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1.  Upregulation of CHOP/GADD153 during Coronavirus Infectious Bronchitis Virus Infection Modulates Apoptosis by Restricting Activation of the Extracellular Signal-Regulated Kinase Pathway 
Journal of Virology  2013;87(14):8124-8134.
Induction of the unfolded protein response (UPR) is an adaptive cellular response to endoplasmic reticulum (ER) stress that allows a cell to reestablish ER homeostasis. However, under severe and persistent ER stress, prolonged UPR may activate unique pathways that lead to cell death. In this study, we investigated the activation of the protein kinase R-like ER kinase (PERK) pathway of UPR in cells infected with the coronavirus infectious bronchitis virus (IBV) and its relationship with IBV-induced apoptosis. The results showed moderate induction of PERK phosphorylation in IBV-infected cells. Meanwhile, activating transcription factor 4 (ATF4) was upregulated at the protein level in the infected cells, resulting in the induction in trans of the transcription factor ATF3 and the proapoptotic growth arrest and DNA damage-inducible protein GADD153. Knockdown of PERK by small interfering RNA (siRNA) suppressed the activation of GADD153 and the IBV-induced apoptosis. Interestingly, knockdown of protein kinase R (PKR) by siRNA and inhibition of the PKR kinase activity by 2-aminopurine (2-AP) also reduced the IBV-induced upregulation of GADD153 and apoptosis induction. In GADD153-knockdown cells, IBV-induced apoptosis was suppressed and virus replication inhibited, revealing a key role of GADD153 in IBV-induced cell death and virus replication. Analysis of the pathways downstream of GADD153 revealed much more activation of the extracellular signal-related kinase (ERK) pathway in GADD153-knockdown cells during IBV infection, indicating that GADD153 may modulate apoptosis through suppression of the pathway. This study provides solid evidence that induction of GADD153 by PERK and PKR plays an important regulatory role in the apoptotic process triggered by IBV infection.
doi:10.1128/JVI.00626-13
PMCID: PMC3700216  PMID: 23678184
2.  Vitamin A Deficiency Impairs Adaptive B and T Cell Responses to a Prototype Monovalent Attenuated Human Rotavirus Vaccine and Virulent Human Rotavirus Challenge in a Gnotobiotic Piglet Model 
PLoS ONE  2013;8(12):e82966.
Rotaviruses (RV) are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD) gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV) vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS) sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFNα) cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12) and 2-3 fold lower anti-inflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented) pigs had significantly higher serum IL12 (PID2) and IFNγ (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more severe rotavirus infection and lower HRV vaccine efficacy.
doi:10.1371/journal.pone.0082966
PMCID: PMC3846786  PMID: 24312675
3.  Genetic Diversity of Hepatitis A Virus in China: VP3-VP1-2A Genes and Evidence of Quasispecies Distribution in the Isolates 
PLoS ONE  2013;8(9):e74752.
Hepatitis A virus (HAV) is the most common cause of infectious hepatitis throughout the world, spread largely by the fecal-oral route. To characterize the genetic diversity of the virus circulating in China where HAV in endemic, we selected the outbreak cases with identical sequences in VP1-2A junction region and compiled a panel of 42 isolates. The VP3-VP1-2A regions of the HAV capsid-coding genes were further sequenced and analyzed. The quasispecies distribution was evaluated by cloning the VP3 and VP1-2A genes in three clinical samples. Phylogenetic analysis demonstrated that the same genotyping results could be obtained whether using the complete VP3, VP1, or partial VP1-2A genes for analysis in this study, although some differences did exist. Most isolates clustered in sub-genotype IA, and fewer in sub-genotype IB. No amino acid mutations were found at the published neutralizing epitope sites, however, several unique amino acid substitutions in the VP3 or VP1 region were identified, with two amino acid variants closely located to the immunodominant site. Quasispecies analysis showed the mutation frequencies were in the range of 7.22x10-4 -2.33x10-3 substitutions per nucleotide for VP3, VP1, or VP1-2A. When compared with the consensus sequences, mutated nucleotide sites represented the minority of all the analyzed sequences sites. HAV replicated as a complex distribution of closely genetically related variants referred to as quasispecies, and were under negative selection. The results indicate that diverse HAV strains and quasispecies inside the viral populations are presented in China, with unique amino acid substitutions detected close to the immunodominant site, and that the possibility of antigenic escaping mutants cannot be ruled out and needs to be further analyzed.
doi:10.1371/journal.pone.0074752
PMCID: PMC3775754  PMID: 24069343
4.  Profiling of Genes Related to Cross Protection and Competition for NbTOM1 by HLSV and TMV 
PLoS ONE  2013;8(9):e73725.
