Severe acute respiratory syndrome coronavirus accessory protein 6 is a virion-associated protein and is released from 6 protein-expressing cells

Analysis of severe acute respiratory syndrome coronavirus (SCoV) by either sucrose gradient equilibrium centrifugation or a virus capture assay using an anti-SCoV S protein antibody demonstrated that the SCoV 6 protein, which is one of the accessory proteins of SCoV, was incorporated into virus particles. Coexpression of the SCoV S, M, E, and 6 proteins was sufficient for incorporation of the 6 protein into virus-like particles. Cells transfected with plasmid expressing the 6 protein released SCoV 6 protein; however, infected cells released SCoV 6 protein only in association with SCoV particles.     

Severe acute respiratory syndrome coronavirus 3a protein is a viral structural protein

The present study showed the association of a severe acute respiratory syndrome coronavirus (SCoV) accessory protein, 3a, with plasma membrane and intracellular SCoV particles in infected cells. 3a protein appeared to undergo posttranslational modifications in infected cells and was incorporated into SCoV particles, establishing that 3a protein was a SCoV structural protein.     

Severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein

Severe acute respiratory syndrome coronavirus (SCoV) 7a protein is one of the viral accessory proteins. In expressing cells, 7a protein exhibits a variety of biological activities, including induction of apoptosis, activation of the mitogen-activated protein kinase signaling pathway, inhibition of host protein translation, and suppression of cell growth progression. Analysis of SCoV particles that were purified by either sucrose gradient equilibrium centrifugation or a virus capture assay, in which intact SCoV particles were specifically immunoprecipitated by anti-S protein monoclonal antibody, demonstrated that 7a protein was associated with purified SCoV particles. Coexpression of 7a protein with SCoV S, M, N, and E proteins resulted in production of virus-like particles (VLPs) carrying 7a protein, while 7a protein was not released from cells expressing 7a protein alone. Although interaction between 7a protein and another SCoV accessory protein, 3a, has been reported, 3a protein was dispensable for assembly of 7a protein into VLPs. S protein was not required for the 7a protein incorporation into VLPs, and yet 7a protein interacted with S protein in coexpressing cells. These data established that, in addition to 3a protein, 7a protein was a SCoV accessory protein identified as a SCoV structural protein.     

Severe acute respiratory syndrome coronavirus protein 7a interacts with hSGT

Severe acute respiratory syndrome coronavirus (SARS-CoV) 7a is an accessory protein with no known homologues. In this study, we report the interaction of a SARS-CoV 7a and small glutamine-rich tetratricopeptide repeat-containing protein (SGT). SARS-CoV 7a and human SGT interaction was identified using a two-hybrid system screen and confirmed with interaction screens in cell culture and cellular co-localization studies. The SGT domain of interaction was mapped by deletion mutant analysis and results indicated that tetratricopeptide repeat 2 (aa 125-158) was essential for interaction. We also showed that 7a interacted with SARS-CoV structural proteins M (membrane) and E (envelope), which have been shown to be essential for virus-like particle formation. Taken together, our results coupled with data from studies of the interaction between SGT and HIV-1 vpu indicated that SGT could be involved in the life-cycle, possibly assembly of SARS-CoV.     

The severe acute respiratory syndrome coronavirus 3a is a novel structural protein

The severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein is one of the opening reading frames in the viral genome with no homologue in other known coronaviruses. Expression of the 3a protein has been demonstrated during both in vitro and in vivo infection. Here we present biochemical data to show that 3a is a novel coronavirus structural protein. 3a was detected in virions purified from SARS-CoV infected Vero E6 cells although two truncated products were present predominantly instead of the full-length protein. In Vero E6 cells transiently transfected with a cDNA construct for expressing 3a, a similar cleavage was observed. Furthermore, co-expression of 3a, membrane and envelope proteins using the baculovirus system showed that both full-length and truncated 3a can be assembled into virus-like particles. This is the first report that demonstrated the incorporation of 3a into virion and showed that the SARS-CoV encodes a novel coronavirus structural protein.     

