Human cytomegalovirus (HCMV) is the major viral pathogen causing severe morbidity and mortality among patients with the acquired immunodeficiency syndrome (AIDS). Ocular HCMV disease may lead to destruction of the retina and blindness in up to 30 percent of those with AIDS. The initial events in virion infectivity and the mechanisms responsible for cell-to-cell spread of virus and fusion of HCMV-infected cells remain unknown. This research program focuses on understanding the functions of the envelope glycoproteins gB and gH in the transmission of HCMV infection in human cells of retinal origin--polarized retinal pigment epithelial (RPE) cells and retinoblastoma cells. These cells will be used as a model system for studying the molecular events in HCMV infection of retinal cell types and will elucidate the role of gB and gH in virion attachment, penetration, and spread of infection from cell to cell in the human retina.
Aim one focuses on analyzing the function of gB by constructing stable glial cell lines that constitutively produce mutated forms of gB. Mutations will be targeted to the functional region that promotes virion entry, spread of virus from cell to cell, and cell fusion. Selected constructs will be used to engineer cell lines; those expressing gB in the plasma membrane will be studied for altered fusion capacity. Viral mutants in which gB and gH have been disrupted, membrane will be studied for altered fusion capacity. Viral mutants in which gB and gH have been disrupted, complemented or replaced with mutant glycoproteins will be used to study the role of these glycoproteins in virion function.
Aim two focuses on studying HCMV infection in polarized RPE cells. The cell surface receptor for HCMV virions will be identified in the apical membrane of polarized RPE cells, using gB as a ligand that bind the surface receptor. To study the receptor, gB will used in protein overlay assays and in an affinity system as a protein that binds the receptor in polarized RPE cells labeled by domain-selective biotinylation. The domain on gB that interacts with the receptor will be studied using mutated forms of gB and viral mutants. Signals for polarized transport of gB to the apical and basolateral surfaces of polarized RPE cells will be investigated by transient expression of mutated gB constructs. Viral mutants in the gB gene will be used to study virion infectivity and the relationship between the polarity of glycoprotein transport and the polarity of virion release from polarized RPE cells. Transmission of infection to polarized RPE cells by HCMV-infected retinoblastoma cells, induced to express the phenotypic properties of differentiated cell types in the retina, will be investigated.
Aim three focuses on analyzing the function and transport of gH in gH-producing cell lines and the role of gH in infection of polarized RPE cells. Epitopes in functional regions will be mapped by site-directed mutagenesis of the gene. Viral mutants in gH will be constructed and recombinants with selected mutations in the gH gene will be analyzed for defects in infectivity and cell- to-cell transmission of infection to polarized RPE cells. Interactions between gH and proteins that aid in its transport to the cell surface will be investigated. Studies in this application will provide new insights into the molecular interactions between important HCMV glycoproteins and the membranes of retinal cells. Our results may lead to the identification of the receptor for HCMV in polarized RPE cells and yield new strategies for blocking the progression of HCMV disease in the eye, based on an understanding of virion infectivity and the transmission of infection in human cells of retinal origin.
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