use prefix []or [-]not [+]and [=]has feature [!]exclude feature ie. 'interleukin-6 -animal +phenotypic =protein !tumor'

Displaying 10 papers, 43 pages, start at 351, 67 Hits
6 section matches

Abstract

Influenza virus is the most frequently reported viral cause of rhabdomyolysis. A 7-year-old child is presented with rhabdomyolysis associated with parainfluenza type 2 virus. Nine cases of rhabdomyolysis associated with parainfluenza virus have been reported. Complications may include electrolyte disturbances, acute renal failure, and compartment syndrome.

Discussion

In two cases of parainfluenza precipitating rhabdomyolysis, a dual infection was detected in postmortem specimens. In a child, viral particles consistent with a picornavirus were detected by electron microscopy in the muscles [7] . Chlamydia pneumoniae was detected by PCR in an adult with hemorrhagic pneumonia [11] . Whether coinfection carries a worse prognosis in the context of rhabdomyolysis remains to be clarified.
Antiviral treatment such as ribavirin in rhabdomyolysis caused by parainfluenza virus was not reported. The efficacy of antivirals such as neuraminidase inhibitors in rhabdomyolysis caused by influenza is unknown. In addition, recurrent rhabdomyolysis and compartment syndrome, first precipitated by parainfluenza and on a second occasion by influenza, have been described in a child unvaccinated for influenza [15] . Recurrent rhabdomyolysis associated with influenza has been reported in 10 children [12] . Our patient's risk of developing recurrent disease upon subsequent viral infection is unknown. His mother agreed for the child to receive the influenza immunization. Influenza vaccination triggering rhabdomyolysis is an extremely rare event and has occurred in adults on statin therapy [16] .

Conclusion

In conclusion, although mild myalgias are commonly reported and are self-limited in infections caused by parainfluenza virus, worsening pains, difficulty in ambulating, exquisite tenderness to palpation, or muscle swelling should prompt further investigations to detect possible rhabdomyolysis, even in the absence of dark urine. Potential complications include acute renal failure, electrolyte disturbances, and compartment syndrome. Although influenza is the most frequently reported viral cause of rhabdomyolysis, the implementation of laboratory methods such as multiplex polymerase-chain reaction assays may lead to enhanced recognition and characterization of rhabdomyolysis associated with parainfluenza or other pathogens.

Introduction

Rhabdomyolysis is characterized by the breakdown of skeletal muscle fibers. It has a diverse etiology including infections, trauma, strenuous exercise, drug reactions, metabolic disorders, and status epilepticus [1] . In contrast with the adults, Mannix et al. in the largest series of pediatric rhabdomyolysis, reported that viral myositis was the most frequent cause, accounting for 38% (73/191) of cases and in particular during the first decade of life [2] . Viruses associated with rhabdomyolysis include influenza types A and B, HIV, enteroviruses, Epstein-Barr, cytomegalovirus, adenovirus, herpes simplex, and varicella virus. Influenza has been considered as the most frequent [1] .

Case Description

White blood cell count was 3.000/mcl, with 60% neutrophils, 32% lymphocytes, and 7% monocytes. The hemoglobin was 13.5 gr/dL, the hematocrit 41%, and the platelet count 244.000/mcl. C-reactive protein and erythrocyte sedimentation rate were normal. His electrolytes were within normal limits; BUN was 13 mg/dL (normal 5-20), and creatinine was 0.44 mg/dL (normal 0.10-0.80). AST was 623 U/L (normal 0-35); ALT was 105 U/L (normal 0-40). Alkaline phosphatase and total bilirubin were normal. A urinalysis obtained by his pediatrician showed pH 6.5, specific gravity 1.037, 2+ protein, and 3+ hemoglobin. Microscopy showed 2 white blood and 1 red blood cells/high-power field. Myoglobin was detected in the urine. Initial serum creatinine phosphokinase (CPK) was 5,614 U/L (normal <226). A nasopharyngeal specimen was tested for various respiratory pathogens by a multiplex nested polymerasechain reaction assay (FilmArray Respiratory Panels, BioFire Diagnostics Inc., Salt Lake City, UT, USA). It was found nonreactive for influenza A and B, adenovirus, rhinovirus/ enterovirus, coronavirus, metapneumovirus, respiratory syncytial virus, Bordetella pertussis, Mycoplasma pneumonia, Chlamydia pneumoniae, and parainfluenza virus types 1, 3, and 4, but it was found positive for parainfluenza type 2. A blood culture drawn on admission remained negative.
6 section matches

Abstract

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease leading to numerous hospitalizations and deaths among the infant and elderly populations worldwide. There is no vaccine or a less effective drug available against RSV infections. Natural RSV infection stimulates the Th1 immune response and activates the production of neutralizing antibodies, while earlier vaccine trials that used UV-inactivated RSV exacerbated the disease due to the activation of the allergic Th2 response. With a focus on Th1 immunity, we developed a DNA vaccine containing the native RSV fusion (RSV F) protein and studied its immune response in BALB/c mice. High levels of RSV specific antibodies were induced during subsequent immunizations. The serum antibodies were able to neutralize RSV in vitro. The RSV inhibition by sera was also shown by immunofluorescence analyses. Antibody response of the RSV F DNA vaccine showed a strong Th1 response. Also, sera from RSV F immunized and RSV infected mice reduced the RSV infection by 50% and 80%, respectively. Our data evidently showed that the RSV F DNA vaccine activated the Th1 biased immune response and led to the production of neutralizing antibodies, which is the desired immune response required for protection from RSV infections.

Introduction

Respiratory syncytial virus (RSV), a member of genus Pneumovirus and classified in the family Paramyxoviridae, is the most common cause of severe disease of the lower respiratory tract in infants and the elderly especially in developing countries [1, 2] . There are also some reports claiming that RSV could lead to severe repeated infections such as recurrent wheezing, pneumonia, or asthma in later childhood [3] . Worldwide, the number of RSV-associated cases is estimated to be 33 million and the number of deaths up to 234,000 in children younger than 5 years old in spite of the fact that those numbers are lower in the USA due to the precautions against RSV [4, 5] . Besides the young children, the hospitalization rate of elderly people above 50 years old may be the same as influenza cases [2] . RSV vaccine development efforts such as inactivated RSV, live-attenuated RSV, or subunit vaccines are underway.

Statistical

Analysis. qPCR, ELISA, and virus neutralization assay data are presented as means and standard deviations; statistical analysis of the data was performed using Sigma plot version 11.0 (Systat Software, Inc., Germany). Differences between the means of the four experimental groups were determined using one-way analysis of variance (ANOVA) Tukey's test with the significance level of 1%.