The severe acute respiratory syndrome coronavirus 3a protein up-regulates expression of fibrinogen in lung epithelial cells

Here we analyzed the gene expression profile of cells that stably express the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein to determine its effects on host functions. A lung epithelial cell-line, A549, was chosen for this study because the lung is the primary organ infected by SARS-CoV and fatalities resulted mainly from pulmonary complications. Our results showed that the expression of 3a up-regulates the mRNA levels of all three subunits, Aalpha, Bbeta, and gamma, of fibrinogen. Consequently, the intracellular levels as well as the secretion of fibrinogen were increased. We also observed increased fibrinogen levels in SARS-CoV-infected Vero E6 cells.     

Mitochondrial location of severe acute respiratory syndrome coronavirus 3b protein

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a distant member of the Group 2 coronaviruses, has recently been identified as the etiological agent of severe acute respiratory syndrome (SARS). The genome of SARS-CoV contains four structural genes that are homologous to genes found in other coronaviruses, as well as six subgroup-specific open reading frames (ORFs). ORF3 encodes a predicted 154-amino-acid protein that lacks similarity to any known protein, and is designated 3b in this article. We reported previously that SARS-CoV 3b is predominantly localized in the nucleolus, and induces G0/G1 arrest and apoptosis in transfected cells. In this study, we show that SARS-CoV 3b fused with EGFP at its N- or C- terminus co-localized with a mitochondria-specific marker in some transfected cells. Mutation analysis of SARS-CoV 3b revealed that the domain spanning amino acids 80 to 138 was essential for its mitochondria localization. These results provide new directions for studies of the role of SARS-CoV 3b protein in SARS pathogenesis.     

Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters

Small animal models are needed in order to evaluate the efficacy of candidate vaccines and antivirals directed against the severe acute respiratory syndrome coronavirus (SARS CoV). We investigated the ability of SARS CoV to infect 5-week-old Golden Syrian hamsters. When administered intranasally, SARS CoV replicates to high titers in the lungs and nasal turbinates. Peak replication in the lower respiratory tract was noted on day 2 postinfection (p.i.) and was cleared by day 7 p.i. Low levels of virus were present in the nasal turbinates of a few hamsters at 14 days p.i. Viral replication in epithelial cells of the respiratory tract was accompanied by cellular necrosis early in infection, followed by an inflammatory response coincident with viral clearance, focal consolidation in pulmonary tissue, and eventual pulmonary tissue repair. Despite high levels of virus replication and associated pathology in the respiratory tract, the hamsters showed no evidence of disease. Neutralizing antibodies were detected in sera at day 7 p.i., and mean titers at day 28 p.i. exceeded 1:400. Hamsters challenged with SARS CoV at day 28 p.i. were completely protected from virus replication and accompanying pathology in the respiratory tract. Comparing these data to the mouse model, SARS CoV replicates to a higher titer and for a longer duration in the respiratory tract of hamsters and is accompanied by significant pathology that is absent in mice. Viremia and extrapulmonary spread of SARS CoV to liver and spleen, which are seen in hamsters, were not detected in mice. The hamster, therefore, is superior to the mouse as a model for the evaluation of antiviral agents and candidate vaccines against SARS CoV replication.     

Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells

The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.     

Characterization of severe acute respiratory syndrome coronavirus membrane protein

The coronavirus membrane protein (M) is the key player in the assembly of virions at intracellular membranes between endoplasmic-reticulum and Golgi-complex. Using a newly established human monoclonal anti-M antibody we detected glycosylated and nonglycosylated membrane-associated M in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infected cells and in purified virions. Further analyses revealed that M contained a single N-glycosylation site at asparagine 4. Recombinant M was transported to the plasma membrane and gained complex-type N-glycosylation. In SARS-CoV infected cells and in purified virions, however, N-glycosylation of M remained endoglycosidase H-sensitive suggesting that trimming of the N-linked sugar side chain is inhibited.     

The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.     

Severe acute respiratory syndrome coronavirus gene 7 products contribute to virus-induced apoptosis

The proteins encoded by gene 7 of the severe acute respiratory syndrome coronavirus (SARS-CoV) have been demonstrated to have proapoptotic activity when expressed from cDNA but appear to be dispensable for virus replication. Recombinant SARS-CoVs bearing deletions in gene 7 were used to assess the contribution of gene 7 to virus replication and apoptosis in several transformed cell lines, as well as to replication and pathogenesis in golden Syrian hamsters. Deletion of gene 7 had no effect on SARS-CoV replication in transformed cell lines, nor did it alter the induction of early apoptosis markers such as annexin V binding and activation of caspase 3. However, viruses with gene 7 disruptions were not as efficient as wild-type virus in inducing DNA fragmentation, as judged by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, indicating that the gene 7 products do contribute to virus-induced apoptosis. Disruption of gene 7 did not affect virus replication or morbidity in golden Syrian hamsters, suggesting that the gene 7 products are not required for acute infection in vivo. The data indicate that open reading frames 7a and 7b contribute to but are not solely responsible for the apoptosis seen in SARS-CoV-infected cells.     

