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Abstract

The replication of coronavirus, a family of important animal and human pathogens, is closely associated with the cellular membrane compartments, especially the endoplasmic reticulum (ER). Coronavirus infection of cultured cells was previously shown to cause ER stress and induce the unfolded protein response (UPR), a process that aims to restore the ER homeostasis by global translation shutdown and increasing the ER folding capacity. However, under prolonged ER stress, UPR can also induce apoptotic cell death. Accumulating evidence from recent studies has shown that induction of ER stress and UPR may constitute a major aspect of coronavirus-host interaction. Activation of the three branches of UPR modulates a wide variety of signaling pathways, such as mitogen-activated protein (MAP) kinase activation, autophagy, apoptosis, and innate immune response. ER stress and UPR activation may therefore contribute significantly to the viral replication and pathogenesis during coronavirus infection. In this review, we summarize the current knowledge on coronavirus-induced ER stress and UPR activation, with emphasis on their cross-talking to apoptotic signaling.

INTEGRATED STRESS RESPONSE PATHWAYS AND INNATE IMMUNITY

Several recent studies have demonstrated the critical roles of cellular stress response pathways in modulating the innate immune activation (Cláudio et al., 2013) . One of the key regulators that bridge stress and innate immunity is GADD34, a negative regulator of eIF2α activation. It has been shown that when stimulated with polyriboinosinic:polyribocytidylic acid (polyI:C), the integrated stress response pathways were activated in dendritic cells (DCs), leading to up-regulation of ATF4 and GADD34 (Clavarino et al., 2012) . Interestingly, GADD34 expression did not significantly affect protein synthesis in DCs, but was shown to be crucial for the production of interferon β (IFN-β) and pro-inflammatory cytokines interleukin-6 (IL-6; Clavarino et al., 2012) . In contrast, GADD34 has also been shown to specify PP1 to dephosphorylate the TGF-β-activated kinase 1 (TAK1), thus negatively regulating the toll-like receptor (TLR) signaling and pro-inflammatory cytokines [IL-6 and TNF-α (Tumor necrosis factor alpha)] production in macrophages (Gu et al., 2014) . The functional disparities of GADD34 in DCs and macrophages indicate that the integrated stress response may be regulated by some other signaling pathways, resulting in celltype specific outcomes in the innate immune activation. Since GADD34 induction was readily observed in cells infected with IBV (Wang et al., 2009 ), it will be intriguing to ask whether GADD34 also contributes to IBV-induced pro-inflammatory Frontiers in Microbiology | Virology cytokine production, and to determine potential cross-talks between the PERK pathway and innate immune activation during IBV infection.

INTRODUCTION

Coronaviruses are a family of enveloped viruses with positive sense, non-segmented, single-stranded RNA genomes. Many coronaviruses are important veterinary pathogens. For example, avian infectious bronchitis virus (IBV) reduces the performance of both meat-type and egg-laying chickens and causes severe economic loss to the poultry industry worldwide (Cavanagh, 2007) . Certain coronaviruses, such as HCoV-229E and HCoV-OC43, infect humans and account for a significant percentage of adult common colds (Hamre and Procknow, 1966; Kaye et al., 1972) . Moreover, in 2003, a highly pathogenic human coronavirus (SARS-CoV) was identified as the causative agent of severe acute respiratory syndrome (SARS) with high mortality rate and led to global panic (Ksiazek et al., 2003) . Later, it was found that the SARS-CoV was originated from bat and likely jumped to humans via some intermediate host (palm civets; Li et al., 2005; Wang and Eaton, 2007) . Recently, a live SARS-like coronavirus was isolated from fecal samples of Chinese horseshoe bats, which could use the SARS-CoV cellular receptor -human angiotensin converting enzyme II (ACE2) for cell entry (Ge et al., 2013) . This indicates that an intermediate host may not be necessary and direct human infection by some bat coronaviruses is possible. Moreover, a novel human coronavirus -the Middle East respiratory syndrome coronavirus (MERS-CoV), emerged in Saudi Arabia in September 2012 (de Groot et al., 2013) . Although the risk of sustained human-to-human transmission is considered low, infection of MERS-CoV causes ∼50% mortality in patients with comorbidities (Graham et al., 2013) . Initial studies had pointed to bats as the source of MERS-CoV (Annan et al., 2013) ; however, accumulating evidence strongly suggested the dromedary camels to be the natural reservoirs and animal source of MERS-CoV (Hemida et al., 2013; Alagaili et al., 2014) . Thus, coronaviruses can cross the species barrier to become lethal human pathogens, and studies on coronaviruses are both economically and medically important.
Taxonomically, the family Coronaviridae is classified into two subfamilies, the coronavirinae and the torovirinae. The coronavirinae is further classified into three genera, namely the Alphacoronavirus, Betacoronavirus, and Gammacoronavirus (Masters, 2006) . The classification was originally based on antigenic relationships and later confirmed by sequence comparisons of entire viral genomes (Gorbalenya et al., 2004) . Almost all Alphacoronaviruses and Betacoronaviruses have mammalian hosts, including humans. In contrast, Gammacoronaviruses have mainly been isolated from avian hosts.
Morphologically, coronaviruses are spherical or pleomorphic in shape with a mean diameter of 80-120 nm. They are characterized by the large (20 nm) "club-like" projections on the surface, which are the heavily glycosylated trimeric spike (S) proteins (Masters, 2006) . Two additional structural proteins are found on the envelope. The abundant membrane (M) proteins give the virion its shape, whereas the small envelope (E) proteins play an essential role during assembly (Sturman et al., 1980; Liu and Inglis, 1991) . Inside the envelope, the helical nucleocapsid is formed by binding of the nucleocapsid (N) proteins on the genomic RNA in a beads-on-a-string fashion. The genome, ranging from 27,000 to 32,000 nucleotides in size, is the largest RNA genomes known to date.
Coronavirus infection starts with receptor binding via the S protein (Figure 1) . The S proteins of most coronaviruses are www.frontiersin.org FIGURE 1 | Schematic diagram showing the replication cycle of coronavirus and the stages in which ER stress may be induced during coronavirus infection. Infection starts with receptor binding and entry by membrane fusion. After uncoating, the genomic RNA is used as a template to synthesize progeny genomes and a nested set of subgenomic RNAs. The replication transcription centers are closely associated with DMVs, which are proposed to be adopted from the modified ER, possibly by the combined activities of non-structural proteins nsp3, nsp4, and nsp6. The S, E, and M proteins are synthesized and anchored on the ER, whereas the N protein is translated in the cytosol. Assembly takes place in the ERGIC and mature virions are released via smooth-walled vesicles by exocytosis. The three stages that presumably induce ER stress are highlighted with numbered star signs, namely: (1) formation of DMVs, (2) massive production and modification of structural proteins, and (3) depletion of ER membrane during budding. cleaved by host protease into two functional subunits: an Nterminal receptor binding domain (S1) and a C-terminal domain (S2) responsible for membrane fusion (Huang et al., 2006; Qiu et al., 2006; Yamada et al., 2009) . The interaction between the cell surface receptor and the S1 subunit is the major determinant of the tropism of coronaviruses (Kuo et al., 2000) . Upon receptor binding of S1, a conformational change is triggered in the S2 subunit, exposing its hidden fusion peptide for insertion into the cellular membrane. This is followed by the packing of the two heptad repeats in the three monomers into a six-helix bundle fusion core. This close juxtaposition of the viral and cellular membrane enables fusion of the lipid bilayers, and the viral nucleocapsid is thus delivered into the cytoplasm (Masters, 2006) .
Using the genomic RNA as a template, the replicase then synthesizes the negative sense genomic RNAs, which are used as templates for synthesizing progeny positive sense RNA genomes. On the other hand, through discontinuous transcription of the genome, the replicase synthesizes a nested set of subgenomic RNAs (sgRNAs; Sawicki et al., 2007) . Replication and transcription of the coronavirus genome involve the formation of the replication/transcription complexes (RTCs), which are anchored to the intracellular membranes via the multi-spanning transmembrane proteins nsp3, nsp4, and nsp6 (Oostra et al., 2007) . Also, inside the infected cells, coronaviruses induce modification of the intracellular membrane network and formation of the double membrane vesicles (DMVs; Knoops et al., 2008) . Several studies have shown that the DMVs are closely associated with the coronavirus RTCs and the de novo synthesized viral RNAs (Gosert et al., 2002; Snijder et al., 2006) .
The virions budded into the ERGIC are exported through secretory pathway in smooth-wall vesicles, which ultimately fuse with the plasma membrane and release the mature virus particles (Krijnse-Locker et al., 1994) . For some coronaviruses, a portion of the S protein escapes from viral assembly and is secreted to the plasma membrane. These S proteins cause fusion of the infected cell with neighboring uninfected cells, resulting in the formation of a large, multinucleated cell known as a syncytium, which enables the virus to spread without being released into the extracellular space (Masters, 2006) .
In this review, current studies on the involvement of the UPR in coronavirus infection and pathogenesis will be summarized. The role of UPR activation in host response, in particular the induction of apoptosis, will also be reviewed.