Introduction

However, despite over five decades of intensive research on developing a RSV vaccine, there is no approved vaccine or drug available [6] . Instead of vaccine, some researchers have been attempting to develop prophylactic antibody therapies targeting RSV F protein [7, 8] . Antiviral drugs such as ribavirin (a nucleoside analog), which targets hepatitis C and other viruses including RSV, ALS-8176 (a new nucleoside analog), and GS5806 (pyrazolo [1,5-a] pyrimidine based RSV fusion inhibitor), and neutralizing monoclonal antibodies such as Palivizumab (Synagis6) and Motavizumab (Numax), are administered to infants at high risk of developing respiratory diseases [9] [10] [11] [12] . As an alternative to expensive therapies, a vaccine conferring long lasting immunity is a less expensive and more efficient option against recurrent RSV infections [10] . Due to frequent antigenic variations of RNA viruses (RSV, influenza virus, and rhinovirus), developing a vaccine with complete protection is challenging. The incomplete immunity in response to natural RSV infections is responsible 2 Advances in Virology for repeated infections. RSV vaccine studies in the 1960s using formalin inactivated RSV (FI-RSV) consisting of the whole virus exacerbated the disease and even in some cases resulted in deaths because of the elevated T helper type-2 (Th2) mediated immune response [1, 13] . In addition, using a vector expressing RSV antigens is found far safer than subunit or inactivated RSV immunization [14] . With these important immunological responses, a safe and stable vaccine with long lasting immunity is an urgent need for the public.
The outer surface glycoproteins, fusion (F) and attachment (G), of RSV are known antigenic proteins that induce the humoral and cellular immune responses and are targets of antigen presenting cells [15] . The RSV F protein mediates the fusion of the virus particle into the host by merging the virion envelope with the host cell membrane following virion attachment using the G protein. In addition, the F protein facilitates fusion of neighboring normal cells with infected cells, thus creating multinuclear giant cells called syncytia, which characterizes RSV infection [9, 16] . The RSV F protein is highly conserved among the different RSV strains compared to other RSV proteins [16] . On the other hand, the variability of the G amino acid sequence among various RSV strains is high [17] . Furthermore, previous reports demonstrated that RSV F vaccines provide protection against both RSV A and RSV B strains by producing neutralizing antibodies [8, 14, 18, 19] , whereas RSV G vaccines prominently induced a Th2 biased immune response, thereby enhancing the severity of the disease in subsequent RSV infections [20, 21] .

Animals and RSV Stock

Preparation. BALB/c female mice (4-6 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). The animals were housed under standard approved conditions with a cycle of 12 h of light and 12 h of darkness and provided daily with sterile food and water ad libitum. For all immunization studies, an approved protocol by the Alabama State University Institutional Animal Care and Use Committee was followed. Human RSV long strain was purchased from the American Type Culture Collection (ATCC, Manassas, VA, ATCC # VR-26) and propagated in HEp-2 cells (ATCC # CCL-23). HEp-2 cells were grown in tissue culture flasks in MEM supplemented with 10% FBS and antibiotics. Human RSV was added to the cell monolayer, and virus adsorption was carried out for 1 h at 37 ∘ C in a humidified atmosphere with 5% CO 2 . MEM with 2% FBS was added to the flask and infection of cells was observed for an additional 3-4 days. RSV infected cells were centrifuged at 3,000 ×g at 4 ∘ C to remove cellular debris, aliquoted, and stored at −80 ∘ C until they were used.
6 section matches

Abstract

The life cycle of the human immunodeficiency virus type 1 (HIV-1) has an absolute requirement for ribosomal frameshifting during protein translation in order to produce the polyprotein precursor of the viral enzymes. While an RNA stemloop structure (the "HIV-1 Frameshift Stimulating Signal", or HIV-1 FSS) controls the frameshift efficiency and has been hypothesized as an attractive therapeutic target, developing compounds that selectively bind this RNA and interfere with HIV-1 replication has proven challenging. Building on our prior discovery of a "hit" molecule able to bind this stem-loop, we now report the development of compounds displaying high affinity for the HIV-1 FSS. These compounds are able to enhance frameshifting more than 50% in a dual-luciferase assay in human embryonic kidney cells, and they strongly inhibit the infectivity of pseudotyped HIV-1 virions.

■ INTRODUCTION

Building on our laboratory's longstanding interest in understanding the factors that drive affinity and sequence selectivity in small molecule recognition of RNA, 33 we previously reported the use of an 11,325-member resin-bound dynamic combinatorial library 34 (designed based on the structure of DNA-binding, bisintercalating peptide antibiotics) to identify a compound (1) able to bind the HIV-1 FSS upper stem-loop with moderate affinity (K D = 4.1 ± 2.4 μM immobilized on an surface plasmon resonance (SPR) chip via one of its amine groups; K D = 350 ± 110 nM in solution as measured by fluorescence 35 ) and good selectivity. 36 Subsequent efforts revealed that replacement of the disulfide bridge with an olefin (to produce compounds 2 and 3) could be accomplished without any reduction in affinity. These studies also indicated that affinity was essentially abolished if the π-surface area of the molecule was reduced, while the peptidic portion of 1 was required for sequence-selective binding. 35 (Right) HTLV-2 FSS stem-loop sequence used as a specificity control in this work. Note that when the slippery sequence occupies the decoding site of the ribosome, the lower stem is unwound and it is the upper stem-loop that acts as the effective frameshift stimulatory signal. While the binding ability of 2 and 3 was intriguing, subsequent preliminary experiments indicated the compounds were unable to inhibit virus in a pseudotyped HIV-1 assay (data not shown). Given these results, we hypothesized that further increases in affinity were essential. We anticipated that increasing the π surface area of 1 could represent a viable strategy for enhancing the affinity for the HIV-1 FSS without significant reductions in selectivity. In particular, incorporation of a benzo[g]quinoline moiety to produce 4 and 5 was viewed as attractive. This hypothesis was supported in part by parallel efforts in our laboratory on the design and synthesis of compounds targeting CUG repeat RNA in which incorporation of a benzo[g]quinoline was found to enhance affinity and to provide compounds with activity in vivo in a mouse model of Type 1 Myotonic Dystrophy. 37 Synthesis of 4 and 5 proceeded via cross-metathesis of half-structure 6, by analogy to our previous work. We also synthesized 7 and 8 ("one-armed" benzo[g]quinoline-bearing structures), as well as compounds 9 and 10, to test the effect of sequential removal of the putative intercalators. Compound 11 was synthesized in order to ascertain the effectiveness of the olefin bioisostere in improving cellular availability and bioactivity relative to an easily reduced disulfide.
Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immune deficiency syndrome (AIDS), remains a significant challenge to global health. 1, 2 Since its initial identification in 1983, HIV-1 infection has reached the status of a pandemic. In 2009 alone, there were approximately 2.7 million new infections and about 2.0 million deaths from AIDS related causes. 3 Currently there is no cure for HIV-1 infection. While progression of the disease can be controlled by highly active antiretroviral therapy (HAART), a combination of drugs designed to inhibit different stages in the virus' life cycle, 4 the complexity of the HAART regimen, and the ability of the virus to evolve resistance suggest that alternative drug targets for HIV-1 treatment and prophylaxis are needed. 5 One potentially attractive target for pharmacological interference in the HIV-1 life cycle is the virus' requirement for a programmed −1 ribosomal frameshift (−1 PRF) in order to express its enzymes. 6 Ribosomal frameshifting is a recoding mechanism common among viruses with polycistronic (multiple open reading frames, or ORFs, in a single gene) genomes. It allows viruses to translate polypeptides in different ORFs by avoiding the stop codon(s) present in the single mRNA transcript. In HIV-1, the pol gene is in the −1 reading frame with respect to gag. Gag, the precusor of the viral structural proteins, is produced via normal translational rules, while Pol, the precursor of the viral enzymes, is synthesized as a fused Gag-Pol polyprotein via −1 PRF. This occurs with a frequency of 5−10% of ribosomes translating the full-length viral mRNA. A critical molar ratio of Gag-Pol to Gag protein is required for HIV-1 replication and infectivity; alterations to this ratio have been shown to be detrimental. 7 −1 PRF in HIV-1 is controlled by two cis acting mRNA elements: a heptameric slippery sequence (U UUU UUA), with the 0 frame indicated by spaces and where the frameshift actually occurs, and a downstream two-stem helix immediately following the slippery sequence, also known as the frameshift stimulatory signal (HIV-1 FSS, Figure 1 ). 8 While several mechanisms have been proposed to account for the frameshift, 9 it is currently hypothesized that this event results from an incomplete translocation for a limited number of ribosomes, due to resistance of the FSS to unwinding. 10−12 These ribosomes then start translation of pol in the new −1 reading frame. Modification of the slippery site or stimulatory sequence (either via natural variation or laboratory mutations) in ways affecting frameshifting efficiency translates to a decrease in viral replication. 13, 14 These and other results have led several groups to propose −1 PRF as a potential target for developing antiretroviral agents for HIV-1. 6,15−17 NMR structural analyses indicate that the HIV-1 FSS RNA consists of a G-C rich upper stem-loop structure, 18 separated from a flexible lower stem by a GGA trinucleotide bulge ( Figure 1 ). 19, 20 The bulge produces a roughly 60°bend between the upper and lower stems. The upper stem-loop is exceptionally stable. This stability is believed to play a vital role in the stimulation of the frameshift, since the ribosome must unwind the stem during translation. The lower stem is thermodynamically less stable. The highly structured ACAA tetraloop is uncommon among tetraloops 21 but is conserved among all HIV-1 group M subtypes except the uncommon H and J subtypes. Likewise, the heptameric slippery sequence is conserved across all HIV-1 group M subtypes. SHAPE analysis of the intact HIV genome suggests a more complex structure for the FSS RNA, although the upper stem-loop is retained. 22, 23 Since other viruses also rely on frameshifting, 24 targeting frameshift-regulating structures may have general utility beyond the context of HIV. For example, human T-cell leukemia virus type 2 (HTLV-2) uses two −1 PRF events similar to HIV-1 in order to synthesize fused Gag-Pro and Gag-Pro-Pol precursor proteins. 25 The RNA responsible for the −1 PRF essential for expression of Gag-Pro in HTLV-2 also consists of two cis-acting RNA elements, a heptanucleotide (AAAAAAC) slippery site and a stem-loop shown in Figure 1 . 26 This HTLV-2 FSS served as a sequence specificity control in the experiments we describe herein.
The first attempted use of a synthetic molecule to alter HIV-1 frameshifting and thereby influence viral replication was reported by Green and co-workers in 1998. 27 The authors showed that 1,4-bis[N-(3-N,N-dimethylpropyl)amidino]benzene tetrahydrochloride, a bis guanidinium-containing compound termed "RG501", was able to stimulate −1 frameshifting, alter the Gag-Pol:Gag ratio, inhibit HIV-1 replication in CEM cells (a lymphocytic cell line), and interfere with the formation of viral particles in chronically infected CH-1 cells (a COS cell line stably transfected with HIV-gpt, an HIV derivative in which the E. coli gpt gene replaces HIV env 28 ). Increases in reverse transcriptase (RT) following treatment with 1.5 mM RG501 were also observed, as would be expected for an increase in Gag-Pol production. Recently, the Butcher group confirmed that this compound indeed binds the HIV-1 FSS RNA with weak affinity (K D ∼ 360 μM), and they carried out NMR structure analysis, indicating that RG501 binds in the major groove of the upper stem-loop. 29 Unfortunately, as noted by the authors, RG501 is a relatively nonselective binder and interacts with other RNAs. It stimulates frameshifting in viruses with different FSS, 29 and likely that also interacts with the ribosome. 6 RG501 is also toxic, 27 a likely result of its lack of selectivity. Moreover, its interference with HIV replication begins at concentrations below those observed to affect frameshifting. 27, 24 Other compounds such as guanidinoneomycin, 30 idarubicin, and doxorubicin 31 have been shown to bind the HIV-1 FSS. Doxorubicin was found to bind with a K D of 2.8 μM, decreased frameshifting in a rabbit reticulocyte assay, and also significantly reduced overall translation. A screen of an Arg-rich peptide library revealed a sequence able to significantly reduce frameshifting, but this displayed no selectivity for the HIV-1 FSS relative to other frameshift-stimulating constructs and likely also interacts with the ribosome. 32 Thus, as far as we are aware, there are no reported examples of synthetic molecules able to alter HIV-1 frameshifting and interfere with viral infectivity via selective, high-affinity binding to the FSS RNA.

