Herpes viruses are important pathogens of humans and animals. They enter eukaryotic cells by fusion of their viral envelope with the cellular membrane, and spread partly by inducing fusion of adjacent cellular membranes. Our long term goal is to understand the mechanisms of viral-entry into cells and virus- induced cell fusion. HSV-1 glycoproteins B(gB), D(gD) and H(gH) and a hydrophobic protein with membrane spanning potential termed the Fusion Factor (FF) are involved in these fusion events. Viruses that carry single mutations in gB (gB-Syn3) and FF (FF- Syn1) cause extensive fusion of infected cells and enter faster than wild-type viruses. We propose to elucidate interactions among these proteins that are important for virus-entry and virus-induced cell fusion. Our approach is two-fold: first, a genetic component focuses on the identification of domains of viral proteins that interact with gB and FF via the isolation of intergenic and intragenic suppressor mutations of the gB-Syn1, and FF-Syn3 mutations and other mutations that enhance fusion at low pH conditions. Intragenic and intergenic suppressor mutations of the Syn1 and Syn3 loci will be isolated from viruses grown in the presence of mutagens. In addition, we will isolate mutations in HFEM(tsB5; gB-Syn3) and mP(MP; FF-Syn1) viruses that enable them to cause extensive fusion at pH 6.5. These mutations will be mapped on the viral genome in marker-rescue and marker- transfer experiments and mutations will be identified by DNA sequencing. Second, a physical component aims to assess the effect of co-expression of gB, gD, gH and FF on their synthesis, subcellular localization and transport to the cell surface and further aims to develop a virus-dependent model system for cell fusion to investigate physical interactions among these proteins. Glycoproteins gB, gB(Syn3), gD, gH, fusion factor(FF), and FF(Syn1) will be cloned into SV40 transient expression vectors and co-expressed in COS cells and Vero cells to define different combinations of these proteins that cause cell fusion. A permanent cell line will be constructed expressing a combination of proteins that cause cell fusion under inducible conditions. The synthesis, maturation and transport of each protein to the cell surface will be examined. Amino-terminal and carboxy- terminal truncations of each of the proteins will be used in complementation experiments to define interacting domains. A cell line transformed with a DNA fragment that codes for the FF protein will be used to isolate a FF-null virus and the intracellular distribution of the FF will be investigated.