Cross protection is the phenomenon through which a mild strain virus suppresses symptoms induced by a closely related severe strain virus in infected plants. Hibiscus latent Singapore virus (HLSV) and Tobacco mosaic virus (TMV) are species within the genus tobamovirus. HLSV can protect Nicotianabenthamiana against TMV-U1 strain, resulting in mild symptoms instead of severe systemic necrosis. The mechanism of cross protection between HLSV and TMV is unknown. In the past, some researchers suggest that the protecting virus strain might occupy virus-specific replication sites within a cell leaving no room for the challenge virus. Quantitative real-time RT-PCR was performed to detect viral RNA levels during cross protection. HLSV accumulation increased in cross protected plants compared with that of single HLSV infected plants, while TMV decreased in cross protected plants. This suggests that there is a competition for host factors between HLSV and TMV for replication. To investigate the mechanism under the cross protection between HLSV and TMV, microarray analysis was conducted to examine the transcriptional levels of global host genes during cross protection, using Tobacco Gene Expression Microarray, 4x44 k slides. The transcriptional level of some host genes corresponded to accumulation level of TMV. Some host genes were up-regulated only by HLSV. Tobamovirus multiplication gene 1 (TOM1), essential for tobamovirus multiplication, was involved in competition for replication by HLSV and TMV during cross protection. Both HLSV and TMV accumulation decreased when NbTOM1 was silenced. A large quantity of HLSV resulted in decreased TMV accumulation in HLSV+TMV (100:1) co-infection. These results indicate that host genes involved in the plant defense response and virus multiplication are up-regulated by challenge virus TMV but not by protecting virus HLSV during cross protection.
doi:10.1371/journal.pone.0073725
PMCID: PMC3762752  PMID: 24023899
5.  Recent Progress in Studies of Arterivirus- and Coronavirus-Host Interactions 
Viruses  2012;4(6):980-1010.
Animal coronaviruses, such as infectious bronchitis virus (IBV), and arteriviruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), are able to manifest highly contagious infections in their specific native hosts, thereby arising in critical economic damage to animal industries. This review discusses recent progress in studies of virus-host interactions during animal and human coronavirus and arterivirus infections, with emphasis on IBV-host cell interactions. These interactions may be directly involved in viral replication or lead to the alteration of certain signaling pathways, such as cell stress response and innate immunity, to facilitate viral replication and pathogenesis.
doi:10.3390/v4060980
PMCID: PMC3397358  PMID: 22816036
virus-host interactions; coronavirus; arterivirus
6.  Binding of the 5′-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription 
Nucleic Acids Research  2012;40(11):5065-5077.
Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5′-untranslated region (5′-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5′-UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem–loop I of IBV 5′-UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.
doi:10.1093/nar/gks165
PMCID: PMC3367200  PMID: 22362731
7.  Up-Regulation of Mcl-1 and Bak by Coronavirus Infection of Human, Avian and Animal Cells Modulates Apoptosis and Viral Replication 
PLoS ONE  2012;7(1):e30191.