FORMATION OF DOUBLE MEMBRANE VESICLES

It is well-known that the replication of many plus-stranded RNA viruses induces modification of cellular membranes (Miller and Krijnse-Locker, 2008) . Among them, coronaviruses have been shown to induce the formation of DMVs in infected cells (David-Ferreira and Manaker, 1965) . Based on immunocytochemistry electron microscopy data, the DMVs co-localize with coronavirus major replicase proteins and are presumably the sites where coronavirus RTCs are located (Gosert et al., 2002; Snijder et al., 2006) . Indeed, DMVs are induced in HEK293T cells coexpressing the SARS-CoV nsp3, nsp4, and nsp6, which are all multispanning transmembrane non-structural proteins (Angelini et al., 2013) . There have been different perspectives regarding the origin of the coronavirus-induced DMVs. The late endosomes, autophagosomes, and the early secretary pathway have all been implicated as the membrane source of DMVs (van der Meer et al., 1999; Prentice et al., 2004; Verheije et al., 2008) . Also, co-localization has been observed between SARS-CoV nonstructural proteins and protein disulfide isomerase (PDI), an ER marker (Snijder et al., 2006) . Using high-resolution electron tomography, Knoops et al. (2008) have shown that infection of SARS-CoV reorganizes the ER into a reticulovesicular network, which consists of convoluted membranes and interconnected DMVs. Recently, Reggiori et al. (2010) have proposed a model in which coronaviruses hijack the EDEMosomes to derive ER membrane for DMV formation. The EDEMosomes are COPIIindependent vesicles that export from the ER, which are normally used to fine-tune the level of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a regulator of ER-associated degradation (ERAD; Calì et al., 2008) . It has been demonstrated that MHV infection causes accumulation of EDEM1 and osteosarcoma amplified 9 (OS-9, another EDEMosome cargo), and that both EDEM1 and OS-9 co-localize with the RTCs of MHV (Reggiori et al., 2010) . These results thus add mechanical evidence to support the ER-origin of the coronavirus-induced DMVs.

GLYCOSYLATION OF CORONAVIRAL STRUCTURAL PROTEINS

Except for the N protein, all coronavirus structural proteins are transmembrane proteins synthesized in the ER. The M protein, which is the most abundant component of the virus particle, is known to undergo either O-linked (for most betacoronaviruses) www.frontiersin.org or N-linked (for all alpha-and gammacoronaviruses) glycosylation in the ER (Jacobs et al., 1986; Cavanagh and Davis, 1988; Nal et al., 2005) . The glycosylation of M protein is proposed to play a certain function in alpha interferon (IFN) induction and in vivo tissue tropism (Charley and Laude, 1988; Laude et al., 1992; de Haan et al., 2003) . The pre-glycosylated S monomers are around 128-160 kDa, whereas sizes can reach 150-200 kDa postglycosylation (exclusively N-linked), indicating that the S protein is highly glycosylated (Masters, 2006) . At least for transmissible gastroenteritis coronavirus (TGEV), glycosylation is presumed to facilitate monomer folding and trimerization (Delmas and Laude, 1990) . Moreover, the glycans on SARS-CoV S proteins have been shown to bind C-type lectins DC-SIGN (dendritic cellspecific intercellular adhesion molecule-3-grabbing non-integrin) and L-SIGN (liver lymph node-specific intercellular adhesion molecule-3-grabbing non-integrin), which can serve as alternative receptors for SARS-CoV independent of the major receptor ACE2 (Han et al., 2007) . The folding, maturation, and assembly of the gigantic S trimeric glycoprotein rely heavily on the protein chaperons inside the ER, such as calnexin. In fact, the N-terminal part of the S2 domain of SARS-CoV S protein has been found to interact with calnexin, and knock-down of calnexin decreases the infectivity of pseudotyped lentivirus carrying the SARS-CoV S protein (Fukushi et al., 2012) . Also, treatment of αglucosidase inhibitors, which inhibit the interactions of calnexin with its substrates, dose dependently inhibits the incorporation of S into pseudovirus and suppresses SARS-CoV replication in cell cultures (Fukushi et al., 2012) . During coronavirus replication, massive amount of structural proteins is synthesized to assembly progeny virions. The production, folding, and modification of these proteins undoubtedly increase the workload of the ER.

DEPLETION OF ER LIPID DURING THE BUDDING OF VIRIONS

Budding of coronaviruses occurs in the ERGIC, which is a structural and functional continuance of the ER. Thus, the release of mature virions by exocytosis in effect depletes the lipid component of the ER. Taken together, coronavirus infection results in: (1) massive morphological rearrangement of the ER; (2) significant increase ER burden for protein synthesis, folding and modification; and (3) extensive depletion of ER lipid component. These factors together may contribute to the coronavirus-induced ER stress.
In the following sections, the activation of the three individual branches of the UPR by coronavirus infection will be discussed in detail.