Dual-Luciferase Frameshift Assay in HEK 293 FT Cells.

Compounds 4 and 5 Decrease Viral Infectivity. In order for the observed −1 frameshift effect of compounds to be significant, it was important that it correlate to an equivalent reduction in viral replication. To assess this, we analyzed the effect of compounds in a single-round infection with pseudotyped HIV in HEK 293T producer cells and TZM-bl target cells (a HeLa cell line). 46 The wild type HIV proviral vector (pDHIV3-GFP) codes for all HIV-1 NL4−3 genes except nef (which is replaced with GFP) and env, thus preserving gag and pol, and the frameshift required for production of the Gag-Pol polyprotein. Preliminary experiments with 2 and 3 showed no activity in this assay. However, we observed a statistically significant (p < 0.001) and concentration-dependent decrease in infectivity of the pseudotyped HIV-1 virions when producer cells were treated with 4 and 5 ( Figure 5 ). The decrease in infectivity was >90% at a concentration of 20 μM 4, and >90% at a concentration of 40 μM for 5. The core peptide alone (10) showed a modest decrease in infectivity at a concentration of 40 . Compounds 4 and 5 increase frameshifting (>50%) in a dual-luciferase assay incorporating the HIV-1 FSS but have no effect on frameshifting in analogous assay incorporating the HTLV-2 FSS. Relative frameshift efficiency in HEK 293FT cells treated with 4, 5 or control peptide 10 after transfection with pDualHIV (−1) or pDualHTLV-2 (−1). The frameshift efficiency was calculated as the ratio of Fluc to Rluc in pDualHIV(−1) or pDualHTLV-2 (−1) transiently transfected cells. This ratio was arbitrarily set to 100% for plasmid-transfected cells, but not exposed to compounds. Error is SEM on three replicates for each concentration. μM, likely via nonspecific effects, given its lack of affinity for the FSS. EC 50 values for 4 and 5 were 3.9 and 25.6 μM, respectively. The protease inhibitor Indinavir was tested in parallel to calibrate the assay; its EC 50 was found to be 14.8 nM, in line with previously reported values. 47 While virus titer was determined in these experiments using an ELISA assay to normalize viral load into target cells, a compound-dependent decrease in viral production was also readily observable via fluorescence microscopy, as the pseudotyped HIV carries GFP as a marker ( Figure 6 ). Phasecontrast images of these cells showed no morphological changes, consistent with WST-1 results (Supporting Information). The concentration range required for a decrease in viral infectivity by 4 and 5 was similar to the range at which a significant increase in −1 frameshift was observed in HEK 293 FT cells, supporting the hypothesis that these compounds exert their antiviral activity primarily by altering −1 PRF. In order to provide further support for this hypothesis, viral particles were isolated from supernatants from these experiments by spinning through a 20% sucrose cushion, and probed via Western blot for the presence of reverse transcriptase (RT). As RT is produced only as part of the Gag-Pol fusion, increasing amounts of RT relative to capsid protein p24 (a structural protein produced as part of Gag) would be required given an increase in frameshifting. Indeed, that is what is observed as a function of added 4 or 5 (Figure 7) . The amount of RT in unprocessed Gag-Pol (p160) relative to mature RT (p66 + p51) also increased significantly; this may indicate that treatment with compounds 4 and 5 also inhibits Pol processing. However, the pattern of p160 cleavage intermediates observed

Journal of Medicinal Chemistry

HIV-1 Infectivity Assay. The antiviral activity of 4, 5, and 10 was measured by single-round infectivity assay with pseudotyped HIV-1 using HEK293T producer cells. The HIV-1 proviral vector (pDHIV3-GFP) codes for all HIV-1 NL4−3 genes except nef (replaced with GFP) and env, thus preserving gag and pol, and the frameshift required for production of the Gag-Pol polyprotein. A single-round infectivity assay was conducted by transient transfection of the viral vector with VSV-G coat protein vector at a ratio of 1:0.5 using Fugene HD (Promega). The virus producer cells were dosed with compounds four hours after transfection, and viral particles were harvested from the media 24 h after transfecting by filtering through a 0.45-μm syringe filter. Viral load was normalized with a p24 ELISA (Perkin-Elmer).
6 section matches

Abstract

We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1κ and one IgG1λ, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common "a" determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log 10 higher titer than the original IgG1λ. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody.

