Severe Acute Respiratory Syndrome (SARS) is a recently emerged viral disease caused by a coronavirus (SARS-CoV). In contrast to the situation with other viral systems, there remain many unanswered questions regarding the process of SARS-CoV entry into host cells, especially with regard to membrane fusion events. While SARS-CoV has not undergone re-emergence since the initial outbreak, there remains a continued threat of further zoonotic outbreaks of SARS-CoV, or other animal coronaviruses. A detailed understanding of virus entry will be essential in our ability to respond to future outbreaks. We propose two specific aims: 1) To determine the role of a proteolytic cleavage site in the SARS-CoV S2 domain controlling membrane fusion. Our Preliminary Results indicate that a novel proteolytic cleavage site 793-KPTKR-797 within the S2 domain of the SARS coronavirus (SARS-CoV) S protein is instrumental in controlling viral fusion. Mutation of basic residues in this region leads to a loss of trypsin-mediated fusion and the introduction of a furin recognition site in this position allows fusion in the absence of ACE2 (the SARS-CoV receptor) suggesting an important role in host range. We have also shown that cleavage at R797 also primes membrane fusion via the lung-expressed serine proteases TMPRSS2 and HAT. We propose a comprehensive mutagenic and biochemical study of the S2 cleavage site (S2'), with the goal of understanding how selective cleavage of the SARS-CoV S protein by different proteases regulate S priming and fusion activation during virus entry. 2) To characterize the role of neutrophil elastase on SARS-CoV activation via cleavage within the S2 domain. It is also known that SARS-CoV entry can be activated by elastase, a mechanism of infection enhancement that is especially important in the context of the pronounced inflammatory response seen in the lungs of SARS-CoV-infected individuals. Our Preliminary Results indicate that neutrophil elastase also cleaves in the vicinity of the S2'cleavage site, at residue T795. We propose a comprehensive study of the elastase cleavage site at S2', with the goal of understanding how cleavage of the SARS-CoV S protein by elastase impacts virus entry and fusion. This project has a focus on the SARS-CoV because of its high priority status;however our work is likely to be directly applicable to other virus systems. Overall our studies will reveal critical features of SARS-CoV fusion activation and host range. Notably the presence of a cleavage site within S2 are very highly conserved across the Coronaviridae, and are likely to be universal features;with the fusion reaction primed by different proteases depending on the individual virus and circumstances. These studies will provide a model for both pathogenesis of SARS-CoV, as well as an understanding how coronaviruses might overcome species-specificity and emerge into new hosts.
Coronaviruses have recently received much attention as agents of infectious disease, due to the outbreak of severe acute respiratory syndrome (SARS) in the spring of 2003. While the SARS-coronavirus has not undergone re-emergence since the initial outbreak, there remains a continued threat of further outbreaks of SARS-CoV, or of other novel coronaviruses. The ability of the SARS-coronavirus to recognize and undergo membrane fusion with host cells is a major factor in its host range. Our studies are designed to elucidate the molecular details of SARS-coronavirus fusion and entry, which will serve as a model for both pathogenesis of SARS-CoV and the development of new antiviral drugs, as well as to help us understand how coronaviruses might overcome species-specificity and emerge into new hosts.