ACTIVATION OF PERK PATHWAY DURING CORONAVIRUSES INFECTION AND ITS INVOLVEMENT IN CORONAVIRUS-INDUCED APOPTOSIS

There have been diverging results on the activation of PKR and/or PERK during coronavirus infection. In an early study, it has been found that there is minimal transcriptional activation of PKR and another IFN-stimulated gene, 2 5 -oligoadenylate synthetase (OAS) in cells infected with MHV-1 (Zorzitto et al., 2006) . In a separate study, phosphorylation of PKR and eIF2α was also not observed in MHV A59-infected cells . However, Bechill et al. (2008) have detected significant eIF2α phosphorylation and up-regulation of ATF4 in cells infected with MHV A59, although no induction of GADD153 and GADD34 was observed. It has been suggested that due to the lack of GADD34-mediated eIF2α dephosphorylation, MHV infection induces sustained translation repression of most cellular proteins (Bechill et al., 2008) . However, the translation of MHV mRNAs seems to be resistant to eIF2α phosphorylation, and the detailed mechanisms for such evasion are yet to be investigated. As for SARS-CoV, PKR, PERK, and eIF2α phosphorylation are readily detectable in virus-infected cells (Krähling et al., 2009 ). However, knock-down of PKR using specific morpholino oligomers did not affect SARS-CoV-induced eIF2α phosphorylation but significantly inhibited SARS-CoVinduced apoptosis (Krähling et al., 2009) . It is possible that eIF2α is phosphorylated by PERK in SARS-CoV-infected cells, but similar loss-of-function experiments have not been performed, although overexpression of SARS-CoV accessory protein 3a has been shown to activate the PERK pathway (Minakshi et al., 2009) .
The discrepancy regarding the activation of PKR/PERK during coronavirus infection may be a result from the different cell culture systems and virus strains used. The interpretation is further complicated by the IFN-inducible nature of PKR. It is generally believed that coronaviruses are poor type I IFN inducers in vitro (Garlinghouse et al., 1984; Spiegel et al., 2005; Roth-Cross et al., 2007) , although the IFN response may be essential for antiviral activities in vivo (Ireland et al., 2008) . Moreover, it is known that coronaviruses employ multiple mechanisms to antagonize the IFN response. For example, the nsp16 has been shown to utilize the 2 -O-methyltransferase activity to modify coronavirus mRNAs, so as to evade from the cytosolic RNA sensor melanoma differentiation-associated protein 5 (MDA5) and type I IFN induction (Roth-Cross et al., 2008; Züst et al., 2011) . Furthermore, the activities of several IFN-induced genes (ISGs) have also been shown to be modulated by coronaviruses during infection. For instance, Zhao et al. (2012) have demonstrated that the MHV accessory protein ns2 cleaves 2 ,5 -oligoadenylate, the product of www.frontiersin.org FIGURE 3 | Working model of PKR/PERK-eIF2α-ATF4-GADD153 pathway activation during coronavirus infection, using IBV as an example. Phosphorylation of eIF2α by PERK and PKR induces the expression of ATF4, ATF3, and GADD153. GADD153 exerts its pro-apoptotic activities via suppressing Bcl2 and ERKs by inducing TRIB3. The potential induction of DUSP1 by ATF3 may modulate phosphorylation of p38 and JNK, thus regulating IBV-induced apoptosis and cytokine production. The translation attenuation due to eIF2α activation can also lead to reduced inhibition of IκBα on NF-κB, which in turn promote cytokine production. Pointed arrows indicate activation, and blunt-ended lines indicate inhibition. The question mark indicates hypothetical mechanism.

INTEGRATED STRESS RESPONSE PATHWAYS AND INNATE IMMUNITY

Besides p38, DUSP1 has also been shown to dephosphorylate c-Jun N-terminal kinase (JNK) and ERK (Sun et al., 1993; Franklin and Kraft, 1997) . It has been long proposed that ERK phosphorylation promotes cell survival, whereas prolonged JNK and p38 phosphorylation is linked to the induction of apoptosis (Xia et al., 1995) . Thus, the induction of DUSP1 by ER stress in coronavirus-infected cells may also contribute to virus-induced apoptosis via modulation of the MAP kinase pathways.

ACTIVATION OF THE IRE1 PATHWAY DURING CORONAVIRUSES INFECTION

The involvement of IRE1-XBP1 pathway during coronavirus infection has been investigated by several studies, using MHV as a model. Either MHV infection or overexpression of the MHV S protein (but not other structural proteins) induces XBP1 mRNA splicing (Versteeg et al., 2007; Bechill et al., 2008) . However, although XBP1 mRNA is efficiently spliced, the protein product of spliced XBP1 cannot be detected in either the whole cell lysate or the nuclear fraction. Moreover, UPR downstream genes known to be activated by XBP1s, such as ER DNA J domain-containing protein 4 (ERdj4), EDEM1, and protein kinase inhibitor of 58 kDa (p58 IPK ), are not significantly induced after infection (Bechill et al., 2008) . Using a luciferase reporter system, it is shown that MHV infection does not inhibit transactivation of unfolded protein response element (UPRE) and ER stress response element (ERSE) promoter by XBP1s. Because MHV infection is associated with persistent eIF2α phosphorylation and host translational repression, it is likely that failure to translate the XBP1s protein may be the main reason why activation of the IRE1 branch does not occur even though XBP1 mRNA splicing is observed. On the other hand, although SARS-CoV belongs to the same genera of Betacoronavirus as MHV, neither infection with SARS-CoV nor overexpression of SARS-CoV S protein induces XBP1 mRNA splicing (Versteeg et al., 2007; DeDiego et al., 2011) . It is www.frontiersin.org FIGURE 4 | Working model of IRE1-XBP1 signaling pathway during coronavirus infection, using IBV as an example. IRE1 mediates XBP1 splicing, which up-regulates UPR target genes to restore ER stress, and the spliced XBP1 may also modulate the IFN and cytokine secretion. IRE1 activation modulates the phosphorylation of Akt and JNK, thus affecting IBV-induced apoptosis. IRE1 is also responsible for basal activity of IKK, which phosphorylates IκBα to remove its inhibition on NF-κB, thus facilitating the production of type I IFN and pro-inflammatory cytokines. Pointed arrows indicate activation, and blunt-ended lines indicate inhibition. The question mark indicates hypothetical mechanism.
Interestingly, a recent report by DeDiego et al. (2011) demonstrates that the coronavirus E protein may modulate the IRE1-XBP1 pathway. Using a recombinant SARS-CoV that lacks the E protein (rSARS-CoV-E), it is found that both XBP1 splicing and induction of UPR genes significantly increase in the absence of E protein. Moreover, E protein also suppresses ER stress induced by RSV and drugs (thapsigargin and tunicamycin; DeDiego et al., 2011) . Whether the UPR modulating activity is related to the viroporin property of E protein remains to be investigated, but this study explains, at least in part, why SARS-CoV lacking the E protein is attenuated in animal models (Liao et al., 2004; DeDiego et al., 2007) .

