Herpes simplex virus infects cells through a multi-step process involving virus attachment to the cell plasma membrane followed by fusion of the virus envelope with the membrane resulting in penetration of the nucleocapsid into the cytoplasm. Two HSV envelope glycoproteins, gB and gD, are required for penetration. Both glycoproteins also play a role in syncytia formation, an aberrant form of virus-mediated cell-cell membrane fusion. The overall goal of this proposal is to fine map mutations in these glycoprotein genes which affect virus penetration and syncytia formation and to identify critical molecular structures which mediate these functions. The genetics of virus penetration and syncytia formation will be explored to determine whether these two processes are different manifestations of the same biological activity. A combination of deletion, linker insertion and bisulfite mutagenesis strategies will be used to saturate biologically important domains in these molecules with mutations. Mutant glycoprotein genes will be introduced into a specialized recipient viral genome using the P1 cre-lox site specific recombination system in a shuttle vector. Lethal and nonlethal mutations will be studied during replication on complementing and noncomplementing cell lines. Studies of gB will define functionally important structures within the external, transmembrane and cytoplasmic domains. Phenotypes to be explored include penetration minus, altered dimer formation, complementation inhibition, altered rate of entry, mab resistance, temperature sensitivity and syncytial plaque formation. Cooperativity among syn gene products will be examined using transient assays. For gD, linker insertion mutagenesis will be initiated across the whole gene to search for functionally important domains. Emphasis will be on mutations which result in the penetration minus phenotype. Virus neutralizing gD-specific mabs block virus penetration. Thus, neutralizing determinants on gD will be mapped by studies of the reactivity patterns of mAbs with a panel of gD truncated polypeptides, linker insertion mutants and by DNA sequencing of mar mutants. Finally, structures within gD that block virus penetration when the glycoprotein is expressed in transformed cell membranes will e defined, using a panel of cell lines transformed with a novel retrovirus vector carrying mutant forms of gD. Together, these studies of gB and gD should genetically map and uncover essential structures that contribute to virus penetration and syncytia formation.
