Characterization of the apoptotic properties of severe acute respiratory syndrome coronavirus (SARS-CoV) structural proteins

by Chow, Yan-ching

(Uncorrected OCR) Abstract of thesis entitled CHARACTERIZATION OF THE APOPTOTIC PROPERTIES OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS (SARS-CoV) STRUCTURAL PROTEINS Submitted by Yan Ching Ken CHOW for the degree of Master of Philosophy at The University of Hong Kong in August 2004 The sudden outbreak of Severe Acute Respiratory Syndrome (SARS) in 2002 prompted the establishment of a global scientific network subsuming most of the traditional rivalries in the competitive field of virology. Although the rapid identification of the causative agent ?SARS-Coronavirus (SARS-CoV), represents an important breakthrough, our understanding of this deadly virus remains limited. For example, infection of SARS-CoV causes extensive cytopathic effects in Vero E6 cells through apoptosis, but the underlying mechanisms and the responsible viral protein(s) remain to be elucidated. As part of this study, screening of putative pro-apoptotic protein(s) encoded by the SARS-CoV genome including all the putative structural genes of the virus was described. In vitro expression of the genes mediated by recombinant vaccinia virus or adenovirus showed that only the full length and the S2 domain of the spike protein induced apoptosis in Vero E6 cells in a time- and dosage-dependent manner, as monitored by cell viability, chromatin condensation and DNA fragmentation of the infected cells. In contrast, expression of S1 protein, E protein, M protein and N protein did not induce measurable apoptosis. Recombinant adenovirus-mediated expression of S2 also induced apoptosis in BHK-21 and primary splenocyte culture of BALB/c mice but not in HeLa cells, indicating that the S2-induced cell death is not universal among different mammalian cell lines. In an attempt to more precisely map the apoptotic domain within the S2 gene, it was found that a deletion of 324 amino acids from the C-terminus did not abolish the S2- induced apoptosis in Vero E6 cells but a further 107 amino acids deletion did, indicating the potential apoptotic domain in S2. More strikingly, bioinformatics analysis revealed that such a region is structurally conserved when comparing with the caspase-recruitment domain (CARD). These data suggested that the SARS-CoV S-gene is a multifunctional viral protein possessing apoptotic property, in addition to its known roles in viral-cell fusion and viral entry. The results of the current study provide valuable information on the molecular pathogenesis of SARS and may therefore permit better strategies on anti-viral therapy and vaccine design against the disease to be developed.