVEC were grown to confluence (approximately 0.5, 0.2, and 1.2 × 10 6 cells/well, respectively) in 500 μL of media in 12 well tissue culture plates. Monocyte and PBMC were incubated in suspension (approximately 1 × 10 6 cells/mL) in 1 mL of media in micro centrifuge tubes.
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Abstract

Despite being simple eukaryotic organisms, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have been widely used as a model to study human pathologies and the replication of human, animal, and plant viruses, as well as the function of individual viral proteins. The complete genome of S. cerevisiae was the first of eukaryotic origin to be sequenced and contains about 6,000 genes. More than 75% of the genes have an assigned function, while more than 40% share conserved sequences with known or predicted human genes. This strong homology has allowed the function of human orthologs to be unveiled starting from the data obtained in yeast. RNA plant viruses were the first to be studied in yeast. In this paper, we focus on the use of the yeast model to study the function of the proteins of human immunodeficiency virus type 1 (HIV-1) and the search for its cellular partners. This human retrovirus is the cause of AIDS. The WHO estimates that there are 33.4 million people worldwide living with HIV/AIDS, with 2.7 million new HIV infections per year and 2.0 million annual deaths due to AIDS. Current therapy is able to control the disease but there is no permanent cure or a vaccine. By using yeast, it is possible to dissect the function of some HIV-1 proteins and discover new cellular factors common to this simple cell and humans that may become potential therapeutic targets, leading to a long-lasting treatment for AIDS.

Introduction

In basic research in Virology, yeast has assisted the elucidation of the function of individual proteins from important pathogenic viruses such as Hepatitis C virus, and Epstein- Barr virus ([11, 12] ; for a review and further references see [13] ). Furthermore, studies of viruses that infect yeast have provided many important contributions to the dissection of the life cycle of many other higher eukaryotes RNA viruses and the host factors involved [14] .
Since the genetics and cell biology of higher eukaryotes are extremely complex, scientists looking for a good model have turned to the use of yeast as a simpler system for the study of various pathologies including virus proliferation and assay new drugs against these pathogenic agents.
An exciting example of the beneficial using this model system is found in two very recent reports, although not related to virus research, on the use of the yeast model to study a severe human pathology. Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's Disease, is a neurodegenerative disease in which the motor neurons of the central nervous system that control muscles die off leading to paralysis and death within 3-5 years of onset. The cause of the disease is unknown, and there is no treatment to stop or slow it. A gene related to ALS, called FUS, has been used to explore its biology in yeast and highlight the potential for modeling elements of complex diseases in this simple eukaryotic cell. Results from both studies suggest 2 Journal of Biomedicine and Biotechnology that defects in RNA processing and transport may be a key element of ALS pathophysiology. In the cytoplasm of motor neurons of ALS patients, proteins aggregate to form insoluble aggregations, called inclusions, that may involve FUS and another ALS causing protein, called TDP-43 [7] . When the two research groups overexpressed human FUS in yeast, they observed cytoplasmic inclusions. In humans, the majority of FUS is found in the nucleus, and some ALSassociated mutations reduce the nuclear/cytoplasmic ratio of the protein, suggesting that transfer to the cytoplasm, rather than mutation, may be important in this pathology. This hypothesis was supported by the observation that decreasing the overexpression of wild-type FUS in the yeast nucleus diminished its toxic effect. Both FUS and TDP-43 are RNA binding proteins. Genome-wide screens to identify yeast genes that specifically decreased cell toxicity were performed and the identified genes included some coding for other DNA/RNA binding proteins. One of these yeast genes, called ECM32, has a human homolog, hUPF1, which was found to eliminate toxicity. Among other functions hUPF1 plays an important role in mRNA quality control, supporting the idea that pathways involving RNA are defective in FUSinduced ALS. Interestingly, expression of hUPF1 was able to rescue FUS toxicity in yeast without driving FUS out of the inclusions or sending it back to the nucleus, suggesting that it may be possible to overcome therapeutically the effects of abnormally localized FUS without addressing the difficult problem of restoring it to its original compartment. Two important consequences may result from this approach. By identifying new genes that can diminish ALS-linked toxicity, they point to RNA processing as a therapeutic target in this disease. It has also been shown that yeast has the potential to be a much simpler and exciting system for modeling aspects of ALS. Since testing ideas about pathogenesis and treatment is much faster and cheaper in yeast, these results may open the way for more rapid progress in understanding the disease, its treatment, and the role of this new gene in ALS development. Important details of other neurodegenerative diseases have been obtained using yeast. For example, protein misfolding associated with many human diseases, including Alzheimer's, Parkinson's, and Huntington's disease, has been studied in yeast. As in the case of ALS mentioned above, protein misfolding often results in the formation of intracellular or extracellular inclusions whose role in protein misfolding diseases is unclear. Studies on the implication of protein aggregation and toxicity in yeast provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between the toxic and protective roles of protein conformation changes. Results from these studies using yeast have the potential to transform basic concepts of protein folding in human diseases and may help in identifying new therapeutic strategies for their treatment [8] .
In virus research, S. cerevisiae is a very useful organism. In terms of public health, the use of yeast to produce vaccines was a remarkable breakthrough. For example, the first recombinant vaccine, the hepatitis B surface antigen expressed in yeast, has become a safe and efficient prophylactic vaccine worldwide [9] . In addition, yeast has proved useful for drug discovery as illustrated by the power of applying genomic approaches using the yeast model to characterize the biological activity of small molecules and to identify their cellular targets, an important step towards understanding the mode of action of human therapeutic agents [10] . This study and other recent reports show that the path from sequence and functional analysis to direct therapeutic applications is not necessarily long, at least using the yeast model, a simple organism, which could well be considered among "man's best friends."
The study of the replication of RNA plant viruses was the first to be attempted using yeast cells. The great majority of RNA+ viruses carry their own RNA polymerase and are replicated in the cytoplasm while entry to the nuclear compartment during the viral cycle is confined to retroviruses (see below). The first higher eukaryotic virus reported to replicate in yeast was Brome mosaic virus, a positive-strand RNA ((+)RNA) virus that infects plants ( [15] , for an excellent review see [16] ).
Since the pioneering work of the Ahlquist laboratory, a growing list of viruses has been reported to undergo replication in yeast. These include RNA and DNA viruses that infect plants, insects, mammals, and humans (Table 1 ) [17] [18] [19] [20] [21] [22] .
This wide range of viruses emphasizes the general applicability of the yeast system. In this paper, we will focus on the contribution of the yeast model to the study of retroviruses with special emphasis on the human immunodeficiency virus type 1 (HIV-1).

