Molecular analysis of cell entry of SARS coronavirus

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SARS coronavirus (SARS-CoV) is believed to bind with its spike (S) to the receptor, angiotensin-converting enzyme2 (ACE2) expressed on susceptible target cells. After binding, virion is transport to an endosome where the S protein is cleaved by proteases, most likely cathepsin-L, which results in the fusogenic activation of the S protein. Then, viral envelope fuses with endosomal membrane and viral genetic material enters into cells. This is an entry mechanism proposed by Bates and his colleagues from the following 2 findings. First, SARS-CoV infection is inhibited by lysosomotropic agent, indicating that. SARS-CoV takes endosomal pathway and low pH environment in the endosome is critical for entry. Secondly, SARS-CoV infected cells undergo fusion when treated by trypsin that induces cleavage of the S protein, however, those cells do not form fusion when treated with low pH buffer. If the hypothesis proposed by Bates et al is correct, then the viruses with cleaved S protein that shows … More the fusion of infected cells can enter into cells from plasma membrane. To verify this possibility, we have made pseudotype VSV that harbors cleaved S protein of SARS-CoV. On the S protein of SARS-CoV, there are 3 regions similar in amino acid sequence to putative sites to be cleaved by furin. We have made mutations in these regions, so that these regions are cleaved by furin and expressed those mutated S proteins on ACE2 expressing cells. Among three mutant S proteins, one with a mutation at amino acid 795-797 of SARS-S protein (C3) induced fusion in cells in the absence of trypsin that induces cleavage of SARS-S protein, though other two mutants and wild type (wt) S failed to do so. One of these mutated S protein as well as wt S protein induced fusion in the presence of trypsin. Pseudotype viruses with C3 or wt S protein were examined for their infection in the presence of bafilomycin, lysosomotropic agent, as well as inhibitors for cathepsin-L The infection of pseudotype with C3 S protein was not inhibited with those agents, but pseudotype with wt S protein was blocked by those reagents. These results suggest that pseudotype with SARS-CoV S protein cleaved at 795-797 could enter into cells directly from cell surface, while wt S pseudotype took an endosomal pathway. These results are not in disagreement with the hypothesis drawn by Bates and his co-workers.Heptad repeat peptide (HRP) of a number of enveloped viruses is generally used to block the virus entry. HRP of SARS-CoV was also reported to exhibit the inhibitory effects upon infection, however, inhibition was not as efficient as that in human immunodeficiency virus (HIV) or even remarkably lower than murine coronavirus MHV infection. Since HRP did not block the SARS-CoV infection via endosomal pathway, we examined in this study whether HRP blocks the entry from cell surface. We have obtained the data that although HRP did not work to prevent the infection via endosomal pathway, it blocked efficiently the infection of SARS-CoV from cell surface. This study showed the possible application of HRP as an anti-viral compound for SARS therapy. Less