Introduction

Hepatitis B virus (HBV) is one of the world's most common infectious agents causing millions of infections each year [1] . Between 500,000 and 700,000 people die each year from chronic infection-related cirrhosis, hepatocellular carcinoma (HCC) or from fulminant hepatitis B [1, 2] . Transmission occurs via percutaneous and mucosal exposure to infectious body fluids. Therefore, the most common route of transmission is sexual transmission. However, infection through blood transfusions and blood products has not been completely eliminated [3] and contaminated injections during medical procedures, sharing of needles, syringes and paraphernalia among intravenous drug users still represent a major public health problem. Vertical transmission is common, especially in Asia, where HBV titers in maternal blood are high, and in developing countries which have not yet implemented hepatitis B vaccination. Further, HBV poses a risk to healthcare workers exposed to accidental needle-stick injuries.
The costs of HBIG treatment and prevention are not the only limitations to its use. Additional limitations include the following: i) supply is limited and depend on vaccinated human donors exhibiting high titer protective anti-HBs, ii) purification is time consuming and must undergo lengthy virus-inactivation procedures; iii) anti-HBs titer is variable and effective virus neutralization efficiency largely unknown being exclusively based on arbitrarily protective anti-HBs serum titers; iv) polyclonal immunoglobulin include several antibody specificities and may select for HBV mutants resistant to currently available antiviral drugs; v) HBIG preparations are currently combined with antiviral drugs to insure complete protection, thus adding to the costs.

Discussion

One potential problem in the prevention of HBV infection or reinfection is the emergence of HBsAg escape mutants, which have been described in a minority of patients on HBIG prophylaxis after liver transplantation or emerging as a result of selection after immunization [22, 23] . This problem can by and large be overcome by maintaining a high titer of neutralizing antibody and this can be achieved with a potent mAb such as the one described herein. The problem may be circumvented by preparing pools of mAbs, although this would be extremely difficult to achieve working with human mAbs since the common "a" determinant is a dominant epitope which tends to monopolize the specificity of immune responses to a complex protein. Indeed, the "original antigenic sin", first described in 1953 [24] , is the phenomenon in which sequential exposure to viral variants induces preferential antibody response to a dominant virus strain encountered in the past. As a result, the response to the minor strains is diminished.

Introduction

Prior to the routine use of HBIG as immunoprophylaxis, recurrence of HBV in the liver allograft occurred in up to 80%, and infrequently was associated with an aggressive fibrosing cholestatic variant that caused progressive graft dysfunction and significant mortality. The subsequent availability of safe and effective antiviral drugs led to additional survival benefits by improving prophylactic efficacy and preventing disease progression in those with recurrence [5] .
HBIG prophylaxis is expensive. HBIG is commonly administered intravenously at high dose, daily for the first week and monthly thereafter, which makes the current costs of management of patients transplanted for HBV-related cirrhosis prohibitive, even for developed countries. Dose reduction has been proposed for cost reduction, either based on a flat dose or on a response-guided basis in order to maintain circulating anti-HBs at a protective level. However, HBIG doses are variable and should be individualized among patients. It has also been proposed to abandon HBIG prophylaxis in favor of using antiviral drugs alone, however this is a very controversial issue [5] .
6 section matches

Abstract

A novel series of {4-[(2-amino-6-chloro-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}alkylphosphonates and {4-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methyl]-1H-1,2,3triazol-1-yl}alkylphosphonates as acyclic analogues of guanosine were synthesized and assessed for antiviral activity against a broad range of DNA and RNA viruses and for their cytostatic activity toward three cancerous cell lines (HeLa, L1210 and CEM). They were devoid of antiviral activity; however, several phosphonates were found slightly cytostatic against HeLa cells at an IC50 in the 80-210 µM range. Compounds (1R,2S)-17k and (1S,2S)-17k showed the highest inhibitory effects (IC50 = 15-30 µM) against the proliferation of murine leukemia (L1210) and human T-lymphocyte (CEM) cell lines.

Antiviral Activity and Cytostatic/Cytotoxic Evaluation

All phosphonates 16a-j and 17a-k were evaluated for their antiviral activities against a wide variety of DNA and RNA viruses using the following cell-based assays: (1) human embryonic lung (HEL) cell cultures: herpes simplex virus-1 (KOS), herpes simplex virus-2 (G), vaccinia virus, vesicular stomatitis virus, herpes simplex virus-1 (TK − KOS ACV r ) and adenovirus-2, cytomegalovirus (AD-169 strain and Davis strain) and varicella-zoster virus (TK + VZV stain and TK − VZV stain); (2) HeLa cell cultures: vesicular stomatitis virus, Coxsackie virus B4 and respiratory syncytial virus; (3) Vero cell cultures: para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus; (4) Crandell-Rees feline kidney (CRFK) cell cultures: feline corona virus (FIPV) and feline herpesvirus (FHV); and (5) Madin-Darby canine kidney (MDCK) cell cultures: influenza A virus H1N1 subtype, influenza A virus H3N2 subtype and influenza B virus. Ganciclovir, cidofovir, acyclovir, brivudine, (S)-9-(2,3dihydroxypropyl)adenine ((S)-DHPA), Hippeastrum hybrid agglutinin (HHA), Urtica dioica agglutinin (UDA), dextran sulfate (molecular weight 5000, DS-5000), ribavirin, oseltamivir carboxylate, amantadine and rimantadine were used as the reference compounds. The antiviral activity was expressed as the EC50: the compound concentration required to reduce virus-induced cytopathogenicity by 50%. Unfortunately, no inhibitory activity against any virus was detected for the evaluated compounds at 250 µM.

Antiviral Activity Assays

The compounds were evaluated against the following viruses: herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK − ) HSV-1 KOS strain resistant to acyclovir (ACV r ), herpes simplex virus type 2 (HSV-2) strains Lyons and G, varicella-zoster virus (VZV) strain Oka, TK − VZV strain 07−1, human cytomegalovirus (HCMV) strains AD-169 and Davis, vaccinia virus Lederle strain, respiratory syncytial virus (RSV) strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, para-influenza 3, influenza virus A (subtypes H1N1, H3N2), influenza virus B, reovirus-1, Sindbis, reovirus-1, Punta Toro, human immunodeficiency virus type 1 strain IIIB and human immunodeficiency virus type 2 strain ROD. The antiviral, other than anti-HIV, assays were based on the inhibition of the virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cells (HeLa) or Madin-Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU) (VZV) in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC50 or compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%.