Human Immunodeficiency Virus Type 1 (HIV-1)

Even though retroviruses carry an RNA+ genome, their replicative cycle involves the cell nucleus. These viruses are characterized by the fact that they carry an RNAdependent DNA polymerase (reverse transcriptase (RT)) that synthesizes a double-strand DNA called proviral DNA in the cytoplasm of the infected cell by using the viral RNA genome as template. Proviral DNA and other viral and cellular factors forming the preintegration complex (PIC) enter the nuclear compartment and integrate the proviral double-stranded DNA in the host genome via an encoded retroviral enzyme, integrase (IN). In this paper we will focus on the use of the yeast model system to study the following proteins encoded in the HIV-1: the enzymes reverse transcriptase, integrase and protease, Vpr, Rev and the translation machinery of HIV-1.

Enzymes Encoded in the HIV-1 Genome

HIV-1 Encoded Enzymes. All retroviruses carry three enzymes, an RNA-and DNA-dependent DNA polymerase or reverse transcriptase (RT), integrase (IN), and protease (PR) (see [29] ). The organization of these proteins in several retroviruses is shown in Figure 2 while their 3D structure is shown in Figure 3 . RT also contains an additional enzymatic activity, RNase H, which has been mapped to a separate, contiguous portion of the polypeptide. First identified in the avian viruses, the retroviral enzymes are organized in domains on the Gag-Pro or Gag-Pro-Pol precursor polypeptide. These domains are not always cleaved into separate mature proteins. In most genera, all enzymes are translated together as a Gag-Pro-Pol precursor, which are processed to yield the mature forms of the enzymes. Whether expression of pro and pol is due to frameshifting or termination suppression, they account for approximately 5% of RT and IN on a molecular basis but are synthesized and packaged in the virion at the same level as the Gag protein. For most retroviruses, the same holds for PR. A scheme describing the retroviral frameshifting process is shown in Figure 7 . Since RT, IN, and PR are essential for viral replication and have characteristics that distinguish them from related cellular enzymes, they have all become privileged targets for drugs against the acquired immunodeficiency syndrome (AIDS).

Reverse Transcriptase (RT).

