Glycoprotein B (gB) of human cytomegalovirus (CMV) is the most abundant component of the virus envelope, a target of neutralizing antibodies and a key gene product that possesses potential fusogenic domains and cellular adhesion sequences. The long term goal of our research is to define the molecular interactions of CMV envelope proteins with cellular components that mediate critical steps in the lifecycle such as entry, cell to cell spread and virion maturation/egress. The specific goal of this proposal is to define the structure/function relationship of CMV gB. In the previous funding period, we learned that gB is an essential gene product required for infection of cells. We also discovered that gB is a authentic ligand for CMV, binding to one or more classes of receptors on the surface of cells. Engagement of gB with its receptor(s) results in dramatic cellular responses such as intracellular signaling and induction of gene expression, all of which are also attributable to the virus. In this funding period, we will extend our novel findings and exploit the collection of reagents and functional assays we have developed. In the first aim, genetic, cell biological and biochemical approaches will be used to identify the gB receptor. In the second aim, we will functionally characterize a large panel of gB mutants produced in the previous funding period. Our collection of linker-insertion mutants which uniformly spans the gB ORF has been characterized with respect to higher order structure, protein folding and intracellular trafficking. Mutants lacking gross structural defects will be tested for their ability to perform ligand, signaling and fusion functions. The mutant proteins will be appraised in isolation, via pseudotyping procedures or by analysis of purified proteins, and they will be assessed in the context of the viral genome by recombining the mutants into a gB null background.
The final aim of the proposal seeks to define gB's structure. The disulfide bond structure will be determined by biochemical means. In addition, resolution of the gB structure will be resolved at the atomic level via a collaboration with Dr. Peter Kim at Massachusetts Institute of Technology. These experiments will yield the first structure/functional map of any CMV envelope protein. Since gB is an identified target of vaccine and antiviral strategies, our research findings will be of great significance in efforts to treat and prevent GMV disease.
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