Preliminary Research On The Structure And Function Of SARS-CoV 3a Protein

Severe acute respiratory syndrome (SARS) is an epidemic of respiratory caused by its pathogen SARS coronavirus (SARS-CoV). SARS-CoV 3a is a structural protein, mainly localizing to Golgi apparatus and co-localizing with SARS-CoV M. Here we observed transient expression of 3 a inhibited cell growth and prevented 5-Bromodeoxyuridine incorporation, suggesting 3a deregulated cell cycle progression. Cell cycle analysis demonstrated that 3 a expression was associated with blockage of cell cycle progression at G1 phase in HEK 293 after 24 to 60h post-transfection. It also found in COS-7 and Vero cells.To define the functional domain of 3 a protein for inducing cell cycle arrest, a series of truncated mutants were constructed. Mutation analysis of 3 a revealed that C-terminal region (176aa～274aa), 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). Finally, we made efforts to further explore the mechanism of 3a-induced G0/G1 cell cycle arrest. Analyzing the cellular proteins involving in regulation of cell cycle progression, we demonstrated that 3 a expression was correlated with a significant reduction of cyclin D3 level and phosphorylation of retinoblastoma (Rb) protein at ser795 and ser809/811, not with the expression of cyclin D1, D2, cdk4 and cdk6 in 293 cells. The reduction of cyclin D3 level and phosphorylation of Rb were further confirmed in SARS-CoV infected Vero cells.In conclusion, 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, and moreover, it may plays an important role in SARS-CoV induced pathogenesis.