The reverse transcriptase enzyme purified from the virus is a heterodimer with subunits of 66 and 51 kDa called p66 and p51 (Figure 3 (a)). p51 subunit is a shortened version of p66 after cleavage by the HIV-1 encoded protease. As with many other recombinant proteins the yeast S. cerevisiae has been used to express HIV-1 RT. Only the HIV-1 gene for the p66 subunit was carried by the expression vector used and surprisingly the recombinant enzyme purified after expression in yeast was the heterodimeric p66/p51 form. Protein sequencing showed that the cleavage to produce the p51 subunit gave a product identical to the native viral enzyme showing that yeast possesses a protease with the same exquisite specificity as the HIV-1 protease [31] .

Protease.

A lethal effect similar to that obtained by the expression of HIV-1 IN in yeast was observed more recently when the retrovirus-encoded protease (HIV-1 PR, Figure 3 (c)) was expressed both in Saccharomyces cerevisiae and in mammalian cells. These findings contribute to a deeper understanding of HIV-1-induced cytopathogenesis. Expression of HIV-1 PR stopped yeast growth followed by cell lysis. The lytic phenotype included loss of plasma membrane integrity and cell wall breakage leading to the release of cell content to the medium. Interestingly, this effect seems to be specific for HIV-1 PR since neither poliovirus 2A protease nor 2BC protein, which are both highly toxic for S. cerevisiae, was able to produce similar effects. Drastic alterations in membrane permeability preceded the lysis in yeast expressing the HIV-1 PR. The morphological changes after expression of HIV-1 PR in yeast and mammalian cells were similar in many aspects [49] .

Expression of Retroviral Enzymes Is Controlled by Frameshifting

During the replication of retroviruses, large numbers of Gag molecules must be generated to serve as precursors to the structural proteins of the virions. However, the enzymes encoded by the pro and pol genes (PR, RT, and IN) are generally needed in lower amounts to carry out their catalytic functions. Retroviruses have developed a mechanism leading to the synthesis of the Gag protein at higher levels relative to the pro and pol gene products, while retaining coregulated expression (Figure 7 ). This is due to the use of the same initiation codon in the same mRNA to express the gag, pro, and pol genes. Translation of this RNA leads occasionally to synthesis of a fusion protein that is usually called the Gag-Pro-Pol precursor. Typically allows the same mechanism that targets the Gag precursor to the site of virion assembly also to direct the Gag-Pro-Pol precursor. In all retroviruses, the gag gene is positioned at the 5 end of the viral genome, upstream of the pro and pol genes. The Gag-Pro-Pol precursor is generated using a strategy in which the termination codon that defines the 3 terminus of the gag reading frame is bypassed, allowing translation to continue into the adjacent pro and pol reading frames. Bypass of the termination codon occurs by one of two mechanisms. The first mechanism (used by the mammalian type-C retroviruses) is read through (termination) suppression, in which the gag termination codon is occasionally misread as a sense codon. Translation then continues past the termination codon and into the pro-pol reading frame. The second mechanism, which is used by most retroviruses, is ribosomal frameshifting. Here, occasional ribosomes slip backward one nucleotide (-1 frameshift, i.e., in the 5 direction) during translation of gag. Thus, the ribosome leaves the gag reading frame (with its downstream termination codon) and shifts into an overlapping portion of the pro-pol reading frame.

4.1.

Vpr. The 14-Kd, 96-amino-acid HIV-1 encoded protein Vpr (viral protein R) [50, 51] , plays several roles in the replication cycle of this retrovirus. Thus, it has been proposed that Vpr regulates the nuclear import of the preintegration complex (PIC) carrying the proviral DNA, the viral integrase, and other viral and cellular proteins. It is also required in the replication of HIV-1 in nondividing cells like the macrophages, and much evidence indicates that it is able to induce the arrest of cell cycle at the G2 step in proliferating cells, the latter effect likely playing an important role in the immunosuppression process in AIDS patients (for a recent review see [52] ).

Summary and Perspectives

In summary, although much of the potential promise of yeast is still to be revealed, it has proved extremely valuable in virus research. In addition to techniques like the double-hybrid system, this simple eukaryotic cell has been very useful for producing recombinant viral proteins whose purification from native virions is difficult or simply impossible. It also allows the mechanism of action of viral proteins to be studied thanks to the close analogy between human and yeast proteins, and this has led to the emergence of new therapeutic targets. By associating the toxic phenotype induced by some viral proteins in yeast cells with the genetic manipulation facilitating the entry of drugs with potential therapeutic properties, it may become possible to establish a simple cheap system allowing faster screening of the antiviral agents of the future. Moreover, future work should lead to the discovery of new cellular factors involved in virus proliferation, thus shortening the time necessary to develop new therapies against current and new viruses. The ultimate information on the behavior of viruses or virus proteins inside the cell should be attained with plant, animal, and human cells or in vivo in whole organisms, although it may be difficult and costly for most laboratories to develop these approaches. Since experiments with yeast will always be technically easier, more rapid, and cheaper than those with human and other complex eukaryote cells, yeast will remain a method of choice for studying virus infections mechanisms and the search for new drug targets.