Virus-induced apoptosis and viral mechanisms that regulate this cell death program are key issues in understanding virus-host interactions and viral pathogenesis. Like many other human and animal viruses, coronavirus infection of mammalian cells induces apoptosis. In this study, the global gene expression profiles are first determined in IBV-infected Vero cells at 24 hours post-infection by Affymetrix array, using avian coronavirus infectious bronchitis virus (IBV) as a model system. It reveals an up-regulation at the transcriptional level of both pro-apoptotic Bak and pro-survival myeloid cell leukemia-1 (Mcl-1). These results were further confirmed both in vivo and in vitro, in IBV-infected embryonated chicken eggs, chicken fibroblast cells and mammalian cells at transcriptional and translational levels, respectively. Interestingly, the onset of apoptosis occurred earlier in IBV-infected mammalian cells silenced with short interfering RNA targeting Mcl-1 (siMcl-1), and was delayed in cells silenced with siBak. IBV progeny production and release were increased in infected Mcl-1 knockdown cells compared to similarly infected control cells, while the contrary was observed in infected Bak knockdown cells. Furthermore, IBV infection-induced up-regulation of GADD153 regulated the expression of Mcl-1. Inhibition of the mitogen-activated protein/extracellular signal-regulated kinase (MEK/ERK) and phosphoinositide 3-kinase (PI3K/Akt) signaling pathways by chemical inhibitors and knockdown of GADD153 by siRNA demonstrated the involvement of ER-stress response in regulation of IBV-induced Mcl-1 expression. These results illustrate the sophisticated regulatory strategies evolved by a coronavirus to modulate both virus-induced apoptosis and viral replication during its replication cycle.
doi:10.1371/journal.pone.0030191
PMCID: PMC3256233  PMID: 22253918
8.  The Cellular RNA Helicase DDX1 Interacts with Coronavirus Nonstructural Protein 14 and Enhances Viral Replication▿  
Journal of Virology  2010;84(17):8571-8583.
The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.
doi:10.1128/JVI.00392-10
PMCID: PMC2918985  PMID: 20573827
9.  Functional and Genetic Studies of the Substrate Specificity of Coronavirus Infectious Bronchitis Virus 3C-Like Proteinase ▿  
Journal of Virology  2010;84(14):7325-7336.
Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.
doi:10.1128/JVI.02490-09
PMCID: PMC2898227  PMID: 20444893
10.  Inhibition of Protein Kinase R Activation and Upregulation of GADD34 Expression Play a Synergistic Role in Facilitating Coronavirus Replication by Maintaining De Novo Protein Synthesis in Virus-Infected Cells▿  
Journal of Virology  2009;83(23):12462-12472.
A diversity of strategies is evolved by RNA viruses to manipulate the host translation machinery in order to create an optimal environment for viral replication and progeny production. One of the common viral targets is the α subunit of eukaryotic initiation factor 2 (eIF-2α). In this report, we show that phosphorylation of eIF-2α was severely suppressed in human and animal cells infected with the coronavirus infectious bronchitis virus (IBV). To understand whether this suppression is through inhibition of protein kinase R (PKR), the double-stranded-RNA-dependent kinase that is one of the main kinases responsible for phosphorylation of eIF-2α, cells infected with IBV were analyzed by Western blotting. The results showed that the level of phosphorylated PKR was greatly reduced in IBV-infected cells. Overexpression of IBV structural and nonstructural proteins (nsp) demonstrated that nsp2 is a weak PKR antagonist. Furthermore, GADD34, a component of the protein phosphatase 1 (PP1) complex, which dephosphorylates eIF-2α, was significantly induced in IBV-infected cells. Inhibition of the PP1 activity by okadaic acid and overexpression of GADD34, eIF-2α, and PKR, as well as their mutant constructs in virus-infected cells, showed that these viral regulatory strategies played a synergistic role in facilitating coronavirus replication. Taken together, these results confirm that IBV has developed a combination of two mechanisms, i.e., blocking PKR activation and inducing GADD34 expression, to maintain de novo protein synthesis in IBV-infected cells and, meanwhile, to enhance viral replication.
doi:10.1128/JVI.01546-09
PMCID: PMC2786722  PMID: 19776135
11.  Diagnostic Approach for the Differentiation of the Pandemic Influenza A(H1N1)v Virus from Recent Human Influenza Viruses by Real-Time PCR 
PLoS ONE  2010;5(4):e9966.
Background
The current spread of pandemic influenza A(H1N1)v virus necessitates an intensified surveillance of influenza virus infections worldwide. So far, in many laboratories routine diagnostics were limited to generic influenza virus detection only. To provide interested laboratories with real-time PCR assays for type and subtype identification, we present a bundle of PCR assays with which any human influenza A and B virus can be easily identified, including assays for the detection of the pandemic A(H1N1)v virus.