Introduction

An effective treatment for viral infections is one of the most difficult goals of contemporary medicine. The discovery of acyclic nucleosides/nucleotides, which act as antimetabolites, had a significant impact on the progress in the therapy of viral infections [1, 2] . Among them, adefovir is active against DNA viruses and retroviruses [3] [4] [5] , whereas tenofovir exhibits high potency and selectivity against HIV-1 and HIV-2 viruses and hepatitis B virus [6, 7] . Ganciclovir [8] [9] [10] and its prodrug with improved oral bioavailability, valganciclovir [11, 12] , are used for the treatment of cytomegalovirus infections. Cidofovir [5, 13, 14] shows activity against herpes viruses, including cytomegalovirus, as well as adenoand pox-viruses. The specificity of the antiviral activity of the compounds already known strongly depends on the structural features of the aliphatic chain installed as a sugar ring replacer, whereas a choice of nucleobases is mostly limited to adenine, guanine and 2,6-diaminopurine. Various guanine-containing analogues of nucleosides have been reported as potent antiviral agents ( Figure 1 ) [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . Moreover, acyclic analogues of nucleotides having guanine and hypoxanthine as a nucleobase with antimalarial activity have also been reported [26, 31] . Phosphorylation of nucleosides and their structural analogues is inefficient, and at the same time, it appears to be one of the most important steps with implications on their activity, since the first step of phosphorylation is carried out by viral kinases. Therefore, several nucleotide analogues have been designed by incorporation of a phosphonate residue ((RO)2P(O)-CH2-) instead of a phosphate group ((RO)2P(O)-O-C(5′)) to avoid the first phosphorylation step and to ensure the stability of phosphonates to enzymatic hydrolysis [32] [33] [34] [35] . In recent years, analogues of nucleotides containing various modifications of an acyclic fragment have been widely studied. Among them, extended linkers, including a 1,2,3-triazole moiety, were synthesized [36] [37] [38] [39] [40] , and several compounds with promising anticancer (1-3) [38] and antiviral (3) (4) (5) (6) (7) (8) [37] [38] [39] properties were found. Although various canonical nucleobases and their mimetics were applied, only a few acyclic guanosine, as well as 2-amino-6-chloropurine analogues containing the 1,2,3-triazole linker (9) (10) (11) (12) (13) have been obtained so far ( Figure 2 ) [36, [40] [41] [42] [43] ; however, among them, only Compound 13 was tested and revealed inhibitory activity against thymidine phosphorylase [40] . As a continuation of our ongoing project directed towards biologically-active acyclic analogues of nucleotides with the 1,2,3-triazole linker, a new series of analogues 16 and 17 containing 2-amino-6-chloropurine and guanine as nucleobases has been designed to study their antiviral and cytostatic properties. To install a guanine moiety at C4 in the 1,2,3-triazole ring, two strategies were used. The dipolar cycloaddition of the respective azidoalkylphosphonates 14 to propargylated guanines should directly lead to Compound 15 or 16 [41] [42] [43] , whereas application of 2-amino-6-chloro-9-propargylpurine as dipolarophile in the reaction with azides 14 should produce Compound 17 to be subsequently transformed into 16 [40, [44] [45] [46] [47] (Scheme 1).

Conclusions

A novel series of {4-[(2-amino-6-chloro-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}alkylphosphonates 17 was synthesized and subsequently transformed into {4-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) methyl]-1H-1,2,3-triazol-1-yl}alkylphosphonates 16 as acyclic analogues of guanosine. Evaluation of the antiviral activity of phosphonates 17a-k, as well as 16a-j was performed against a broad variety of DNA and RNA viruses; however, none of them was found active at concentrations up to 250 μM. The cytostatic properties of Compounds 17a-k and 16a-j were studied on L1210, CEM and HeLa cell lines. Among them, Compounds (1R,2S)-17k and (1S,2S)-17k were moderately active toward L1210 and CEM cells (IC50 in the 16-30 µM range).

Scheme 1.

Generally, in order to secure sufficient bioavailability of active phosphonate nucleotide analogues, they are administered as prodrugs, namely the respective phosphonate esters or amides [33] [34] [35] [48] [49] [50] [51] [52] [53] . For this reason, we designed guanosine analogues 16 and 17 as the respective phosphonate esters to ensure sufficient membrane permeability. Moreover, our recent experiences clearly supported the strategy to prepare diesters rather than free phosphonic acids, which are completely ionized at physiological pH. Indeed, we found several examples of active diesters in the class of 1,2,3-triazole phosphonates, whereas the respective free acids appeared inactive [38, 39, 54] .
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Abstract

Analogues or isosteres of α,γ-diketoacid (DKA) 1a show potent inhibition of hepatitis C virus (HCV) NS5B polymerase through chelation of the two magnesium ions at the active site. The anti-HCV activity of the flavonoid quercetin (2) could partly be attributed to it being a structural mimic of DKAs. In order to delineate the structural features required for the inhibitory effect and improve the anti-HCV potency, two novel types of quercetin analogues, 7-O-arylmethylquercetins and quercetin-3-O-benzoic acid esters, were designed, synthesized and evaluated for their anti-HCV properties in cell-based assays. Among the 38 newly synthesized compounds, 7-O-substituted derivative 3i and 3-O-substituted derivative 4f were found to be the most active in the corresponding series (EC50 = 3.8 μM and 9.0 μΜ, respectively). Docking studies suggested that the quercetin analogues are capable of establishing key coordination with the two magnesium ions as well as interactions with residues at the active site of HCV NS5B.

Introduction

It was estimated that about 170 million people worldwide were chronically infected by hepatitis C virus (HCV) and were consequently at risk of developing liver cirrhosis and/or hepatocellular carcinoma [1] . A protective vaccine is not yet available and the previous standard of care (SOC) , which is pegylated-interferon, combined with ribavirin, is often difficult to tolerate and results in a sustained viral response (SVR) in only 50% of patients infected with the predominant genotype 1 [2, 3] . Recent advances in the development of direct acting antivirals (DAAs) have significantly improved SVR in patients, providing a new hope for cure in infected patients. Three NS3/4A protease inhibitors were approved by FDA for the treatment of genotype 1 hepatitis C, and in late 2013, sofosbuvir, a first-in-class NS5B polymerase inhibitor, was launched and became a cornerstone of recommended HCV therapy against almost all HCV genotypes [4] [5] [6] [7] . However, due to the potential of drug resistant strains [8] and the high price of the new HCV drugs [9] , the development of novel anti-HCV agents is still an urgent necessity.

Cells and Viruses Preparations

The biological assays were performed following a previously reported procedure [23] . The Huh7.5.1 cell line and the HCV infectious virus J399EM were kindly provided by Xin-Wen Chen, Wuhan Institute of Virology, Chinese Academy of Sciences. Huh7.5.1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum, 0.5 mg/mL G418, 100 IU/mL penicillin, and 100 mg/mL streptomycin.
J399EM is an infectious HCV virus derived from the JFH1 virus (genotype 2a) by insertion of enhanced green fluorescent protein (EGFP) into the HCV NS5A region. The establishment of the infectious J399EM HCV virus has been described previously [24] . J399EM was propagated in Huh7.5.1 cells. Briefly, Huh7.5.1 cells were infected with J399EM at MOI = 0.1 and cultured for 4 days. Culture supernatant was collected and filtered through a 0.45 μm membrane and stored at −80 °C as virus stock. Virus titer was tested in Huh7.5.1 cells. Stocks were serially diluted, and the foci forming units (FFU) were determined as the number of fluorescent colonies formed in infected Huh7.5.1.

