Human cytomegalovirus (HCMV) is a ubiquitous, usually benign virus. Nevertheless, HCMV frequently contributes to rejection of organs in transplant patients and causes systemic disease and defects in the development of the CNS in neonates. HCMV infects many different cell types including epithelial and endothelial, glial cells, fibroblasts and monocyte-macrophages. This broad tropism is facilitated by a capacity to enter different cell types via distinct entry pathways involving different viral glycoproteins including: gH/gL/UL128-131, denoted the pentamer, gH/gL/gO, the trimer. Our model for how HCMV enters epithelial and endothelial cells suggests that HCMV trimers bind to cell surface receptors, e.g. PDGFRα viruses are internalized and pentamer acts in endosomes to promote gB-mediated fusion in fibroblasts, then of the virion envelope with cellular membranes. There is a third form of gH/gL, a complex of gH/gL with gB, gB-gH/gL and we do not know whether gB-gH/gL promotes in virus entry. Given their importance in virus entry, trimer and pentamer are also important targets of antibodies (Abs) and are considered key players in the design of HCMV vaccines. Four aims are proposed: Aim 1. To characterize pathways of HCMV entry into fibroblasts and epithelial and endothelial cells and determine where trimer and pentamer function. This aim will test the hypothesis that trimer binding to cellular receptors leads to cell surface traffic followed by internalization of virus particles into cells and downstream pentamer-mediated effects promoting virus exit from endosomes into the cytoplasm. Aim 2. To determine the structures of trimer and trimer:PDGFRα, define trimer structure/function relationships and identify other trimer receptors. We have a preliminary structure of trimer with its receptor PDGFR We will extend these structural studies and use site directed mutants to test function. Other studies will identify trimer receptors important for entry into epithelial and endothelial cells. Aim 3. To investigate how HCMV gB-gH/gL complexes function. We will test the hypothesis that gB- gH/gL is important for HCMV entry by using mutant forms of gB and gH/gL to block assembly of gB-gH/gL. Aim 4. To characterize trimer- specific Abs in human sera and compare to pentamer Abs. We made striking observations that trimer- and pentamer-specific Abs in human sera synergize to neutralize HCMV. We will extend these studies characterize the prevalence and potency of trimer-specific Abs and identify the epitopes in trimer recognized by these Ab.