THE ATF6 BRANCH OF UPR

Compared with the PERK and IRE1 pathway, the induction of ATF6 pathway during coronaviruses infection has not been deeply investigated. In MHV-infected cells, significant cleavage of ATF6 could be detected starting from 7 h post-infection (Bechill et al., 2008) . However, the levels of both full length and cleaved ATF6 protein diminished at later time points during infection. Moreover, activation of ATF6 target genes was not observed at the mRNA level, as determined by luciferase reporter constructs under the control of ERSE promoters (Bechill et al., 2008) . It is also unlikely that MHV infection suppresses downstream signaling of the ATF6 pathway, because the reporter induction by overexpressed ATF6 was not inhibited by MHV infection. The authors thus conclude that global translation shutdown via eIF2α phosphorylation prevents accumulation of ATF6 and activation of ATF6 target genes (Bechill et al., 2008) . The involvement of ATF6 pathway during infection of other coronaviruses has not been well characterized.
Although the spike proteins of coronaviruses have been considered as the major contributor in ER stress induction, overexpression of SARS-CoV spike protein fails to activate ATF6 reporter constructs (Chan et al., 2006) . On the other hand, the accessory protein 8ab of SARS-CoV has been identified to induce ATF6 activation (Sung et al., 2009 ). The 8ab protein was found in SARS-CoV isolated from animals and early human isolates. In SARS-CoV isolated from humans during the peak of the epidemic, there is a 29-nt deletion in the middle of ORF8, resulting in the splitting of ORF8 into two smaller ORFs, namely ORF8a and ORF8b, which encode two truncated polypeptides 8a and 8b (Guan et al., 2003) . ATF6 cleavage and nuclear translocation was observed in cells transfected with SARS-CoV 8ab (Sung et al., 2009 ). Physical interaction between 8ab and the luminal domain of ATF6 was also demonstrated by co-immunoprecipitation. However, similar experiments have not been performed for the 8a and 8b proteins. Also, further studies using recombinant SARS-CoV lacking 8a, 8b, or 8ab would be required.

CONCLUSION

Coronaviruses constitute human and animal pathogens that are medically and economically important. Much remains unknown regarding the host-virus interactions during infection. Recent studies have demonstrated that coronaviruse infection induces ER stress in infected cells and activates the UPR. Activation of the PERK pathway (possibly in synergy with PKR and/or other integrated stress response kinases) leads to phosphorylation of eIF2α and a global translation shutdown. At late stage of infection, up-regulation of transcription factor GADD153 likely contributes to coronaviruses induced apoptosis. Activation of the IRE1 pathway induces XBP1 mRNA splicing and expression of downstream UPR genes. Interestingly, IRE1 but not XBP1 is also shown to modulate the JNK and Akt kinase activities, thus protecting infected cells from virus induced apoptosis. The ATF6 pathway is also activated in coronavirus-infected cells, resulting in the up-regulation of chaperon proteins to counteract ER stress.
However, many questions remain to be addressed. First, although the coronaviruses spike proteins are demonstrated to induce ER stress and UPR, detailed mechanisms regarding www.frontiersin.org molecular interactions between the spike proteins and PERK/IRE1/ATF6 have not been determined. Second, it should be noted that the phenotypes observed in cells overexpressing viral proteins may not essentially reflect their physiological functions in the setting of a real infection. Further experiments using recombinant viruses with deletion of or modification in the target viral proteins should be performed to validate these findings (DeDiego et al., 2011) . Last but not the least, the three branches of UPR should not be considered functionally independent, but rather as an integrated regulatory network (Ron and Walter, 2007) . For example, besides being spliced by IRE1, XBP1 is also transcriptionally activated by PERK and ATF6 (Yoshida et al., 2001a; Calfon et al., 2002) . Also, it is difficult to separate the translation shutdown effect mediated by PERK and the induction of UPR genes by PERK and the other two ER stress sensors, as in the studies with MHV (Bechill et al., 2008) .
Nonetheless, there are scientific and clinical significance for studies on ER stress and UPR induction during infection with coronaviruses and other viruses. As an evolutionarily conserved and well-characterized stress response pathway, it serves as a perfect model to study host-virus interactions and pathogenesis. Moreover, besides apoptosis, UPR has been recently demonstrated to crosstalk with other major cellular signaling pathways, including MAP kinases pathways, autophagy, and innate immune responses (Yoneda et al., 2001; Ogata et al., 2006; Martinon et al., 2010; Hu et al., 2011; Clavarino et al., 2012) . Thus, further investigations on coronavirus-induced UPR may also help identifying new targets for antiviral agents and developing more effective vaccines against coronaviruses.

CORONAVIRUS INFECTION AND ER STRESS

Global proteomic and microarray analyses have shown that the expression of several genes related to the ER stress, such as glucoseregulated protein 94 (GRP94) and glucose-regulated protein 78 (GRP78, also known as immunoglobulin heavy chain-binding protein, or BiP), is up-regulated in cells infected with SARS-CoV or in cells overexpressing the SARS-CoV S2 subunit (Jiang et al., 2005; Yeung et al., 2008) . Using a luciferase reporter system, Chan et al. (2006) found that both GRP94 and GRP78 were induced in SARS-CoV-infected FRhK4 cells. Consistently, the mRNA level of homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1 (HERPUD1), an ER stress marker, was up-regulated in L cells infected with mouse hepatitis virus (MHV) or SARS-CoV (Versteeg et al., 2007) . Data from this group have shown a similar induction of ER stress in IBV-infected Vero, H1299, and Huh-7 cells (unpublished observations). Although no parallel studies have been performed on Alphacoronaviruses, it is likely that all three genera of coronaviruses may induce ER stress in the infected cells. Current evidence suggests the following three main mechanisms.

ACTIVATION OF PERK PATHWAY DURING CORONAVIRUSES INFECTION AND ITS INVOLVEMENT IN CORONAVIRUS-INDUCED APOPTOSIS

an ISG called OAS. This results in the suppression of the cellular endoribonuclease RNase L activity and facilitates virus replication in vitro and in vivo (Zhao et al., 2011 (Zhao et al., , 2012 . Thus, similar uncharacterized mechanisms may be used by MHV and other coronaviruses to block the activation and/or downstream signaling of PKR. In this regard, the activation of PERK via ER stress seems to be an alternative pathway to activate eIF2α, although coronaviruses may counteract by directly targeting eIF2α, as described below.
Studies done by this group have shown that, phosphorylation of PKR, PERK, and eIF2α was detectable at early stage of IBV infection (0-8 hpi) but diminished quickly later (Wang et al., 2009; Liao et al., 2013) . The rapid de-phosphorylation of eIF2α is likely due to the accumulation of GADD34, which is a component of the PP1 complex and a downstream target gene induced by GADD153 (Wang et al., 2009 ). Despite of the rapid de-phosphorylation of eIF2α, significant induction of GADD153 was observed at late stage of infection (16-24 h) at both mRNA and protein levels (Liao et al., 2013) . The up-regulation of GADD153 was likely mediated by both PKR and PERK, since knock-down of either PKR or PERK by siRNA reduces IBV-induced GADD153 (Liao et al., 2013) . The up-regulation of GADD153 promotes apoptosis in IBV-infected cells, possibly via inducing the pro-apoptotic protein tribbles-related protein 3 (TRIB3) and suppressing the pro-survival kinase extracellular signal-related kinase (ERK; Liao et al., 2013) . Based on the findings so far obtained, it is safe to conclude that the PERK/PKR-eIF2α-ATF4-GADD153 pathway is activated by some, but not all, coronaviruses. In the infected cells, this pathway is activated at an early stage but quickly modulated by feedback de-phosphorylation. The PERK/PKR-eIF2α-ATF4-GADD153 most likely plays a pro-apoptotic function during coronavirus infection.

