Polyprotein 1a and 1b are cleaved by the internally encoded 3C-like protease (3CL) to release functional proteins necessary for virus replication 13. Coronavirus replicase gene encompasses two overlapping translation products, polyprotein 1a ( в€ј450 kD) and 1b ( в€ј750 kD), both of which are conserved in length and amino acid sequence with other coronavirus replicase proteins. Another major biological function of E protein is that it can induce apoptosis in E-protein-expressing cells 12.

Coexpression of coronavirus M protein and E protein results in the production of virus-like particles (VLPs), while expression of M protein alone does not produce VLPs 10, 11. These features make it to be a suitable candidate for raising neutralizing antibodies and diagnosis applications 8, 9.Д® protein is associated with coronavirus envelope formation. In addition, N protein in many coronaviruses is highly conserved, immunogenic, and abundantly expressed during infection. Nucleocapid (N) protein has multiple functions that are involved in providing nuclear-import signals, interfering cell process, virus replication and RNA package. However there is no experimental evidence to indicate that whether the spike protein of SARS-CoV is cleaved. The spike proteins of some coronaviruses have to be cleaved to become fusion competent (S1 and S2 subunits), but some others do not 7. Variations of S protein among strains of coronavirus are responsible for host range and tissue tropism 6. The first step of the infectious cycle of enveloped viruses is the interaction with cellular receptors and subsequent fusion with the cell membrane, which is mediated by spike protein. The spike protein, forming the в€ј180 kD glycoprotein projection on the viral surface, is crucial for viral attachment and entry into the host cell. All of them are crucial to form the architecture of SARS CoV, especially S and N protein, which may be the best candidates for therapeutic target of SARS. The structural proteins of SARS- CoV include: the surface spike (S) glycoprotein, the membrane (M) protein, the small envelope (E) glycoprotein and the nucleocapsid (N) protein 4, 5.

The genome of SARS-CoV has 29,727 nucleotides in length containing 11 open reading frames. SARS-CoV is now identified as a member of the coronavirus family of enveloped, positive-stranded RNA viruses, which have a broad host range. Accurate diagnosis and explorirg a tool to understand the pathogenesis of the disease are in urgent need 3. SARS spread has caused the unstability for the globe society and dramatically decreased the economy development over the world. More than 8000 probable SARS cases and 900 related deaths were reported. It apparently began in Guangdong province of China in November of 2002 and has spread to several countries 2. The severe acute respiratory syndrome (SARS), caused by SARS-associated coronavirus (SARS-CoV) 1, was recently identified as a new clinical entity. The data demonstrated that the cleavage of S protein was observed in the lysate, indicating that proteolytic processing of S protein is present in host cells. When S2 Ab was used to perform immune precipitation with lysate of SARS-CoV infected cells, a cleaved S2 fragment was detected with S2-specific mAb by Western blot analysis. Furthermore, the anti-S1 and S2 Abs were proved to be capable of binding to SARS CoV under electron microscope observation. Our results showed that the antibodies could efficiently and specifically bind to their corresponding proteins from E.coli expressed or lysate of SARS-CoV infected Vero-E6 cells by Western blot analysis. In order to investigate the maturation and proteolytic processing of the S protein of SARS CoV, we generated S1 and S2 subunit specific antibodies (Abs) as well as N, E and 3CL protein-specific Abs. Many studies have shown that the cleavage of spike protein seriously affects its function. In contrast, TGV, FIPV and HCoV-229E are not. Some spike proteins of coronavirus, such as MHV, HCoV-OC43, AIBV and BcoV, are proteolytically cleaved into two subunits, S1 and S2. It is essential for the interaction of the virons with host cell receptors and subsequent fusion of the viral envelop with host cell membrane to allow infection. Spike protein is one of the major structural proteins of severe acute respiratory syndrome-coronavirus.

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