Principal Findings
The assays show optimal performance characteristics in their validation on plasmids containing the respective assay target sequences. All assays have furthermore been applied to several thousand clinical samples since 2007 (assays for seasonal influenza) and April 2009 (pandemic influenza assays), respectively, and showed excellent results also on clinical material.
Conclusions
We consider the presented assays to be well suited for the detection and subtyping of circulating influenza viruses.
doi:10.1371/journal.pone.0009966
PMCID: PMC2848602  PMID: 20376359
12.  Binding of Herpes Simplex Virus Type-1 Virions Leads to the Induction of Intracellular Signalling in the Absence of Virus Entry 
PLoS ONE  2010;5(3):e9560.
The envelope of HSV-1 contains a number of glycoproteins, four of which are essential for virus entry. Virus particles lacking gB, gD, gH or gL are entry-defective, although these viruses retain the ability to bind to the plasma membrane via the remaining glycoproteins. Soluble forms of gD have been shown to trigger the nuclear translocation of the NF-κB transcriptional complex in addition to stimulating the production of Type I interferon. By taking advantage of the entry-defective phenotype of glycoprotein-deficient HSV-1 virus particles, the results presented here show that binding of virions to cellular receptors on the plasma membrane is sufficient to stimulate a change in cellular gene expression. Preliminary microarray studies, validated by quantitative real-time PCR, identified the differential expression of cellular genes associated with the NF-κB, PI3K/Akt, Jak/Stat and related Jak/Src pathways by virions lacking gB or gH but not gD. Gene induction occurred at a few particles per cell, corresponding to physiological conditions during primary infection. Reporter assay studies determined that NF-κB transcriptional activity is stimulated within an hour of HSV-1 binding, peaks between two and three hours post-binding and declines to background levels by five hours after induction. The immediate, transient nature of these signalling events suggests that HSV-1 glycoproteins, particularly gD, may alter the cellular environment pre-entry so as to condition the cell for viral replication.
doi:10.1371/journal.pone.0009560
PMCID: PMC2832691  PMID: 20221426
13.  Proteolytic Activation of the Spike Protein at a Novel RRRR/S Motif Is Implicated in Furin-Dependent Entry, Syncytium Formation, and Infectivity of Coronavirus Infectious Bronchitis Virus in Cultured Cells▿  
Journal of Virology  2009;83(17):8744-8758.
The spike (S) protein of the coronavirus (CoV) infectious bronchitis virus (IBV) is cleaved into S1 and S2 subunits at the furin consensus motif RRFRR537/S in virus-infected cells. In this study, we observe that the S2 subunit of the IBV Beaudette strain is additionally cleaved at the second furin site (RRRR690/S) in cells expressing S constructs and in virus-infected cells. Detailed time course experiments showed that a peptide furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, blocked both viral entry and syncytium formation. Site-directed mutagenesis studies revealed that the S1/S2 cleavage by furin was not necessary for, but could promote, syncytium formation by and infectivity of IBV in Vero cells. In contrast, the second site is involved in the furin dependence of viral entry and syncytium formation. Mutations of the second site from furin-cleavable RRRR/S to non-furin-cleavable PRRRS and AAARS, respectively, abrogated the furin dependence of IBV entry. Instead, a yet-to-be-identified serine protease(s) was involved, as revealed by protease inhibitor studies. Furthermore, sequence analysis of CoV S proteins by multiple alignments showed conservation of an XXXR/S motif, cleavable by either furin or other trypsin-like proteases, at a position equivalent to the second IBV furin site. Taken together, these results suggest that proteolysis at a novel XXXR/S motif in the S2 subunit might be a common mechanism for the entry of CoV into cells.
doi:10.1128/JVI.00613-09
PMCID: PMC2738192  PMID: 19553314
14.  Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel 
PLoS Pathogens  2009;5(7):e1000511.
The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.