Antiviral Assays in Infected Target Cells

All the compounds were first dissolved in DMSO at 50 mM, and then diluted to various concentrations (50 μM, 25 μM, 12.5 μM, 6.3 μM and 3.1 μM). Approximately 1 × 10 4 Huh7.5.1 cells per well were plated in an opaque 96-well tissue culture plate (Costar 3904). The next day, the medium was replaced by J399EM virus at an MOI of 0.3. Four hours later, the virus inoculum was removed and medium containing different concentrations of compounds was added. After an additional 72 h of incubation, the culture medium was removed and the EGFP autofluorescence was measured by using a luminometer with excitation at 488 nm and emission at 516 nm. Compounds and controls were performed in duplicate and each experiment repeated independently at least twice.

Introduction

Quercetin (2, Figure 2 ) is a naturally occurring flavonoid with various biological activities such as antioxidant, antiviral, anticancer, antimicrobial and anti-inflammatory properties; hence, quercetin and its derivatives have potential as drug development leads [15] . As a part of our studies on anti-HCV agents, quercetin was identified as an HCV inhibitor with moderate antiviral activity (EC50 = 19.8 μM) [16] . Furthermore, it had been reported that quercetin could inhibit HCV RdRp at low micromolar concentration (83.7% inhibition at the concentration of 20 μM in an enzyme assay) [17] . We reasoned that the adjacent carbonyl and hydroxyl groups on A and C rings of quercetin make it a perfect DKAs mimic to chelate the two metal ions. In consideration of the structures of 1a and 1b (Figure 1 ), introduction of a 2-cyano benzyl ether group at the aromatic meta-position relative to the chelating moieties was shown to significantly improve the antiviral activity. Thus, we anticipated that substituents of quercetin at the 7-O position (7-O-arylmethyl quercetins 3, Figure 2 ) would mimic the meta-substituted DKA 1b and enhance the anti-HCV activities. Herein, we report the design and synthesis of novel 7-O-arylmethyl quercetin derivatives with various aromatic substituents and the evaluation of their anti-HCV activities. Moreover, it can be hypothesized that another introduced carbonyl group at 3-position of quercetin, together with the adjacent carbonyl and hydroxyl group, would be capable of chelating the two metal ions. Hence, this paper also described a series of novel quercetin-3-O-benzoic acid esters (compounds 4, Figure 2 ) in an attempt to investigate whether the introduction of another carbonyl group at 3-position would result in an improved anti-HCV activity. In addition, several quercetin derivatives with different aliphatic groups substituted at the 3-position were also synthesized for further SAR investigations.
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Abstract

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) induces one of most important devastating disease of swine worldwide, and the current methods poorly control it. Previous studies have indicated that the nonstructural protein 11 (nsp11) of PRRSV may be an important protein for the immune escape of PRRSV. Results: Here, we firstly explored the effect of over-expression of nsp11 on PRRSV infection and found that over-expression of nsp11 enhanced the PRRSV titers while the small interfering RNA (siRNAs) specifically targeting nsp11 could reduce the PRRSV titers in MARC-145 cells. Conclusion: In conclusion, PRRSV nsp11 promotes PRRSV infection in MARC-145 cells and siRNAs targeting nsp11 may be a potential therapeutic strategy to control PRRSV in future.

Background PRRSV, a positive sense and single-stranded RNA virus, is a member of family Arteriviridae [1] . Since it was emerged in the United States in 1987 and in Europe in 1990, PRRSV has rapidly spread in the swine producing regions and became one of the most important devastating diseases of swine worldwide. It can cause severe reproductive failure in sows and respiratory distress in young growing pigs [2] . Infection with PRRSV also made pigs easy to secondary infection by other pathogens [3] . Up to date, since there is no efficient method or drugs against PRRSV, it is very important and urgent to develop the effective therapeutic strategies to control PRRS.

Discussion

Identification of and targeting viral important components is useful for developing viral vaccine and controlling the virus. For example, both the nonstructural protein 1 of influenza virus and the nonstructural protein 1 of mouse hepatitis virus (MHV) were important for the viral virulence respectively, and both the modified live-viral vaccines that deletion of nonstructural protein 1 resulted in complete protection against challenge with influenza virus infection and MHV infection respectively [15] [16] [17] .

Conclusion

Abbreviations EAV, equine arteritis virus; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GFP-nsp11, pcDNA 3.1-GFP-nsp11; MHV, mouse hepatitis virus; MOI, multiplicity of infection; NLRP3, NLR family pyrin domain-containing 3; nsp11, nonstructural protein 11; ORF, open reading frame; PRRSV, porcine reproductive and respiratory syndrome virus ; PVDF, polyvinylidene difluoride; qRT-PCR, quantitative real-time RT-PCR; RNAi, RNA interference; siRNA, small interfering RNA; TCID50, 50 % tissue culture infected dose

The PRRSV genome has nine open reading frames (ORFs) composed of ORF1a, ORF1b, ORF2a, ORF2b, and ORF3-7. ORF1a and ORF1b could produce 16 nonstructural proteins (nsp1α, nsp1β, nsp2 etc.) [4] [5] [6] [7] . Previous studies have shown that the nsp11 of equine arteritis virus(EAV), which is another member of family Arteriviridae, may play a key role in viral RNA synthesis and additional functions in the viral life cycle [8] . Other and our previous work also demonstrated that PRRSV nsp11 inhibited the host innate immune responses such as the transcription of type I interferon [7] , the RNAi innate immune response [9] and the NLR family pyrin domain-containing 3 (NLRP3)-mediated production of IL-1β [10] , which indicated that PRRSV nsp11 may play an important role in PRRSV infection. So the purpose of present study is to explore the effect of over-expression of nsp11 on PRRSV infection and whether the siRNAs targeting the PRRSV nsp11 could influence PRRSV infection.

Discussion

RNA interference (RNAi) is an exciting method to silence viral genes. Inhibition of specific genes by siRNA has proven to be a potential therapeutic strategy against viral infection [18] , especially for the positive single stranded RNA viruses since their genomes function as both the mRNA and the replication template [19, 20] . Up to date, RNAi has been used against several viruses such as hepatitis B virus, foot-and-mouth disease virus, dengue virus and so on [20] [21] [22] . In this work, we also explore whether the siRNA, which targeted the nucleic acid sequence of nsp11, influenced the titers of PRRSV, The endoribonuclease activity of nsp11 was important for nsp11 to enhance the PRRSV titers. MARC-145 cells cultured in 24-well plate were transfected with pcDNA3.1-FLAG-nsp11 (nsp11) (800 ng/well), pcDNA3.1-FLAG-nsp11 H129A (nsp11 H129A) (800 ng/ well) or pcDNA 3.1-FLAG (Con). And 6 h later, the cells were infected with PRRSV at an MOI of 0.1 or mock infected, and 24 h later, the cells were lysed by freezing and thawing three times in three cycles, then the viral titers were measured by TCID50. Data represented means of three replicates, and experiments were repeated three times. Error bars represented the standard deviations. *: P <0.05 compared with the results in control. MOI, multiplicity of infection and the results showed that siRNA targeting nsp11 significantly reduced the titers of PRRSV (Fig. 3) . A recent improved live PRRSV vaccine has indicated that the ORF1a and ORF1b were the virulence determinants of PRRSV [23] . In addition, our recent work also show that RNAi innate immune response was an antiviral response to PRRSV and PRRSV inhibited this response by PRRSV nsp1α and nsp11, which indicated that targeting nsp11 would be useful for RNAi innate immunity against PRRSV [9] . So it is reasonable to propose that our present results gave a new clue for generating the new PRRSV vaccine by targeting PRRSV nsp11.
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Introduction