Introduction

Owing to the high conservation of fundamental biochemical pathways, yeast has been used as a model to unravel biological processes in many higher eukaryotes (for a short review see [2] ). A very important tool facilitating the use of these cells is the availability of yeast libraries in which each nonessential gene has been deleted. It has been used for multiple studies including genome wide screenings for human disease genes and host factors that support virus replication [3] [4] [5] . The above collection is commercially available and covers more than 90% of all yeast genes [6] .

Reverse Transcriptase (RT).

The RT activity of these hybrid Ty1/HIV-1 elements can be monitored by using a simple genetic assay. The reverse transcription of yeast carrying this hybrid RT depends on both the DNA polymerase and RNase H domains of HIV-1 RT. Most HIV-1 RT inhibitors can be divided into two classes, nucleoside analog RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). NRTIs such as AZT (3-azido-3deoxythymidine) and ddI (dideoxyinosine) inhibit reverse transcription by a chain-termination mechanism; that is, when they are added to a growing DNA chain they block further synthesis while the NNRTIs act by blocking the synthesis by binding specifically an hydrophobic pocket of the HIV-1 RT. The reverse transcriptase activity measured in yeast cells was shown to be sensitive to inhibitors of HIV-1 RT like 8Cl-TIBO, a well-characterized nonnucleoside RT inhibitor of HIV-1 RT while the hybrid constructions that express NNRTI-resistant RT variants of HIV-1 are insensitive to8Cl-TIBO demonstrating in yeast the specificity of inhibition in this assay. These hybrid Ty1/HIV-1 (his3AI/AIDS RT), called HART, elements carrying NNRTI-resistant variants of HIV-1, RT were used to identify compounds that are active against drug-resistant viruses [30] .

Integrase. HIV-1 IN catalyzes the insertion of proviral

DNA into the host-cell genome (for general reviews on HIV-1 integrase and further references see [32] [33] [34] [35] [36] ). In the first step of the integration reaction, termed 3 -end processing, two nucleotides are removed from each 3 -end of the double strand viral DNA to produce new "hydroxyl ends" (CA-3 OH). This reaction occurs in the cytoplasm within a large viral nucleoprotein complex (PIC). After entering the nucleus via the PIC multimeric complex, the 5 -ends of processed viral DNA are joined covalently to the host Journal of Biomedicine and Biotechnology Figure 3 (b)) is characterized by three highly conserved amino acid residues, D64, D116, and E152, forming the catalytic triad DD(35)E. Each one of these residues is essential for enzyme activity. The Cterminal region is the least conserved domain and is involved in nonspecific DNA binding. The 3D structure of the three domains of HIV-1 IN has been determined by RMN or X-ray crystallography. Despite considerable efforts, the low solubility of the native HIV-1 integrase has hampered the determination of the crystallographic structure of the entire protein. The three HIV-1 IN domains are required for in vitro 3 -end processing and DNA strand transfer. Mutation analyses of the viral integrase gene showed that this enzyme is required for retroviral replication and that it is a legitimate target for the design of antiretroviral drugs. Recently, the 3D structure of the human foamy retrovirus integrase was determined, taking profit of the increased solubility and yield of this recombinant retroviral enzyme, and confirming a tetramer quaternary structure [37] .

Expression of HIV-1 IN in the Yeast Saccharomyces cerevisiae.

