Herpes simplex virus 1 (HSV-1) is a pathogen which primarily infects the oral mucosa. Glycoprotein B (gB) plays an essential role in HSV infection and is highly conserved among different herpesviruses. It is component of the virion envelope, elicits high titers of neutralizing antibody, functions in penetration of virions into host cells (ts mutation), and evidence suggests that gB forms complexes with submembrane proteins (syn mutation). Analyses of the nucleotide sequence and hydropathicity of gB predict that it contains an extracellular hydrophilic domain, a hydrophobic transmembrane region, and a charged carboxyl terminus projecting into the cytoplasm. Studies are designed to elucidate the requirements for the various functions of gB and to verify aspects of the structure of the molecule focusing on the extracellular region and the carboxyl terminus. 1) We are continuing to map antigenic and functional domains on the hydrophilic region of gB by marker rescue of mutants resistant to potent neutralizing monoclonal antibodies. Cognitive sites of antibodies will be mapped by reconstructing the gB with small, in-frame deletions using a panel of DNaseI deletions clones. 2) We will map the boundies of the domain of the syn locus at the charged carboxyl terminus of mutants in gB. By inducing compensatory mutations across the genome, genes encoding proteins which form complexes with gB will be identified by marker rescue with wild type DNA fragments. 3) Contributions of specific N- linked glycosylation sites to the structure, function, and transport of gB will be studied using site-directed mutagenesis to alter the consenus sequence for carbohydrate addition. gB constructs will be expressed in cells containing a resident HSV-1 alpha 4 gene by transfecting with DNA of plasmids carrying the gB gene linked to the dihydrofolate reductase gene resistant to methotrexate. Analysis of gB functions in virions will be carried out by superinfecting cells producing gB constitutively with conditional lethal mutants in gB or by recombining gB constructs into the viral genome. Ultimately, these data will contribute to our understanding of the functional topology of the gB molecule.
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