INTEGRATED STRESS RESPONSE PATHWAYS AND INNATE IMMUNITY

The massive production of pro-inflammatory cytokines (cytokine storm) has been associated with the immunopathogenesis and high mortality rate of SARS-CoV (Perlman and Dandekar, 2005) . The transcription factor nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) is a master regulator of pro-inflammatory response and innate immunity (Hayden and Ghosh, 2012) . It has been well established that NF-κB is required for the induction of pro-inflammatory cytokines (such as IL-6 and IL-8) and the early expression of IFN-β during RNA virus infection (Libermann and Baltimore, 1990; Kunsch and Rosen, 1993; Wang et al., 2010; Balachandran and Beg, 2011; Basagoudanavar et al., 2011) . Interestingly, induction of TNF-α, IL-6, and IL-8 has been detected in cells overexpressing the spike protein of SARS-CoV via the NF-κB pathway Dosch et al., 2009) . Thus, it is intriguing to consider the involvement of ER stress in activating the NF-κB pathway during coronavirus infection. In its inactive form, NF-κB is sequestered in the cytoplasm by inhibitor of NF-κB alpha (IκBα), which masks the nuclear localization signal of NF-κB (Karin and Ben-Neriah, 2000) . The basal level of IκBα is maintained by constitutive synthesis and degradation of the protein (Kanarek et al., 2010) . Under various stress conditions, phosphorylation of eIF2α leads to global translation repression and a net decrease in IκBα protein level (Jiang et al., 2003) . This then results in the activation of NF-κB and induction of pro-inflammatory response (Figure 3) . Nonetheless, further studies are needed to characterize the actual contributions of ER stress in NF-κB-mediated cytokine induction during coronavirus infection.

ACTIVATION OF THE IRE1 PATHWAY DURING CORONAVIRUSES INFECTION

Result from this group has also shown that the IRE1-XBP1 pathway is activated in cells infected with IBV. In IBV-infected Vero cells, significant splicing of XBP1 mRNA was detected starting from 12 to 16 h post-infection till the late stage of infection. The mRNA levels of XBP1 effector genes (EDEM1, ERdj4, and p58 IPK ) were up-regulated in IBV-infected Vero cells. The activation of IRE1-XBP1 pathway was also detectable, though at a lower level, in other cell lines such as H1299 and Huh-7 cells. Treatment of IRE1 inhibitor effectively blocked IBV-induced XBP1 mRNA splicing and effector genes up-regulation in a dosage-dependent manner. Consistently, knockdown of IRE1 inhibited IBV-induced XBP1 mRNA splicing, whereas overexpression of wild-type IRE1 (but not its kinase dead or RNase domain deleted mutants) enhanced IBV-induced XBP1 mRNA splicing. These results suggest that the IRE1-XBP1 pathway is indeed activated in cells infected with IBV. Interestingly, an earlier onset and more significant apoptosis induction in IRE1-knockdown IBV-infected cells was observed, which is associated with hyper-phosphorylation of pro-apoptotic kinase JNK and hypo-phosphorylation of pro-survival kinase RAC-alpha serine/threonine-protein kinase (Akt). Taken together, IRE1 may modulate IBV-induced apoptosis and serve as a survival factor during coronavirus infection.

IRE1 PATHWAY AND INNATE IMMUNITY

Similarly to the integrated stress response, the IRE1 pathway has also been implicated in the innate immune response (Cláudio et al., 2013) . Martinon et al. (2010) have shown that in murine macrophages, the IRE1-XBP1 pathway is specifically activated by TLR4 and TLR2. Interestingly, the ER stress and TLR activation synergistically activate IRE1 and induce the production of pro-inflammatory cytokines such as IL-1β and IL-6 Frontiers in Microbiology | Virology (Martinon et al., 2010) . Consistently, Hu et al. (2011) have demonstrated that the IRE1-XBP1 pathway is also involved in IFN-β and pro-inflammatory cytokines production in murine DCs induced by polyI:C. Significantly, it has been shown that overexpression of the spliced form of XBP1 enhanced IFN-β production in DCs and significantly suppressed vesicular stomatitis virus infection (Hu et al., 2011) . Preliminary results from this group have also found that the activation of IRE1-XBP1 pathway is required for IL-8 induction in cells infected with IBV (unpublished data). On the other hand, the kinase but not the RNAse activity of IRE1 has been associated with ER-stress-induced NF-kB activation (Tam et al., 2012) . Under ER stress, IRE1 has been shown to phosphorylate TRAF2, which activates the IκB kinase (IKK) and contributes to its basal activity (Figure 4) . IKK in turn phosphorylates IκBα and promotes its proteasomal degradation, releasing NF-κB to activate downstream genes (Tam et al., 2012) . Taken together, these findings suggest that IRE1 may act synergistically with players in innate immunity and serve as a supplementary sensor and/or signaling factors during coronavirus infection.
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Abstract

Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV) isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus.

Sequence Analysis

Recombination in the 1ab ORF area, which encodes the nonstructural proteins involved in the viral replication complex, has the potential to alter the pathogenicity of the virus [28] . The nsp 2 contains hydrophobic residues that likely anchor the replication complex to the Golgi [29] . The nsp 3 encodes the protease PLP2 which cleaves nsps 2, 3, and 4 and an area with ADP-ribose 1'-phosphatase (ADRP) activity. The protease PLP2 has been shown to have deubiquinating-like activity [30] and also to be a type I interferon (IFN) antagonist [31] . Changes in the amino acid composition of this area could affect the ability of the virus to replicate in a variety of cell types. The ADRP region of nsp 3 is conserved among coronaviruses [32, 33] , and a recent study suggested a biological role for the coronavirus ADRP in modulating the expression of pro-inflammatory immune modulators such as tumor necrosis factor alpha and interleukin-6 [34] . Recombination in this area could alter the pathogenicity of the virus by modulating host cytokine expression. The nsp16 is reported to be an S-adenosyl-L-methionine (AdoMet)-dependent RNA (nucleoside-2'O)-methyltransferase (2'O-MTase) responsible for capping the viral mRNA nascent transcripts [32] . An alteration in the efficiency of this protein could profoundly decrease not only viral replication but also pathogenicity. The spike glycoprotein of IBV on the surface of the virus plays a role in attachment to host cell receptors, membrane fusion and entry

Introduction

Avian infectious bronchitis virus (IBV) is a gamma-coronavirus in the family Coronaviridae, the order Nidovirales, and the genus Coronavirus that causes a highly contagious upper-respiratory disease of domestic chickens. In layer type birds it can cause a drop in egg production and some strains are nephropathogenic. Infectious bronchitis remains one of the most widely reported respiratory diseases of chickens worldwide despite the routine usage of attenuated live vaccines to control the disease. Control of IBV is difficult because there is little to no cross-protection between the numerous different serotypes of the virus.
In this study we sequenced and analyzed the entire genome of eight IBV strains that represent different serotypes that have not been previously sequenced, and we compared these sequences with other gamma-coronavirus full-length genome sequences available in GenBank for evidence of recombination [16] . Different serotypes of field viruses and vaccine type viruses were selected to provide a wide variety of sequences potentially capable of contributing gene fragments to recombinants.

Sequence Analysis

The full-length genomes were aligned and phylogenetic trees were constructed using the Neighbor-joining, Minimum Evolution, Maximum Parsimony and UPGMA programs in MEGA4 [17] . The trees all had similar topology and bootstrap support, and a representative tree is shown in Figure 1 . The feline coronavirus FCoV/FIPV/WSU-79-1146 and the beluga whale virus BelugaWhaleCoV/SW1/08 were included as out-groups. The wild bird viruses isolated from a munia (MuniaCoV/HKUY13/09), thrush (ThrushCoV/HKU12/09) and bulbul (BulBulCoV/HKU11/09) formed a unique clade, which is not surprising as this group might represent a new coronavirus genus provisionally designated Deltacoronavirus [18] . The remaining viruses separated into clades consisting of IBV isolates from the US and vaccine viruses, TCoV isolates, an IBV isolate from West Africa and IBV isolates from China and Taiwan.

