): The murine coronaviruses include a large collection of different strains, with each strain infecting different tissues and thus causing a unique disease pattern. Diseases include hepatitis, gastroenteritis, and chronic encephalomyelitis, the latter serving as a model for human neurodegenerative disease. Tropism of the different viruses for distinct tissues is controlled in large part by structural variations within the virion spike glycoprotein. The spike is a complex oligomeric structure that mediates virus binding to specific cellular receptors as well as fusion of viral and cellular membranes. Fulfillment of these functions delivers viral genomes into cells, thereby establishing infection and subsequent disease. Because spike is central to several essential steps in viral infection, tropism and pathogenesis, the long-term objective is to describe the molecular mechanisms by which spike proteins carry out the functions required for genome delivery. The investigator will investigate how naturally occurring variation in the coronavirus spike protein impacts the efficiency of oligomerization, incorporation into virions, and ultimate genome delivery functions. Various spikes will be synthesized from cDNA in conjunction with components required for pseudovirion assembly and virion incorporation efficiencies will be measured. The applicant will determine how the same structural variations alter the kinetics of spike binding to its cellular receptor and spike-mediated membrane fusion. Using soluble forms of the cellular receptor, he will attempt to trigger conversion of the spikes into a fusion-active conformation. Fusion conformations will be identified by liposome binding assays, and putative conformational changes will be probed using spike-specific monoclonal antibodies. The use of site-directed mutants will allow them to identify regions on the receptors that are required to stimulate the formation of fusion conformations. These findings will be used to build a model depicting various stages of coronavirus entry into cells. Such models help to understand this complex biological process and additionally can be used to reveal new targets for therapeutic antiviral agents.
|Lewicki, Daniel N; Gallagher, Thomas M (2002) Quaternary structure of coronavirus spikes in complex with carcinoembryonic antigen-related cell adhesion molecule cellular receptors. J Biol Chem 277:19727-34|
|Krueger, D K; Kelly, S M; Lewicki, D N et al. (2001) Variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion. J Virol 75:2792-802|