Study on molecular mechanisms of herpesvirus entry into host cells

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1. Using a cell-cell fusion assay and a VSV pseudotyping assay, it was demonstrated that herpes simplex virus type 1 (HSV-1) gB, gH, gL, gD were necessary for virus entry. Under the same condition, any combinations of glycoproteins encoded by cytomegalovirus (CMV) and varicella-zoster virus (VZV) did not yield positive results. However, any of CMV or VZV glycoproteins inhibited the HSV-1 glycoprotein mediated fusion or infection with pseudotyped VSV.2. To analyze epitomes on glycoproteins associated neutralization, a new assay using VSV pseudotyped with MLV env protein and the target glycoprotein.3. Inhibition of attachment with heparin-like molecules and inhibition of immediate early phase of infection with kinase inhibitors were evaluated by using reporter cell lines for VZV and CMV.4. A filter-based real-time PCR assay was developed for identification of CMV-positive clinical materials. CMV DNA specimens identified by the assay were used for genotyping of glycoprotein genes. There was a linkage among gH, gO, and gN genotypes, although there was no linkage of gB genotypes with those of other glycoproteins. One hypothesis is that pressure on gB by neutralization relates to the lack of linkage and alternative hypothesis is that particular combination of gH, gO, and gN are required for their functions in virus entry.5. Promoter region for activation of interferon stimulating gene 54K (ISG54K) promoter by CMV infection was delineated. The activation was dependent on CMV IE gene expression.