Conclusions

In this study, evidence was obtained that recombination is occurring among avian coronavirus IBV isolates across their entire genome. Every sequence included in the analysis was recognized as a potential recipient of horizontally acquired sequences at some point in its viral evolutionary past. The nsp2, nsp3, nsp16 were associated with the greatest number of transferred fragments. In addition, the area immediately upstream of the spike gene had the highest number of recombination breakpoints. Breakpoints in the 1ab polyprotein gene have the potential to alter pathogenicity of the virus, and breakpoints near or in spike have the potential to lead to the emergence of new serotypes of IBV or new coronaviruses. Although the spike region determines the serotype of the virus, the remainder of the genome may be a mosaic of sequence fragments from a variety of gamma-coronaviruses. The only evidence of a gamma-coronavirus possibly recombining with an alpha or beta-coronavirus was the The PCR reaction was run on the same machine as the RT step and included a one-time initial denaturation step of 94 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 3 min.

Introduction

Infectious bronchitis virus is an enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 27 kb. The 3' end of the genome encodes four structural proteins; spike (S), envelope (E), membrane (M) and nucleocapsid (N) as well as several non-structural proteins [1] . The S glycoprotein of IBV forms projections on the surface of the virion. Spike is post-translationally cleaved into S1 and S2 subunits with the S1 subunit forming the outermost portion and S2 forming a stalk-like structure that is embedded in the viral membrane. The S1 subunit contains hypervariable regions that play a role in attachment to host cell receptors, and it contains conformationally-dependent virus-neutralizing and serotype-specific epitopes [2, 3] . Spike is also involved in membrane fusion and viral entry into the host cell. The E and M proteins are integral membrane proteins involved in assembly of the virus. The N protein is closely associated with the viral genome and plays a role in replication. The 5' two-thirds of the genome, approximately 21 kb, encodes two polyproteins 1a and 1ab. A minus one frame-shift mechanism is used to translate the 1ab polyprotein. The polyproteins are post-translationally cleaved into 15 non-structural proteins (nsps), nsp 2-16 (IBV does not have an nsp1) that make up the replication complex. Key nsps encoded, include a papain-like protease 2 (PLP2) within nsp 3, a main protease (Mpro) within nsp 5, and the RNA-dependent RNA-polymerase (RdRp) within nsps 11 and 12. Genetic diversity in coronaviruses is due to adaptive evolution driven by high mutation rates and genetic recombination [4] . High mutation rates are attributed to minimal proof reading capabilities associated with the RdRp. Recombination is thought to be due to a unique template switching "copy-choice" mechanism during RNA replication [5] . Evidence of recombination among strains of IBV has been observed both experimentally and in the field [6] [7] [8] [9] [10] [11] . The emergence of several alpha-and beta-coronaviruses has been attributed to recombination [12, 13] but only recently was recombination shown to be the mechanism behind the emergence of a novel gamma-coronavirus, turkey coronavirus (TCoV) [14] . Although "hot spots" of recombination in the genome of IBV have been reported [9, 15] , a thorough study of recombination using multiple different strains across the entire genome has not been conducted.
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Abstract

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one-third of human patients. Recent data indicate that dromedaries represent an important source of infection, although information regarding viral cell tropism and pathogenesis is sparse. In the current study, tissues of eight dromedaries receiving inoculation of MERS-Coronavirus (MERS-CoV) after recombinant Modified-Vaccinia-Virus-Ankara (MVA-S)-vaccination (n = 4), MVAvaccination (mock vaccination, n = 2) and PBS application (mock vaccination, n = 2), respectively, were investigated. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence, and scanning electron microscopy.
macrophages. The acute disease was further accompanied by ciliary loss along with a lack of dipeptidyl peptidase 4 (DPP4), known to mediate virus entry. DPP4 was mainly expressed by human lymphocytes and dromedary monocytes, but overall the expression level was lower in dromedaries. The present study underlines significant species-specific manifestations of MERS and highlights ciliary loss as an important finding in dromedaries. The obtained results promote a better understanding of coronavirus infections, which pose major health challenges. Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 SciEntiFic REpORTS | (2018) 8:9778 |

Discussion

The detection of MERS-CoV nucleocapsid antigen in the cytoplasm of a limited number of CD204/Iba-1 positive staining cells within the lamina propria of the nasal turbinates of mock-vaccinated dromedaries at 4 dpi is similar to previous investigations which detected viral antigen and RNA in mononuclear cells and stellate cells of mediastinal lymph nodes in experimentally infected rhesus macaques 9 and rarely within large mononuclear cells in the tracheal lymph node of infected dromedaries 22 . Moreover, infection of human monocyte-derived dendritic cells and monocyte-derived macrophages has been described in vitro and was accompanied by release of viral particles 38, 39 . The infection of these cell types is supposed to be mediated by DPP4, expressed on the cell surface of human macrophages 38, 40 , and leads to suppression of the innate immunity by reduced expression of tumor necrosis factor (TNF) and interleukin-6 (IL-6) 41 . In contrast, it remains so far uncertain whether the intracytoplasmic detection of MERS-CoV nucleocapsid antigen in dromedary macrophages represents a true productive or abortive infection or whether it is related to phagocytosis of MERS-CoV fragments. Since the number of positive staining macrophages was very low and DPP4 was not detectable on dromedary tonsils and hardly on dromedary lymphocytes and macrophages in lymph nodes it is not unlikely that the intrahistiocytic detection of MERS-CoV nucleocapsid antigen is related to phagocytosis of viral particles or infected cellular components. Nonetheless the staining of the antigen within affected cells was brightly and diffusely distributed in the cytoplasm and the detection of viral antigen in phagosomes would be suspected to rather appear as discrete spots 42, 43 . It might therefore been speculated that the viral antigen detection indeed represents virus infection of dromedary macrophages. However, further investigations have to elucidate whether it is a productive or abortive infection.

In June 2012 a novel lineage C betacoronavirus (HCoV-EMC) was identified in a patient from the Kingdom of Saudi Arabia who suffered from acute pneumonia and renal failure 1 . Subsequently, the virus was named Middle East respiratory syndrome coronavirus (MERS-CoV) in accordance with the geographical area of its first description and main occurrence 2 . Until today, MERS-CoV represents an existential threat to global health since the virus spread to 27 countries and caused more than 2000 laboratory confirmed cases in humans including 730 fatal cases, which equals approximately one third of all affected patients (World Health Organization (2017) Middle East respiratory syndrome coronavirus, available at http://www.who.int/emergencies/mers-cov/en/, accessed October 27, 2017) .
The sequence of MERS-CoV was determined to be closely related to other betacoronaviruses isolated from bats and therefore a bat origin has been proposed early after genomic characterization [3] [4] [5] [6] [7] [8] . However, transmission of MERS-CoV to humans was suspected to occur via an intermediate mammalian host, since the majority of human Middle East respiratory syndrome (MERS) patients did not state any direct contact to bats prior to disease onset 6, 9 . Similarly, severe acute respiratory syndrome coronavirus (SARS-CoV), a betacoronavirus of the lineage B, originated from bats 10 and spread from palm civets to humans in 2002/2003 11 .