Author Summary
Coronaviruses are viral pathogens that cause a variety of lethal diseases in birds and mammals, and common colds in humans. In 2003, however, an animal coronavirus was able to infect humans and produced severe acute respiratory syndrome (SARS), causing a near pandemic. Such events are likely to reoccur in the future, and new antiviral strategies are necessary. A small coronavirus protein called ‘envelope’ is important for pathogenesis, affecting the formation of the viral envelope and the distribution of the virus in the body. In vitro studies have shown that synthetic coronavirus envelope proteins have channel activity that in some cases has been inhibited by the drug hexamethylene amiloride, but not by amiloride. In the present paper, we have characterized the structure responsible for this channel activity. We have also determined the binding site of the drug hexamethylene amiloride in the channel, and shown that amiloride has only a mild effect on the NMR signals from the protein. The validity of these results is supported using mammalian cells expressing full length SARS-CoV E, where channel activity was inhibited by hexamethylene amiloride, but only mildly by amiloride. The structural model described for this channel provides a valuable insight into coronavirus envelope protein ion channel activity, and could serve as a platform for the development of novel anti-viral drugs.
doi:10.1371/journal.ppat.1000511
PMCID: PMC2702000  PMID: 19593379
15.  Importance of SARS-CoV Spike Protein Trp-rich Region in Viral Infectivity 
SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalanine substitutions. Our results show that individually-substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10–25% infectivity comparing to the wild type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity.
doi:10.1016/j.bbrc.2008.04.044
PMCID: PMC2519895  PMID: 18424264
Trp-rich region; SARS-CoV; Spike protein; membrane fusion; Alanine scan; mutant; infectivity
16.  Acquisition of Cell–Cell Fusion Activity by Amino Acid Substitutions in Spike Protein Determines the Infectivity of a Coronavirus in Cultured Cells 
PLoS ONE  2009;4(7):e6130.
Coronavirus host and cell specificities are determined by specific interactions between the viral spike (S) protein and host cell receptor(s). Avian coronavirus infectious bronchitis (IBV) has been adapted to embryonated chicken eggs, primary chicken kidney (CK) cells, monkey kidney cell line Vero, and other human and animal cells. Here we report that acquisition of the cell–cell fusion activity by amino acid mutations in the S protein determines the infectivity of IBV in cultured cells. Expression of S protein derived from Vero- and CK-adapted strains showed efficient induction of membrane fusion. However, expression of S protein cloned from the third passage of IBV in chicken embryo (EP3) did not show apparent syncytia formation. By construction of chimeric S constructs and site-directed mutagenesis, a point mutation (L857-F) at amino acid position 857 in the heptad repeat 1 region of S protein was shown to be responsible for its acquisition of the cell–cell fusion activity. Furthermore, a G405-D point mutation in the S1 domain, which was acquired during further propagation of Vero-adapted IBV in Vero cells, could enhance the cell–cell fusion activity of the protein. Re-introduction of L857 back to the S gene of Vero-adapted IBV allowed recovery of variants that contain the introduced L857. However, compensatory mutations in S1 and some distant regions of S2 were required for restoration of the cell–cell fusion activity of S protein carrying L857 and for the infectivity of the recovered variants in cultured cells. This study demonstrates that acquisition of the cell–cell fusion activity in S protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins.
doi:10.1371/journal.pone.0006130
PMCID: PMC2700284  PMID: 19572016
17.  A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets 
PLoS ONE  2009;4(6):e6032.
There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP) as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1). In this study, a seasonal trivalent VLP vaccine (TVV) formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI) antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV). Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.
doi:10.1371/journal.pone.0006032
PMCID: PMC2698286  PMID: 19554101
18.  Interaction of the Coronavirus Infectious Bronchitis Virus Membrane Protein with β-Actin and Its Implication in Virion Assembly and Budding 
PLoS ONE  2009;4(3):e4908.
Coronavirus M protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. The M protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. In this study, we showed co-purification of the M protein from coronavirus infectious bronchitis virus (IBV) with actin. To understand the cellular factors that may be involved in virion assembly, budding and maturation processes, IBV M was used as the bait in a yeast two-hybrid screen, resulting in the identification of β-actin as a potentially interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids A159 and K160 on the M protein abolished the interaction. Introduction of the A159-K160 mutation into an infectious IBV clone system blocks the infectivity of the clone, although viral RNA replication and subgenomic mRNA transcription were actively detected. Disruption of actin filaments with cell-permeable agent cytochalasin D at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. However, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. This study reveals an essential function of actin in the replication cycle of coronavirus.
doi:10.1371/journal.pone.0004908
PMCID: PMC2653722  PMID: 19287488
19.  Coronavirus Spike Protein Inhibits Host Cell Translation by Interaction with eIF3f 
PLoS ONE  2008;3(1):e1494.
