Characterization Of Domains Within The Coronavirus Spike Glycoprotein Utilized For Entry

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Enveloped viruses utilize their spike glycoproteins to mediate entry into the target cells. The first step requires binding of the viral spike to the receptor on the surface of the target cells. The binding of the virus to the host receptor is further enhanced in the presence of attachment factors. The final process in the entry event requires the fusion of the host and cell membranes to deliver the viral payload. This is mediated by the fusion machinery of the viral spike and can be triggered by receptor binding, proteolytic activity and/or low pH. The virus family Coronavirdae is a representative of enveloped viruses that cause respiratory disease in a wide range of animal hosts. Coronaviruses like the severe acute respiratory syndrome (SARS) virus and the infectious bronchitis virus (IBV) represent some of the common coronaviruses that have had a negative impact on the health of humans and the poultry industry. It is therefore key that we understand the mechanisms by which these viruses gain entry into their hosts in order to develop specific therapeutics in the event of an outbreak. In this dissertation, we analyzed two domains within the S2 domain of the severe acute respiratory syndrome (SARS) virus spike protein. We employed a series of mutagenic analysis as well as fusion and pseudoviral infection assays to observe that not only are these domains conserved across the Coronaviridae, but they may function as a potential viral fusion peptide in one domain and a critical member of the fusion signaling process in another domain. We also investigated the entry requirements of the infectious bronchitis virus (IBV). This work combined FACS, fluorescence and competitive assays to observe the use of heparan sulfate as an attachment factor for the Beaudette strain of IBV. This work may further add to the rationale for the promiscuity of this lab-adapted strain of IBV because the virus can infect primary chicken cells as well as most cells exposed to the virus.

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