Discussion

The present study demonstrates that acute MERS-CoV infection in dromedaries is accompanied by severe ciliary loss and concomitant lack of DPP4 on infected cells. Adjacent cells in which MERS-CoV antigen is not detectable retain positive staining for DPP4. Ciliary loss and consequent disturbances of mucociliary clearance are a major issue in several viral infections and can foster the development of severe secondary bacterial disease 34 . For instance, common cold in humans is accompanied by a massive loss of cilia and ciliated cells 35 . Similarly, human coronavirus infection of the upper respiratory tract has been described to be associated with migration of axonemes and basal bodies into the cell body (internalization) complemented by loss of cilia on the apical cell surface of infected cells. For the human disease it has been suggested that replicated virions are stored in apical vesicles before they are released. These vesicles may dislocate the basal body and withdraw the cilia into the cell 36 . In dogs, canine respiratory coronavirus infection is also associated with loss and damage to tracheal cilia, accompanied by inflammation 37 . Similar mechanisms might also play a role in MERS-CoV in dromedaries and would at least explain the massive loss of cilia which appears not to be accompanied by massive cell death or other profound histological and ultrastructural alterations in the majority of affected epithelial cells. Interestingly, ciliary loss is accompanied by lack of DPP4, which serves as a cell entry receptor for MERS-CoV in dromedaries. Therefore, the authors suggest that the mild and transient disease in dromedaries is, at least in part, attributable to the downregulation of its own cell entry receptor. Further studies need to be performed to elucidate underlying mechanisms of DPP4 loss in MERS-CoV-infected CK18 positive staining cells of dromedaries. The remaining expression on adjacent MERS-CoV negative cells suggests a potential direct virus mediated mechanism.
In summary, the present study highlights new and important differences between MERS-CoV infection in humans and dromedaries. Most importantly, ciliary loss and reduction of DPP4 expression represent important features of the disease in dromedaries which will deepen our understanding of MERS-CoV. Further investigations need to elucidate the underlying mechanisms of ciliary loss to gain insights into the pathogenesis of this emerging and life-threatening disease. Just recently a novel alphacoronavirus has been detected in Asian house shrews 46 and future zoonotic transmissions of such novel and well-known coronaviruses will require a profound understanding of their pathogenesis in different host species to achieve better preparedness.

Evaluation, quantification and statistical analysis. For quantification of inflammatory cells and

For visualization of the results of scanning electron microscopy a digital scanning microscope (DSM 940, Carl Zeiss Jena GmbH) was used. Per localization and time point post infection eight images were taken at 1000x magnification and the percentage of ciliated area was estimated. Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc.). Mann Whitney-U-Test was applied and results were considered statistically significant at p-value < 0.05. Table 1 . Antigen, clonality, species, source, antigen retrieval, dilution and secondary antibodies used for immunohistochemistry and immunofluorescence. Ab, antibody; CD, cluster of differentiation; CEACAM5, carcinoembryonic antigen related cell adhesion molecule 5; CK, cytokeratin; DPP4, Dipeptidylpeptidase 4; GAM-b, goat anti-mouse IgG, biotinylated; GAR, goat anti-rabbit, biotinylated; Iba-1, ionized calcium-binding adapter molecule 1; Immunoglobulin G (IgG); mc, monoclonal; MERS-CoV, Middle East respiratory syndrome coronavirus; n.a., not applied; pc, polyclonal; RAG-b, rabbit anti-goat IgG, biotinylated; RAR, rabbit anti-rat IgG, biotinylated.
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Abstract

Coronavirus (CoV) envelope (E) protein is a small structural protein critical for virion morphogenesis and release. The recently characterized E protein ion channel activity (EIC) has also been implicated in modulating viral pathogenesis. In this study, we used infectious bronchitis coronavirus (IBV) as a model to study EIC. Two recombinant IBVs (rIBVs) harboring EIC-inactivating mutations -rT16A and rA26F -were serially passaged, and several compensatory mutations were identified in the transmembrane domain (TMD). Two rIBVs harboring these putative EICreverting mutations -rT16A/A26V and rA26F/F14N -were recovered. Compared with the parental rIBV-p65 control, all four EIC mutants exhibited comparable levels of intracellular RNA synthesis, structural protein production, and virion assembly. Our results showed that the IBV EIC contributed to the induction of ER stress response, as up-regulation of ER stress-related genes was markedly reduced in cells infected with the EIC-defective mutants. EIC-defective mutants also formed smaller plaques, released significantly less infectious virions into the culture supernatant, and had lower levels of viral fitness in cell culture. Significantly, all these defective phenotypes were restored in cells infected with the putative EIC revertants. EIC mutations were also implicated in regulating IBV-induced apoptosis, induction of pro-inflammatory cytokines, and viral pathogenicity in vivo. Taken together, this study highlights the importance of CoV EIC in modulating virion release and various aspects of CoV -host interaction.

INTRODUCTION

Coronaviruses (CoVs) are a group of enveloped viruses with non-segmented, single-stranded, and positive-sense RNA genomes (Masters, 2006) . Besides infecting a wide range of domesticated and laboratory vertebrates, six human CoVs have been identified, causing respiratory diseases with mild to severe outcomes. Among these, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are both zoonotic and highly pathogenic CoVs that have emerged in recent epidemic and/or pandemic outbreaks (Li et al., 2005; de Groot et al., 2013) .
Coronaviruses have huge RNA genomes ranging from 27,000 to 32,000 nucleotides. The first 2/3 of the genome encodes the viral replicase, while the remaining 1/3 contains coding sequence for the accessory proteins and the four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) protein (Masters, 2006) . Among them, the S protein is responsible for binding to the cognate receptor and mediating membrane fusion Zheng et al., 2018) ; the M protein is crucial for the assembly and morphogenesis of mature virions (De Haan et al., 2000; Liang et al., 2019) ; the N protein binds to the RNA genome in a beads-on-a-string fashion to form the helically symmetric nucleocapsid (Masters, 2006) .
Infectious bronchitis virus (IBV) is a gammacoronavirus infecting chicken. Apart from the E protein, no other IBV protein has been reported to encode ion channel (IC) activity. Two point mutations in the IBV E protein -T16A and A26F -are homologous to the N15A and V25F in the SARS-CoV E protein, respectively (Ruch and Machamer, 2012) . The T16A mutation abolished the ability of IBV E protein to disrupt the Golgi complex and host secretory pathway, but did not impact virus assembly as determined by the level of VLP production (Ruch and Machamer, 2012) . In contrast, IBV E A26F disrupted cellular secretory pathway, but did not support VLP production (Westerbeck and Machamer, 2015) . Using a pH-indicating fluorescent protein pHluorin, it was also found that transfection of IBV E or E A26F , but not E T16A , correlated with an increase of Golgi luminal pH (Westerbeck and Machamer, 2019) . The authors thus concluded that IBV EIC facilitated virion release, presumably by neutralizing the Golgi lumen to protect the S protein from premature proteolytic cleavage (Westerbeck and Machamer, 2019) . However, these studies were based on overexpression experiments and the IBV EIC was not investigated by reverse genetics in the setting of actual infections. Recently, we reported the successful recovery of recombinant IBVs (rIBVs) harboring these two mutations . Compared with the parental control, rT16A and rA26F exhibited similar levels of intracellular viral replication and assembly, but the levels of virion release were significantly reduced .

