Glycoprotein B (gB) of human cytomegalovirus (HCMV) 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. Envelope glycoproteins are primary determinants of tissue tropism and the mediators of virus entry, cell-cell spread and egress/maturation of virions. The long term goal of our research is to define the molecular interactions with HCMV envelope proteins and cellular components that mediate critical steps in the lifecycle such as entry, cell to cell spread and virion maturation/egress. We learned that gB is an essential gene product required for infection of cells. We discovered that gB is a authentic ligand for HCMV, binding to one or more classes of receptors on the surface of cells. The interaction of gB with its receptor(s) is biologically relevant since virus entry was blocked when gB binding sites were occupied. 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. We will further characterize the nature of the gB signaling pathway by defining the composition of signaling effectors activated in response to gB and the virus. These experiments will disclose receptor candidates that will be functionally tested using a newly identified HCMV entry-defective cell line. Expression cloning strategies are also designed to isolate a cDNA clone encoding an HCMV entry receptor. In preparation for structure/function analysis, we produced a large panel of generalized mutations throughout gB. All the mutants were characterized with respect to higher order structure, protein folding and intracellular trafficking. We have also developed a novel and innovative expression strategy to functionally test the mutants. A modified vesciular stomatitis virus lacking its single envelope gene and encoding a green fluorescent protein can be pseudotyped with gB and gB mutants. The concentrated, membrane-anchored display of this expression system has many experimental advantages and will be used to functionally characterize gB mutants in terms of ligand, signaling and fusogenic activities. These experiments will yield the first structure/functional map of any HCMV envelope protein. A third line of investigation will further characterize the role of a known HCMV and gB binding partner, annexin II. Annexin II lacks a demonstrated role in virus entry and we will test the hypothesis that the gB: annexin II interaction is important for egress and virion maturation using trans-dominant cell lines that are annexin II functionally deficient. 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 HCMV disease.
