Functional Study of the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus

This dissertation, "Functional Study of the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus" by Lanying, Du, 杜蘭英, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled FUNCTIONAL STUDY OF THE SPIKE PROTEIN OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS Submitted by DU Lanying For the Degree of Doctor of Philosophy At the University of Hong Kong In July 2007 Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by SARS coronavirus (SARS-CoV). Although SARS appears to have been successfully contained, there is still a risk for its reemergence due to sporadic laboratory accidents or the presence of the natural reservoir for SARS-CoV-like virus. Therefore, the development of effective vaccines and antiviral therapeutics against SARS-CoV continues to be the current focus of SARS research. Among the four structural proteins of SARS-CoV, the spike (S) protein plays pivotal roles in viral infection and pathogenesis by recognizing and binding to host receptors. The conformational changes induced in this viral protein then facilitate fusion between the viral envelope and the host cell membranes, resulting in viral entry into the host cells. Thus, the aims of this study are to identify functional regions of the S protein and to develop SARS vaccines and antiviral agents based on the identified region(s). Three functional regions of the S protein have been identified as potential targets for development of vaccines and antiviral agents. They are: (1) the receptor binding domain (RBD) in S1, (2) the S1 and S2 potential cleavage region, and (3) the heptad repeat 2 (HR2) in S2. Based on a protein or a viral vector encoding the RBD of SARS-CoV S, two candidate vaccines, respectively named RBD-Fc and RBD-rAAV, were constructed and their immune responses and protective immunity were evaluated in a mouse model. It was shown that both RBD-Fc and RBD-rAAV elicited systemic and/or local antibody immune responses with neutralizing activity, thereby protecting vaccinated mice from subsequent SARS-CoV challenge. This study indicates the possibility of developing vaccines based on the RBD of SARS-CoV S protein. It was also demonstrated that a protease inhibitor, Ben-HCl, inhibited the infection of SARS-CoV and its pseudovirus. In addition, the protease Factor Xa, a target of Ben-HCl, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits and the cleavage correlated with the infectivity of the pseudovirus. Furthermore, two synthetic peptides, which correspond to the sequences proximal to the potential S1 and S2 cleavage site, inhibited SARS-CoV infectivity by over 99.99%, indicating that this critical region may be influential in viral infection. The above results suggest that compounds targeting the cleavage region, such as protease inhibitors, antiviral peptides and others, can be developed into effective anti-SARS- CoV agents. It was further discovered that one synthetic peptide derived from the HR2 region of SARS-CoV S protein was able to effectively suppress SARS-CoV infection. The S2 domain of SARS-CoV S protein contains HR1 and HR2 regions, which are proposed to form a coiled-coil structure important for virus-host cell membrane fusion. The antiviral activity mediated by this synthetic peptide maybe due to the peptide blocking the interaction of HR1 and HR2, thereby interrupting the membrane fusion. Taken together, this study has provided a rationale for development of novel SARS vaccines and antiviral therapies in the preventio