As mentioned above a nucleoprotein complex (PIC) comprising the proviral DNA, the IN, and other viral and cellular proteins is formed in the cytoplasm and enters the nucleus by an unknown mechanism where the retroviral DNA is inserted into the host nuclear genome. The exact composition of the PIC is a controversial matter since different laboratories have described various viral and cellular proteins. The yeast two-hybrid system was used to identify a human gene product that binds tightly to the human immunodeficiency virus type 1 (HIV-1) integrase in vitro and stimulates its DNA-joining activity. This protein has been suggested as being part of the PIC. The sequence of the gene suggests that the protein is a human homolog of yeast SNF5, a transcriptional activator required for highlevel expression of many genes. The gene, termed INI1 (for integrase interactor 1), may encode a nuclear factor that promotes integration and targets incoming viral DNA to active genes [25] . The analogy of the yeast and human orthologs prompted study of whether yeast SNF5 is involved in the lethal effect of HIV-1 IN expression in yeast. The effect of the inactivation of the yeast gene encoding for SNF5 on the lethality induced by the yeast expression of HIV-1 IN has been described. Results showed that the retroviral IN is unable to perform its lethal activity in cells where the SNF5 gene has been disrupted, suggesting that SNF5 may play a role in the lethal effect induced by IN in yeast [42] . SNF5 inactivationdoes not affect neither yeast viability nor expression of HIV-1 IN. Given the homology between SNF5 and its human counterpart INI1, these results suggest that this factor may be important for IN activity in infected cells. Moreover, given the important role proposed for this transcription factor in the integration step and the fact that it is not essential for cell viability, the interaction between INI1/ySNF5 and HIV-1 IN should become a potential target in the search for new antiretroviral agents. Given the proximity of many human and yeast proteins the expression of the human INI1 rescued the lethal phenotype in yeast cells where SNF5 was inactivated, again revealing the functional analogy of many human and yeast genes [42] .

Rev.

The 116-amino-acid HIV-1 Rev, an 18 kDa phosphoprotein discovered in 1986, is capable of being imported into the nucleus and binding specially to the Rev responsive element (RRE) a viral sequence-specific RNA. Rev is able to form multimers and direct the nuclear export of large RRE-containing RNP complexes. Rev activity is crucial in the nuclear export of intron-containing HIV-1 RNAs [55] [56] [57] . Rev shuttles between the nucleus and the cytoplasm and has a nuclear localization signal (NLS) as well as a nuclear export signal (NES). These essential peptide motifs have now been shown to function by accessing cellular signal-mediated pathways for nuclear import and nuclear export. In addition to NLS and NES a nuclear import inhibitory signal (NIS) that inhibits the entry of low molecular weight proteins has been described in Rev [58] . HIV-1 Rev is therefore an excellent system to study aspects of transport across the nuclear envelope [59] . Human immunodeficiency virus type 1 (HIV-1) replication requires the expression of two classes of viral mRNA. The early class of HIV-1 transcripts is fully spliced and encodes viral regulatory gene products. The functional expression of Rev induces the cytoplasmic expression of the unspliced or incompletely spliced mRNAs that encode the viral structural proteins, including Gag and Env. Based on experiments that indicate a similar function of Rev in the yeast S. cerevisiae, a yeast protein interacting with the effector domain of Rev was found [60] . This protein called Rip1p is a novel small nucleoporin-like protein, some of which is associated with nuclear pores. Its closest known yeast relative is a nuclear pore component also involved in mRNA transport from nucleus to cytoplasm. Analysis of yeast strains that overexpress Rip1p or which are deleted for the RIP1 gene show that Rip1p is important for the effect of Rev on gene expression, indicating that the physical interaction is of functional significance in vivo. These results suggest that Rev directly promotes the cytoplasmic transcripts transport by targeting them to the nuclear pore. The NES domain of the Rev protein is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae [61] . As mentioned above Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay. Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs. Rev is able to interact with multiple FG repeat-containing nucleoporins from both S. cerevisiae and mammals. Moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast, Xenopus oocytes, and mammalian cells [62] .

Yeast and HIV-1 Translation (for a Recent Review See [66])

