Herpesviruses are cofactors in the etiology of certain malignant proliferative diseases. In addition, most people become infected with one or more herpesviruses, which can persist indefinitely in quiescent state but then reactivate in immunosuppressed individuals, including cancer patients, to cause significant disease. The studies described here are directed toward understanding basic mechanisms of herpesvirus infection and spread and should lead to novel therapeutic approaches. The outer envelope of a herpesvirus is a membrane containing viral proteins. Virus entry into cells requires fusion of this membrane with a cell membrane. Spread of infection can occur via infectious virus or virus-induced cell fusion. The ultimate objective of studies described here is to understand how herpesviruses, specifically herpes simplex viruses (HSV), induce membrane fusion. Four HSV glycoproteins are required to induce membrane fusion. By use of appropriate expression plasmids, we have determined that transfection of cells with plasmids expressing these four glycoproteins (gB, gD, gH and gL) results in cell fusion with high efficiency provided (i) all four glycoproteins are expressed and (ii) the cells express a receptor for gD, such as HveC (also called nectin-1), a membrane glycoprotein that interacts with actin filaments through afadin. Wild-type viruses expressing these four glycoproteins do not always induce cell fusion, however, because other viral proteins somehow prevent cell fusion. Our hypothesis is that cell fusion requires interactions of gD with gB, gH, or gL, that these interactions are usually triggered by binding of gD to a receptor and that, under certain conditions, other viral proteins can also interact with one of more of these glycoproteins to block membrane fusion or modulate events required for merging the contents of two cells.
Our aims are to (i) identify domains in gD that are required for cell fusion but not for receptor binding; (ii) determine whether in (i); (iii) identify viral proteins that, when expressed with gB, gD, gH, and gL, can prevent cell fusion and characterize their interactions with these glycoproteins; (iv) investigate the effects of HveC-afadin interactions on HSV-1-induced cell fusion. Experimental approaches will include screening of gD mutants and chimeric molecules for function in the cell fusion assay, co-immunoprecipitation following transfection and exposure of gD to receptor, screening for viral proteins that can inhibit cell fusion, and confocal microscopy to localize viral proteins and cell structures in cells that are undergoing fusion that are prevented from fusing despite presence of the fusogenic viral glycoproteins.
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