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G0/G1 arrest and apoptosis induced by SARS-CoV 3b protein in transfected cells
© Yuan et al; licensee BioMed Central Ltd. 2005
Received: 30 April 2005
Accepted: 17 August 2005
Published: 17 August 2005
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 [1–3]. SARS was found to be caused by a novel coronavirus which was designated as SARS coronavirus (SARS-CoV), and the genome of SARS-CoV contains 11 to 14 open reading frames (ORF) and 5 to 8 potential non-structural 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 . With bioinformatics analysis, using the PSORT II server, it was shown that C- or N-terminal signal peptide, coiled-coil regions and trans-membrane region allocation were not detected, however, two potential nuclear localization signals (NLS) were predicted. The cellular localization of 3b protein by confocal microscopy was performed and the nucleolus localization was confirmed. And the nucleolus localization signal sequences of 3b protein may localize in the C-terminal regions from 134 to 154 amino acids .
Next, using similar method, we investigated the effects of p3b/EGFP-N1 on cell cycles in 293 and Vero cells. In these cells, the p3b/EGFP-N1 positive cells were arrested at G0/G1 phase compared with negative ones after transfection (55.25% vs. 37.17% in 293 cells and 82.27% vs. 56.00% in Vero cells at 48 h), but the percentages of sub-G1 phase in positive and negative cells were all too low and comparable. These data indicated that the role of 3b protein in inducing cell cycle G0/G1 phase arrest was a conserved character, but the apoptosis induction of 3b protein might be a cell type specific.
Published data showed that massive necrosis was found in lung, spleen and lymph nodes in SARS patients. As compared with normal tissues, apoptosis cells increased significantly in the spleen, liver, lung, and lymph nodes of SARS patients. The apoptosis cells were further demonstrated to be pneumocytes, lymphocytes, and monocytes [10, 11]. Taken together, the data we presented here, as well as the apoptosis and necrosis data of SARS patients, suggest that 3b is an apoptosis-related gene in SARS genome, which induce cell or tissue specific apoptosis in transfected cells.
We kindly thank Prof. Wei Kang for reading of the manuscript and Dr. Liu Hong-yan, Dr. Li Su-yan, Yao Zhen-yu, Wu Jie and Li Jian-yong for construction of some plasmids.
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