Severe acute respiratory syndrome coronavirus membrane protein interacts with nucleocapsid protein mostly through their carboxyl termini by electrostatic attraction

The severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein is an abundant virion protein, and its interaction with the nucleocapsid protein is crucial for viral assembly and morphogenesis. Although the interacting region in the nucleocapsid protein was mapped to residues 168-208, the interacting region in the membrane protein and the interaction nature are still unclear. In this work, by using yeast two-hybrid and surface plasmon resonance techniques, the residues 197-221 of the membrane protein and the residues 351-422 of the nucleocapsid protein were determined to be involved in their interaction. Sequence analysis revealed that these two fragments are highly charged at neutral pH, suggesting that their interaction may be of ionic nature. Kinetic assays indicated that the endodomain (aa102-221) of the membrane protein interacts with the nucleocapsid protein with high affinity (K(D)=0.55+/-0.04 microM), however, this interaction could be weakened greatly by acidification, higher salt concentration (400 mM NaCl) and divalent cation (50 mM Ca2+), which suggests that electrostatic attraction might play an important role in this interaction. In addition, it is noted that two highly conserved amino acids (L218 and L219) in the membrane protein are not involved in this interaction. Here, we show that electrostatic interactions between the carboxyl termini of SARS-CoV membrane protein and nucleocapsid protein largely mediate the interaction of these two proteins. These results might facilitate therapeutic strategies aiming at the disruption of the association between SARS-CoV membrane and nucleocapsid proteins.     

Severe acute respiratory syndrome coronavirus protein 6 accelerates murine hepatitis virus infections by more than one mechanism

The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes numerous accessory proteins whose importance in the natural infection process is currently unclear. One of these accessory proteins is set apart by its function in the context of a related murine hepatitis virus (MHV) infection. SARS-CoV protein 6 increases MHV neurovirulence and accelerates MHV infection kinetics in tissue culture. Protein 6 also blocks nuclear import of macromolecules from the cytoplasm, a process known to involve its C-terminal residues interacting with cellular importins. In this study, protein 6 was expressed from plasmid DNAs and accumulated in cells prior to infection by wild-type MHV. Output of MHV progeny was significantly increased by preexisting protein 6. Protein 6 with C-terminal deletion mutations no longer interfered with nuclear import processes but still retained much of the capacity to augment MHV infections. However, some virus growth-enhancing activity could be ascribed to the C-terminal end of protein 6. To determine whether this augmentation provided by the C terminus was derived from interference with nuclear import, we evaluated the virus-modulating effects of small interfering RNAs (siRNAs) directed against importin-beta mRNAs, which down-regulated classical nuclear import pathways. The siRNAs did indeed prime cells for enhanced MHV infection. Our findings indicated that protein 6-mediated nuclear import blocks augmented MHV infections but is clearly not the only way that this accessory protein operates to create a milieu conducive to robust virus growth. Thus, the SARS-CoV protein 6 accelerates MHV infections by more than one mechanism.     

Severe acute respiratory syndrome coronavirus protein 6 mediates ubiquitin-dependent proteosomal degradation of N-Myc(and STAT) interactor

Severe acute respiratory syndrome coronavirus(SARS-Co V) encodes eight accessory proteins, the functions of which are not yet fully understood. SARS-Co V protein 6(P6) is one of the previously studied accessory proteins that have been documented to enhance viral replication and suppress host interferon(IFN) signaling pathways. Through yeast two-hybrid screening, we identified eight potential cellular P6-interacting proteins from a human spleen c DNA library. For further investigation, we targeted the IFN signaling pathway-mediating protein, N-Myc(and STAT) interactor(Nmi). Its interaction with P6 was confirmed within cells. The results showed that P6 can promote the ubiquitin-dependent proteosomal degradation of Nmi. This study revealed a new mechanism of SARS-Co V P6 in limiting the IFN signaling to promote SARS-Co V survival in host cells.