It has been more than 30 years since the first incidence of human immunodeficiency virus (HIV) was discovered in the USA in 1981. At that time, little was known about the disease. There was much stigmatization and discrimination as it was a fatal, infectious disease with a rapidly declining illness trajectory and no known cure. Moreover, HIV mainly affected stigmatized groups such as gay men and intravenous drug users. During the 1990s, significant medical advances ushered in a new era and transformed HIV from a fatal to a manageable chronic disease (Mocroft et al., 1998) . Not only have medical advances enabled people with HIV to have an increased life expectancy, but studies have shown that with the use of anti-retroviral therapy (ART), those with undetectable viral load pose a significantly lower risk of sexual transmission of HIV to others (Bavinton et al., 2014; Cohen et al., 2011) . With transmissibility significantly reduced, they can have sexual relationships, get married, and have HIV-negative children, which are possibilities they may not have had pre-ART (Seeley et al., 2009) . Thus, ART allows people with HIV to regain some semblance of normality with socially normative roles (Persson & Richards, 2008) . Sontag (1989) had earlier predicted that HIV would be treated as another chronic disease once it was better understood and when treatment became available. In the years following the advent of ART, there were attempts to reframe HIV from a health crisis requiring "exceptional" intervention strategies to a chronic disease which should be treated "like any other" (Moyer & Hardon, 2014) . However, owing to pervasive stigma and discrimination, there is literature which argues against the notion that HIV has been normalized as another disease (Moyer & Hardon, 2014; Persson, 2013) .

Impact of initial diagnosis

Then, our sex life, from that day on, stopped until now. Firstly, it's also because I feel I'm a whole virus, a sick person, a poison . . . a person with a virus. I don't dare to touch her, . . . It's like a disease demon (病魔). (Bryan) Their view and experience of HIV as different from other medical conditions were not echoed by the healthcare narrative. When Bryan was initially diagnosed, he was told "it's not a death sentence . . . With medication, you can still live the life that you want to . . .". John was also told by his doctor that "After you take the drugs, you can be normal, become a normal person . . . Because I also want to regain my health, hoping that I can work". While Bryan did not believe that life could be normal with HIV, John hoped that taking ART would help him to regain his health so that he could work and thus, lead him towards "becoming a normal person". The healthcare providers equated better health outcomes and longer life expectancy to achieving normality.
At least, if you have cancer, people will say this is cancer. If I tell people that I'm an HIV patient, what do you think their response will be? Nobody will feel sorry for you. They will say that you are promiscuous. (Mary) Three out of the four participants stated that they would rather be diagnosed with cancer than HIV. With cancer, they would gain sympathy from others, but HIV is feared and could result in discrimination and rejection. Bryan described HIV in Chinese as a "poison" (毒). He seemed to suggest that he was contaminated by the virus and that he was unclean, and if he touched his wife, he might contaminate her as well. Even though his wife did not reject him, he reported that their relationship had changed and they were sexually more distant after his diagnosis.

Discussion

In the aftermath of an HIV diagnosis, the infected people confronted a future where their previously familiar and benign world has turned hostile against them and challenged their right to coexist with uninfected people. HIV is much more than a medical condition. It possesses undesirable meanings influenced by its recent tumultuous history as a deadly disease sweeping across the world, and people diagnosed with HIV are judged to deserve their pain and suffering. The challenge is for those living with the virus to emerge from under the shadow of its dominant narrative and transcend it by reconstructing identities which will enable living to be more manageable and bearable. It seems that even in a Chinese majority society like Singapore, the four participants, who are ethnically Chinese, managed to reconstruct "liveable" identities despite the shame of having HIV.

Study aim

Singapore is a low-prevalence country for HIV, with most acquiring the infection through heterosexual transmission, although in recent years men who have sex with men (MSM) constituted about half of the newly diagnosed cases (Ministry of Health, 2016) . HIV has become a manageable chronic disease with the accessibility to and availability of ART (Lee, 2015) . Although the drugs are not subsidised, financial assistance is available to citizens who are unable to afford treatment (Ng, 2012) . People with HIV in Singapore can also access generic ART, which is available in other countries (Lee, 2015) .
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In the absence of a curative treatment, the highly active antiretroviral therapy (HAART) keeps the HIV-1 virus of AIDS patients under control. HAART combines drugs targeting different stages of viral replication including the integration step catalyzed by the integrase protein (IN) (1) . Integration of viral DNA into host genome involves two steps catalyzed by IN: (i) cleavage of a dinucleotide from each 3'-end of the viral DNA (3'processing), and (ii) insertion of this processed viral DNA into the host DNA (strand-transfer) (2) . Clinical IN strand transfer inhibitors (INSTIs) target the catalytic site of the enzyme to specifically inhibit the DNA joining reaction, however, as with all anti-AIDS treatments, the continued success of these drugs is persistently disrupted by resistance mutations (1, 2) . Although 3'-processing can be carried out by monomeric IN (3) , the assembly of IN functional multimers is imperative for the strand-transfer activity (4) (5) (6) (7) (8) , and for virus particle maturation and production (reviewed in (9, 10) ). In the continued quest to identify and develop new drugs, allosteric inhibitors that bind sites outside the catalytic core and disrupt IN multimerization are emerging with potent therapeutic potential (11) (12) (13) (14) .

DISCUSSION

The fact that IN regulates not only viral integration but also the assembly and maturation of virus particles (9, 22) , accentuates the prominence of this target. Whereas the peptide used in this study may not, per se, provide a lead hit for this anti-IN strategy, our work underscores the overlooked SH3-docking platform of HIV-1 IN as a potential therapeutic target for future anti-IN allosteric inhibitors.

Tetramers of IN are formed by the reciprocal swapping of the three, N-terminal, catalytic core and Cterminal, canonical domains of IN. The two internal IN protomers, where catalytic binding of viral and host DNA takes place, make up the majority of the tetramer interface. The outer two protomers, and other surrounding units in higher order assemblies, provide supportive domains indispensible for the assembly of the tetrameric cores (4) (5) (6) (7) (8) . We recently showed that targeting the interface between the N-terminal domain (NTD) and catalytic core domain (CCD) of IN, using a specific antigen-binding fragment (Fab), inhibits IN tetramer formation and consequent enzymatic activity and virus particle production (9) . Disrupting the dimerization of the elementary dimeric blocks building IN tetramers has also been explored as a strategy to inhibit IN activity (15) .
Hindering the assembly of IN functional multimers is only one side of the coin. Allosteric interference has also been shown to promote the formation of aberrant IN multimers and aggregates. The potential of allosteric IN inhibitors has been demonstrated through the thorough characterization of the "LEDGF pocket" formed at the dimer interface of IN and the development of LEDGIN (or ALLINI) inhibitors that bind to it (11) (Figure 1A) . Although less investigated, other IN pockets capable of allosteric inhibitor binding have also been identified ( Figure 1A) : binding of the "Y3" molecule to a pocket near the N-terminal end of CCD α-helix 4, designated Y3pocket, has been shown to inhibit 3'-processing and strand-transfer activities (16) ; the "sucrose" binding pocket found along the CCD dimer interface and flanked by two LEDGF pockets (17, 18) has recently been targeted by the natural product kuwanon-L, which inhibited IN activity in a pattern similar to LEDGINs (19) . Another fragment-binding pocket "FBP" has also been identified at the CCD dimer interface (20) . Although inhibitory profiles for this FBP pocket have not been shown, our previous work on IN from the feline virus showed that a single Phe187Lys mutation (Phe185 in HIV-1 IN) around this site inhibits dimer formation (3) . Fragment-based screening and structural studies have revealed the capability of all three pockets (Y3, FBP and LEDGF) to bind numerous small molecules (21) . Most recently, a new class of LEDGINs has been shown to distinctively bind to undefined interfaces of both CCD and CTD of IN (22) . Therefore, IN harbors several allosteric hotspots that can be explored for novel anti-multimerization intervention strategies.