In response to viral infection, the expression of numerous host genes, including predominantly a number of proinflammatory cytokines and chemokines, is usually up-regulated at both transcriptional and translational levels. It was noted that in cells infected with coronavirus, transcription and translation of some of these genes were differentially induced. Drastic induction of their expression at the transcriptional level was observed in cells infected with coronavirus. However, induction of the same genes at the translational level was usually found to be minimal to moderate. To investigate the underlying mechanisms, yeast two-hybrid screen was carried out using SARS-CoV proteins as baits, revealing that a subunit of the eukaryotic initiation factor 3 (eIF3), eIF3f, may interact with the N-terminal region of the SARS-CoV spike (S) protein. This interaction was subsequently confirmed by co-immunoprecipitation and immunofluorescent staining. Meanwhile, parallel experiments confirmed that eIF3f could also interact with the S protein of another coronavirus, the avian coronavirus infectious bronchitis virus (IBV). These interactions led to the inhibition of translation of a reporter gene in both in vitro expression system and intact cells. Interestingly, IBV-infected cells stably expressing a Flag-tagged eIF3f showed much higher translation of IL-6 and IL-8, suggesting that the interaction between coronavirus S protein and eIF3f plays a functional role in controlling the expression of host genes, especially genes that are induced during coronavirus infection cycles. This study reveals a novel mechanism exploited by coronavirus to regulate viral pathogenesis.
doi:10.1371/journal.pone.0001494
PMCID: PMC2204050  PMID: 18231581
20.  Structure of the SARS coronavirus main proteinase as an active C2 crystallographic dimer 
An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme.
The 34 kDa main proteinase (Mpro) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV Mpro is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P21212 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.
doi:10.1107/S1744309105033257
PMCID: PMC1978130  PMID: 16511208
protease; crystallographic dimer; SARS coronavirus
21.  Matrix Metalloproteinase 3 Promotes Cellular Anti-Dengue Virus Response via Interaction with Transcription Factor NFκB in Cell Nucleus 
PLoS ONE  2014;9(1):e84748.
Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation.
doi:10.1371/journal.pone.0084748
PMCID: PMC3885614  PMID: 24416274
22.  Complete Genome Sequence of the First Chinese Virulent Infectious Laryngotracheitis Virus 
PLoS ONE  2013;8(7):e70154.
Infectious laryngotracheitis (ILT) is an acute respiratory disease caused by infectious laryngotracheitis virus (ILTV). The complete genome sequences of five attenuated ILTV vaccine strains and six virulent ILTV strains as well as two Australian ILTV field strains have been published in Australia and the USA so far. To provide the complete genome sequence information of ILTVs from different geographic regions, the whole genome of ILTV LJS09 isolated in China was sequenced. The genome of ILTV LJS09 was 153,201 bp in length, and contained 79 ORFs. Most of the ORFs had high sequence identity with homologous ORFs of reference strains. There was a large fragment deletion within the noncoding region of unique long region (UL) of ILTV LJS09 compared with SA2 and A20 strains. Though the origin binding protein of ILTV LJS09 existed, there was no AT-rich region in strain LJS09. Alignments of the amino acid sequences revealed seven mutations at amino acids 71 (Arg → Lys), 116 (Ala → Val), 207 (Thr → Ile) and 644 (Thr → Ile) on glycoprotein B, 155 (Phe → Ser) and 376 (Arg → His) on glycoprotein D and 8 (Gln→Pro) on glycoprotein L of ILTV LJS09 compared to those of virulent strain (USDA) as ILTV LJS09 did not grow on chicken embryo fibroblasts, suggesting the role of the key seven amino acids in determination of the cell tropism of ILTV LJS09. This is the first complete genome sequence of the virulent strain of ILTV in Asia using the conventional PCR method, which will help to facilitate the future molecular biological research of ILTVs.
doi:10.1371/journal.pone.0070154
PMCID: PMC3726392  PMID: 23922947
23.  Involvement of MicroRNAs in Infection of Silkworm with Bombyx mori Cytoplasmic Polyhedrosis Virus (BmCPV) 
PLoS ONE  2013;8(7):e68209.