EIC Mutations Modulate the Induction of Apoptosis in IBV-Infected Cells

Infectious bronchitis coronavirus infection induces caspasedependent and p53-independent apoptosis in the infected cells Li et al., 2007) . Moreover, IBV-induced apoptosis FIGURE 2 | EIC contributes to the upregulation of ER stress-related genes and proinflammatory cytokines during IBV infection. (A) Vero cells were infected with the five rIBVs at MOI ∼ 2. Cell lysates were harvested by three freeze/thaw cycles at 24 and 32 h post infection (hpi). Virus titers were expressed in the unit of log TCID50 per ml. Cell lysates were subjected to RNA extraction. Equal amounts of total RNA were reverse transcribed. The levels of IBV genomic RNA (IBVgRNA) were determined by quantitative PCR and normalized to that of the rIBV-p65-infected 24 hpi sample. Cell lysates were also subjected to SDS-PAGE and immunoblotting with antisera against IBV S, M, and N protein. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. The experiment was repeated three times with similar results, and the result of one representative experiment is shown. S*, glycosylated IBV S protein. Comparing with rIBV-p65-infected sample of the same set: ns, non-significant. (B) Cell lysates harvested in (A) were subjected to RNA extraction and RT-qPCR analysis to determine the mRNA expression levels of GRP78, GRP94, HERPUD1, total XBP1, spliced XBP1, ERdj4, p58 IPK , PERK, and CHOP. GAPDH was used as the internal control. Fold changes were determined by normalizing to the rIBV-p65-infected 24 hpi sample. The experiment was repeated three times with similar results, and the result of one representative experiment is shown. Comparing with rIBV-p65-infected sample of the same set: *p < 0.05; **p < 0.01; ns, non-significant.
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Cytokines

Airway epithelial cells secrete a host of cytokines, chemokines, antimicrobial peptides and other factors in response to viral infection. Cytokines beyond the IFNs produced by airway epithelium include interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), granulocyte colony stimulating factor (G-CSF), and granulocyte macrophage-CSF (GM-CSF). IL-6 and TNF-α are potent proinflammatory cytokines that modulate many types of immune cells. IL-6 facilitates the transition from the innate to adaptive immune response by driving down neutrophil activity while concurrently promoting the recruitment, differentiation, and activity of monocytes and T cells [10] . High IL-6 levels correlate with disease severity, but ablation of IL-6 signaling can lead to uncontrolled virus replication resulting in greater mortality [11] . TNF-α impairs viral replication, enhances cytotoxic activity, and cytokine production by leukocytes and activates endothelial cells [12] . Elevated levels of TNF-α have been associated with greater morbidity during infection with highly pathogenic virus, and blocking activity of TNF-α attenuates immunemediated pathology [11] .

Infection of the upper respiratory tract (URT)

Often the URT is the initial site of viral replication since many respiratory viruses are inhaled or transferred by contact to the nasal mucosa. In healthy patients without pre-existing conditions, infections with common strains of rhinovirus, coronavirus, and adenovirus are typically limited to the upper airways. Symptomatic viral infection of the URT (coryza, rhinorrhea, cough, and sore throat) ( Table 1 ) reflects loss of cellular tight junctions, vascular leakage and edema, increased mucus production, and apoptosis, necrosis, and sloughing of epithelial cells [71] [72] [73] . Recruitment of neutrophils and mononuclear cells into the URT further propagates edema and hypersecretion of mucus, exacerbating nasal congestion, sneezing, and coughing in patients [71] [72] [73] .

Pneumonia

While viruses such as RSV and Enterovirus D68 are capable of reaching the terminal airways, influenza viruses and the SARS-CoV and MERS-CoV coronaviruses more frequently reach the terminal airways, and thus are more likely to disrupt pulmonary function and cause pneumonia [72] . The terminal airways are the site of gas exchange and make up the majority of the airway surface of the lung. During infection, loss of tight junctions, vasculature leak, buildup of edematous fluid and fibrin in the alveolar airspaces can result in necrosis of the alveolar cells and hyaline membrane formation. If severe, these changes can lead to diffuse alveolar damage (DAD), manifested clinically as ARDS and respiratory failure. Inflammatory processes and immune responses as described previously compromise the function and properties of terminal airways and terminal airway epithelial cells in a fashion analogous to that of conducting airways. Furthermore, a loss of surface tension and surfactant resulting in alveolar collapse represent additional threats to pulmonary function in the terminal airways. The terminal airways of survivors of acute viral pneumonia can result in alteration of pulmonary elasticity due to organization of inflammatory processes during the resolution phase which may have lasting consequences and put the host at risk for superinfections [72, 73, 75] . Although not discussed here, bacterial superinfection or co-infection are important sequelae of virus infection in the LRT particularly during influenza virus infection.

Introduction

The cells that line the respiratory tract are continually exposed to the external environment, making the lungs a particularly vulnerable site for infection. Respiratory infections represent a major disease and economic burden worldwide. According to the CDC, influenza virus infection and associated complications are one of the top ten causes of death and result in millions of hospitalizations, costing over $10 billion each year in the USA [1] . Other respiratory virus such as highly pathogenic avian influenza and Severe Acute Respiratory Syndrome (SARS-CoV) and Middle Eastern Respiratory Syndrome (MERS-CoV) coronaviruses represents everpresent threats to human health globally. Therefore, understanding the factors, both virus-dependent and host-dependent, that regulate the development and severity of respiratory virus infections is critical for both the prevention and treatment of virus-associated disease in the respiratory tract.
2 section matches

Introduction

There are two subsets of peripheral blood DCs in humans, myeloid (mDCs) and plasmacytoid (pDCs), with distinct functions [12, 13] . Previous studies on DCs and MS revealed that monocyte-derived DCs from patients produce higher levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) than healthy subjects [14] . In line with these observations, mDCs obtained from MS patients produce higher levels of IL-12 and IL-6 in response to a TLR-7/8 agonist [15] . Conversely, pDCs in MS patients produce lower levels of interferon (IFN)-α compared to healthy subjects [15] . Furthermore, it has been shown that upregulation of the costimulatory molecules upon stimulation with IL-3 and CD40L is significantly delayed in pDCs from MS patients compared to healthy donors [16] . The inefficient maturation of pDCs observed in this last paper could be associated with the specific stimulus. Indeed, the impaired expression of CD40 in pDCs from MS patients could impair the responsiveness to a CD40L stimulus [16] . Moreover, this study is not exhaustive because it is known that microbe-activated pDCs have features distinct from those of cytokine-activated DCs [17] .
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS), and is the most common neurological cause of debilitation in adults, with an incidence of 0.1%. MS is currently believed to be an immune-mediated disorder where the immune response attacks the CNS and the pathology is directly mediated by autoantigen-specific T cells [1] . Cross-reactivity between nonself proteins, such as those from bacteria or viruses, and self proteins, termed molecular mimicry, has been proposed to be a possible mechanism for the onset of autoreactive T-cell responses [2] [3] [4] . In fact, the infectious etiology of MS has been suspected for a long time and by a large number of viruses: DNA viruses, such as the Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6) [5] , and RNA viruses, such as human endogenous retroviruses [6] [7] [8] , and the coronavirus [9] .