The genome of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains four structural genes that are homologous to genes found in other coronaviruses, and also contains six subgroup-specific open reading frames (ORFs). Expression of one of these subgroup-specific genes, ORF7a, resulted in apoptosis via a caspase-dependent pathway. Here, we observed that transient expression of ORF7a protein fused with myc or GFP tags at its N or C terminus inhibited cell growth and prevented BrdU incorporation in different cultural cells, suggesting that ORF7a expression may regulate cell cycle progression. Analysis by flow cytometry demonstrated that ORF7a expression was associated with blockage of cell cycle progression at G0/G1 phase in HEK 293 cells after 24 to 60 h post-transfection. Similar results were observed in COS-7 and Vero cells. Mutation analysis of ORF7a revealed that the domain spanning aa 44-82 of 7a protein was essential for its cytoplasmic localization and for induction of the cell cycle arrest. After analyzing the cellular proteins involving in regulation of cell cycle progression, we demonstrated that ORF7a expression was correlated with a significant reduction of cyclin D3 level of mRNA transcription and expression, and phosphorylation of retinoblastoma (Rb) protein at ser795 and ser809/811, not with the expression of cyclin D1, D2, cdk4 and cdk6 in HEK 293 cells. These results suggest that the insufficient expression of cyclin D3 may cause a decreased activity of cyclin D/cdk4/6, resulting in the inhibition of Rb phosphorylation. Accumulation of hypo- or non-phosphorylated pRb thus prevents cell cycle progression at G0/G1 phase.

SARS-CoV 3a protein was a unique protein of SARS coronavirus (SARS-CoV), which was identified in SARS-CoV infected cells and SARS patients' specimen. Recent studies revealed that 3a could interact specifically with many SARS-CoV structural proteins, such as M, E and S protein. Expressed 3a protein was reported to localize to Golgi complex in SARS-CoV infected cells. In this study, it was shown that 3a protein was mainly located in Golgi apparatus with different tags at N- or C-terminus. The localization pattern was similar in different transfected cells. With the assay of truncated 3a protein, it was shown that 3a might contain three transmembrane regions, and the second or third region was properly responsible for Golgi localization. By ultra-centrifugation experiment with different extraction buffers, it was confirmed that 3a was an integral membrane protein and embedded in the phospholipid bilayer. Immunofluorescence assay indicated that 3a was co-localized with M protein in Golgi complex in co-transfected cells. These results provide a new insight for further study of the 3a protein on the pathogenesis of SARS-CoV.

SARS-CoV 3a is a structural protein, mainly localizing to Golgi apparatus and co-localizing with SARS-CoV M in co-transfected cells. Here we observed that transient expression of 3a inhibited cell growth and prevented 5-bromodeoxyuridine incorporation, suggesting that 3a deregulated cell cycle progression. Cell cycle analysis demonstrated that 3a expression was associated with blockage of cell cycle progression at G1 phase in HEK 293, COS-7, and Vero cells 24-60 h after transfection. Mutation analysis of 3a revealed that C-terminal region (176 aa approximately 274 aa), including a potential calcium ATPase motif, was essential for induction of cell cycle arrest. Topological analysis showed that 3a predominantly located in Golgi apparatus, with its N-terminus residing in the lumen (Nlum) and C-terminus in the cytosol (Ccyt). Analyzing the cellular proteins involving in regulation of cell cycle progression, we demonstrated that 3a expression was correlated with a significant reduction of cyclin D3 level and phosphorylation of retinoblastoma (Rb) protein at Ser-795 and Ser-809/811, not with the expression of cyclin D1, D2, cdk4, and cdk6 in 293 cells. Increases in p53 phosphorylation on Ser-15 were observed in both SARS-CoV M and 3a transfected cells, suggesting that it might not correlate with the 3a-induced G0/G1 phase arrest. The reduction of cyclin D3 level and phosphorylation of Rb were further confirmed in SARS-CoV infected Vero cells. These results indicate that SARS-CoV 3a protein, through limiting the expression of cyclin D3, may inhibit Rb phosphorylation, which in turn leads to a block in the G1 phase of the cell cycle and an inhibition of cell proliferation.

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Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), cause of the life-threatening atypical pneumonia, infects many organs, such as lung, liver and immune organ, and induces parenchyma cells apoptosis and necrosis. The genome of SARS-CoV, not closely related to any of the previously characterized coronavirus, encodes replicase and four major structural proteins and a number of non-structural proteins. Published studies suggest that some non-structural proteins may play important roles in the replication, virulence and pathogenesis of viruses. Among the potential SARS-CoV non-structural proteins, 3b protein (ORF4) is predicted encoding 154 amino acids, lacking significant similarities to any known proteins. Till now, there is no report about the function of 3b protein. In this study, 3b gene was linked with the EGFP tag at the C-terminus. Through cell cycle analysis, it was found that over-expression of 3b-EGFP protein in Vero, 293 and COS-7 cells could induce cell cycle arrest at G0/G1 phase, and that especially in COS-7 cells, expression of 3b-EGFP was able to induce the increase of sub-G1 phase from 24 h after transfection, which was most obvious at 48 h. The apoptosis induction of 3b fusion protein in COS-7 cells was further confirmed by double cell labeling with 7-AAD and Annexin V, the function of 3b protein inducing cell G0/G1 arrest and apoptosis may provide a new insight for further study on the mechanism of SARS pathogenesis.The outbreak of Severe Acute Respiratory Syndrome (SARS) posed a great global threat. SARS is a system disease which impairs many organs, such as lung, liver and immune organ. Respiratory distress and decreased immune function are the main causes of SARS patient death [1][2][3]. SARS was found to be caused by a novel coronavirus which was designated as SARS coronavirus (SARSCoV), and the genome of SARS-CoV contains 11 to 14 open reading frames (ORF) and 5 to 8 potential nonstructural proteins [4,5]. The virus non-structural proteins, which vary widely among different coronavirus species, are dispensable for virus replication. It has been known that some non-structural proteins play important roles in virulence and pathogenesis, such as X protein of hepatitis B virus and ORF 8 protein of bovine herpes virus 1U(S) [6,7]. SARS-CoV 3b (ORF4) (ZJ01, AY297028) encodes a 154-amino-acid protein, lacking significant similarities to any previously known proteins [8]. With bioinformatics analysis, using the PSORT II server, it was shown that C-or Nterminal signal peptide, coiled-coil regions and transmembrane region allocation were not detected, however, two potential nuclear localization signals (NLS) were Induction of cell cycle arrest and cell apoptosis by 3b protein expression Figure 1 Induction of cell cycle arrest and cell apoptosis by 3b protein expression. p3b/EGFP-N1 plasmid was transfected into COS-7 cells, and the DNA contents of cells were measured by flow cytometry. EGFP expression positive and negative cells were gated with forward scatter (on the left row). The midd...