The peptide targets two SH3-docking sites at the CCD/CTD interfaces

To characterize the peptide binding site(s), we solved the 2.0 Å crystal structure of HIV-1 IN CCD in complex with the peptide (table-1). Except for the Cterminal pair of residues (Ser-Tyr), well-defined electron densities show binding of the peptide molecule to two sites, which we call site-1 and site-2 ( Figure 2A ). By examining available structures of various IN functional multimers (4) (5) (6) (7) (8) we underscore the conservation of these two CCD/CTD docking platforms ( Figure 2B ). Site-1 is at the tetrameic interface and is formed by a CTD from a flanking protomer docking at the carboxyl-terminus of CCD α4 of an inner protomer ( Figure 2B & 1B) . Forming site-2 is the docking of a CTD of another flanking protomer into the amino-terminus of the same α4 ( Figure 2B & 2C). The surface area buried upon peptide binding at site-2 is ~ 100 Å 2 larger than that at site-1 (744 as compared to 650 Å 2 ). Peptide binding at site-2 also involves a higher number of interacting residues at the interface (26 as compared to 17 at site-1). Side chains of two residues of the peptide (Trp1 and Tyr3 at site-1 and Ser10 and Tyr14 at site-2) participate in direct hydrogen bonds with the CCD (to side chains of Glu69 and Asp167 at site-1, and to carbonyl oxygen of Val79 and Ala80 at site-2). Additional hydrogen bonds were directly formed with the backbone of the peptide (one in site-1 and three in site-2), and water molecules mediated additional interactions at each site (two in site-1 and one in site-2) ( Figure 2D ). Interestingly, Ala80 carbonyl oxygen of CCD at site-2, which makes a hydrogen bond with the peptide, also makes a hydrogen bond (2.93 Å) with Lys266 of CTD in the HIV-1 IN functional multimers (discussed below and Figure 4B ). It has been shown that the positive charge at this Lys266 position is crucial for viral replication in which a Lys266Ala or Lys266Glu produced replication defective viruses (23) .

Implications for the CTD interdomain interactions

The inhibitory effect of CTD, as part of the CCDCTD, was diminished upon separating the CTD segment (residues 210-288) and supplementing it to the CCD protein in the reaction mixture ( Figure 4A , CCD/CTD). Furthermore, the addition of CTD to a reaction containing the separate CCD and NTD (residues 1-55), which by itself ( Figure 4A , CCD/NTD) was found more than 3 folds superior to full-length or conjugate NTDCCD, resulted in a significant 33% inhibition ( Figure 4A , CCD/NTD/CTD). Therefore, while NTD positively interferes with the CCD in the 3'-processing activity, the CTD appears to have a negative effect. The contribution of the NTD appears necessary to support appropriate CTD interactions with the CCD. Structural analysis shows that the NTD linker segment (residues 50-58) contributes 55% (725 Å 2 ) of the total 1310 Å 2 buried CCD/CTD area at site-2 ( Figure 4B) . Interestingly, the carbonyl oxygen of Val54 of NTD linker forms a hydrogen bond (2.91 Å) with the side chain of CTD Arg228 (NH2) (Figure 4B ), mutating of which to Arg228Ala resulted in defective HIV-1 viruses (23) . The side chain of NTD Gln53 (NE2) forms a hydrogen bond (2.50 Å) with the carbonyl oxygen of CTD Asp229 (Figure 4B) , and also interacts with the side chain of Lys264 (3.6 Å). Both Lys264 and Lys266, which hydrogen bond to Ala80, have been shown to play an important role in the LEDGINs induced aberrant multimerization (27) . Indeed, the fundamental role of CTD . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/301721 doi: bioRxiv preprint in aberrant IN aggregation has previously been emphasized (10, 27, 28) . CTD interactions with the NTD linker (residues 44-53) at site-1, which provides the main docking template for CTD at this site, buries a total of 944 Å 2 ( Figure 4C) . Therefore, the NTD linker appears to play a crucial role in modulating the CCD/CTD docking platform and functional IN mutlimerization and is worth exploring in future studies.
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T he recent paper by Totura and colleagues (1) revealed that Toll-like receptor 3 (TLR3) signaling contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection. Despite the importance of SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV) as public health threats, there are currently no drugs available to treat these coronaviruses, with current evidence suggesting that the antiviral drugs ribavirin and interferon (IFN) are only slightly efficacious in ameliorating SARS-CoV or MERS-CoV infections (2) . Now human-to-human infections of MERS-CoV are more frequently reported, with a total case fatality rate of 37.7% (2). Therefore, a feasible but effective treatment is needed urgently, especially treatment with FDA-approved drugs, including some over-the-counter (OTC) drugs.
Like avian influenza viruses (AIVs), MERS-CoVs set off a cytokine storm (3), which is likely to lead to the subsequent acute respiratory distress syndrome (ARDS). Therefore, some immunomodulatory therapies were suggested for respiratory virus infections, such as celecoxib, mesalamine, cyclosporine, and the TLR4 antagonist eritoran (4). TLRs play a critical role in the recognition of pathogens and induction of the innate immune response to many viruses. The TLR3 agonist poly(I·C) and the TLR4 agonist lipopolysaccharide (LPS) are protective against SARS-CoV infection in mice (1). TLR4 Ϫ/Ϫ mice are resistant to acute lung injury via AIV infections (5) . However, TLR4 Ϫ/Ϫ mice have significantly more disease resulting from SARS-CoV infection than wild-type mice (1). The protective signaling via TLR4 pathways may be a unique feature in the pathogenesis of coronaviruses compared to other respiratory pathogens, such as influenza viruses (1) .
Downstream of TLR-MYD88 pathways, activation of NF-B is a hallmark of coronavirus infections, and inhibition of NF-B reduced lung infection and significantly increased mouse survival after SARS-CoV infection (7) .
Despite the opposite functions of TLR4s in AIV and SARS-CoV infections, MYD88 (myeloid differentiation primary response 88; downstream of TLRs) plays an important role in the survival of respiratory virus infections (Fig. 1) . The MYD88 gene was observed to be highly induced by SARS-CoV infection (1). Interestingly, either excessive expression of MYD88 (1) or a deficiency in MYD88 expression (6) resulted in high mortality rates after MERS-CoV infections, implying that a balanced immune response is crucial for survival of respiratory virus infections.
Among TLR-MYD88 antagonists, statins are the most common FDA-approved drugs (atorvastatin will be sold as an OTC drug). Statins do not affect the MYD88 level significantly under normal conditions but maintain (stabilized) MYD88 at the normal level during hypoxia or after hydrogen peroxide treatments (8, 9) . Furthermore, atorvastatin at 10 M significantly attenuated NF-B activation within 24 h, whereas at lower doses of 0.1 and 1 M, the treatment time had to be prolonged for up to 48 h for a significant inhibition to occur (10) . Thus, an early and high dose of a statin (such as a single dose of 40 mg atorvastatin per day, equaling a 0.1 M plasma concentration) might be an idea for treatment of MERS-CoV infections. Given that 3-to 10-timeshigher levels of inflammatory cytokines and chemokines were observed after MERS-CoV infection than after no infection (3), statins may not be very effective for late-stage patients. Timely administration of statins may be crucial to surviving MERS-CoV infection.