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the most important pathogens of silkworm. MicroRNAs (miRNAs) have been demonstrated to play key roles in regulating host-pathogen interaction. However, there are limited reports on the miRNAs expression profiles during insect pathogen challenges. In this study, four small RNA libraries from BmCPV-infected midgut of silkworm at 72 h post-inoculation and 96 h post-inoculation and their corresponding control midguts were constructed and deep sequenced. A total of 316 known miRNAs (including miRNA*) and 90 novel miRNAs were identified. Fifty-eight miRNAs displayed significant differential expression between the infected and normal midgut (P value < = 0.01 and fold change > = 2.0 or < = 0.5), among which ten differentially expressed miRNA were validated by qRT-PCR method. Further bioinformatics analysis of predicted target genes of differentially expressed miRNAs showed that the miRNA targets were involved in stimulus and immune system process in silkworm.
doi:10.1371/journal.pone.0068209
PMCID: PMC3699532  PMID: 23844171
24.  Signal Peptide Cleavage from GP5 of PRRSV: A Minor Fraction of Molecules Retains the Decoy Epitope, a Presumed Molecular Cause for Viral Persistence 
PLoS ONE  2013;8(6):e65548.
Porcine reproductive and respiratory syndrome virus (PRRSV) is the major pathogen in the pig industry. Variability of the antigens and persistence are the biggest challenges for successful control and elimination of the disease. GP5, the major glycoprotein of PRRSV, is considered an important target of neutralizing antibodies, which however appear only late in infection. This was attributed to the presence of a “decoy epitope” located near a hypervariable region of GP5. This region also harbors the predicted signal peptide cleavage sites and (dependent on the virus strain) a variable number of potential N-glycosylation sites. Molecular processing of GP5 has not been addressed experimentally so far: whether and where the signal peptide is cleaved and (as a consequence) whether the “decoy epitope” is present in virus particles. We show that the signal peptide of GP5 from the American type 2 reference strain VR-2332 is cleaved, both during in vitro translation in the presence of microsomes and in transfected cells. This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains. The exact signal peptide cleavage site was elucidated by mass spectrometry of virus-derived and recombinant GP5. The results revealed that the signal peptide of GP5 is cleaved at two sites. As a result, a mixture of GP5 proteins exists in virus particles, some of which still contain the “decoy epitope” sequence. Heterogeneity was also observed for the use of glycosylation sites in the hypervariable region. Lastly, GP5 mutants were engineered where one of the signal peptide cleavage sites was blocked. Wildtype GP5 exhibited exactly the same SDS-PAGE mobility as the mutant that is cleavable at site 2 only. This indicates that the overwhelming majority of all GP5 molecules does not contain the “decoy epitope”.
doi:10.1371/journal.pone.0065548
PMCID: PMC3675037  PMID: 23755249
25.  Human Rotavirus VP6-Specific Antibodies Mediate Intracellular Neutralization by Binding to a Quaternary Structure in the Transcriptional Pore 
PLoS ONE  2013;8(5):e61101.
Several live attenuated rotavirus (RV) vaccines have been licensed, but the mechanisms of protective immunity are still poorly understood. The most frequent human B cell response is directed to the internal protein VP6 on the surface of double-layered particles, which is normally exposed only in the intracellular environment. Here, we show that the canonical VP6 antibodies secreted by humans bind to such particles and inhibit viral transcription. Polymeric IgA RV antibodies mediated an inhibitory effect against virus replication inside cells during IgA transcytosis. We defined the recognition site on VP6 as a quaternary epitope containing a high density of charged residues. RV human mAbs appear to bind to a negatively-charged patch on the surface of the Type I channel in the transcriptionally active particle, and they sterically block the channel. This unique mucosal mechanism of viral neutralization, which is not apparent from conventional immunoassays, may contribute significantly to human immunity to RV.
doi:10.1371/journal.pone.0061101
PMCID: PMC3650007  PMID: 23671563

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