Retroviral frameshifting (see above) is a change in reading frame during gene expression and is a mechanism that allows to keep at a low level the synthesis rate of the functional proteins relative to that of the structural proteins. Using S. cerevisiae to decipher viral frameshifting mechanisms Wilson et al. were the first to provide an in vivo demonstration of a frameshifting event, in S. cerevisiae [67] . They inserted the Gag-Pol fragment containing the potential frameshifting site of HIV-1 (without the stimulatory element) into a yeast expression plasmid, upstream from the interferon (IFN) cDNA. They monitored production of the frameshifted protein by Western blotting. It is now clear that a stimulatory secondary structure is required for maximal frameshifting efficiency although the precise nature of this structure remained unclear for a long time. Results using a dual reporter system in yeast showed that there is a direct correlation between HIV frameshifting efficiency and the stability of the stem loop [68] . The stem loop analyzed in these studies was the upper part of the complete stimulatory element observed by NMR. Under these conditions, the stability of this structure is clearly linked to frameshifting efficiency. A structure was recently identified on the basis of the complete yeast genome sequence, and it seems interesting to explore frameshifting efficiency with the complete sequence. The structure of the tetraloop is similar to the motif found in the RNase III recognition site from S. cerevisiae [69] . As the ACAA motif in the tetraloop is poorly recognized by RNase III, the possibility of engineering the S. cerevisiae RNase III for selective targeting of the HIV-1 tetraloop followed by the expression of this protein in HIV-1-infected cells has been suggested. This hypothetical approach remains to be confirmed but would provide an interesting therapeutic strategy, derived from experiments in yeast, to limit HIV-1 proliferation. The similar slippage efficiencies of the HIV frameshifting site in vivo in yeast and in vitro in a mammalian system demonstrate the high level of conservation of frameshifting mechanisms. Bidou et al. [66] suggested that the demonstration that frameshifting is conserved from yeast to humans paves the way for the use of yeast mutants to analyze retroviral frameshifting as already reported by several groups for other viruses. For instance, SARS coronavirus (sometimes shortened to SARS-CoV) is the virus that causes severe acute respiratory syndrome. SARS-CoV carries a frameshifting signal [70] . The minimal frameshifting signal in this virus is a U UUA AAC slippery sequence and a stimulatory structure folding into a pseudoknot [71] . This pseudoknot has several unusual features, including the third stem in loop 2 and the presence of two unpaired adenosine residues within the structure ( [70, 72] . Plant and Dinman [70] demonstrated the ability of this new site to frameshift in S. cerevisiae. The frequency of frameshifting in yeast was much lower (3%) than for other coronavirus sites tested in yeast (12% for infectious bronchitis virus (IBV)). This may indicate the existence of subtle differences in terms of frameshifting mechanisms. Indeed, the importance of the unpaired adenosine residues remains unclear as this part of the pseudoknot is thought to lie outside the ribosome. It would be interesting to investigate the possible binding of a transacting factor, although the binding of such a factor has never been detected with the well-studied IBV coronavirus pseudoknot. The yeast mak8-1 mutant is known to have a specific defect in frameshifting [73] . It carries an altered form of ribosomal protein L3 in the ribosomal peptidyl-transferase center. This strain was reported to have a slightly higher SARS-CoV frameshifting efficiency than wildtype strains [70] , in the first demonstration that this newly discovered frameshifting site is used.

Expression of HIV-1 IN in the Yeast Saccharomyces cerevisiae.

Based on results dating from 1985 where the expression of the bacterial EcoR1 restriction endonuclease expressed in yeast cells led to the appearance of a lethal phenotype [38] the idea emerged that the expression of retroviral IN also carrying an endonuclease activity may produce a similar phenotype. The idea behind this approach was that the feasibility of such a system may allow the setting up of an easy and rapid procedure for screening antiretroviral drugs. However, the weak entrance of molecules through the yeast wall and cell membrane hampered this project. The yeast model has been extremely useful to study many aspects of the integration step in retroviral replication. An expression plasmid containing the retroviral integrase gene under the control of the inducible promoter ADH2/GAPDH regulated by the glucose concentration of the medium was constructed. Haploid yeast strain W303-1A did not appear to be clearly sensitive to HIV-1 integrase expression ( Figure 4 ). However, disruption of the RAD 52 gene, which is involved in the repair of double-strand DNA breaks, strongly increased the deleterious effects of the retroviral enzyme in this yeast strain. The diploid strain constructed with W303-1A and an isogenic strain of the opposite mating type also showed strong sensitivity to the HIV-1 IN. The lethal phenotype was suppressed by missense integrase mutations in the catalytic domain that are known to abolish HIV-1 IN activities in vitro ( Figure 4 ) [39] . Subsequent studies were performed in order to determine the critical amino acid(s) and/or motif(s) required for the induction of the lethal phenotype in the [40] . The question remained whether the lethal effect was related to the nonspecific endonuclease activity of the viral IN or whether the mechanism involved was due to a pleiotropic effect of this protein. Lethality in yeast seems to be related to the mutagenic effect of the recombinant HIV-1 IN, most probably via the non-sequence-specific endonuclease activity carriedout by this enzyme. This nonsequence-specific endonuclease activity was further characterized. Although the enzyme was active on DNA substrates devoid of viral long terminal repeat (LTR) sequences characteristic of the retroviral proviral genome, the presence of LTR regions significantly stimulated this activity. Genetic experiments showed that both the mutagenic effect and the level of recombination events were affected in cells expressing the active retroviral enzyme, while expression of the mutated inactive IN D116A had no significant effect [41] .