Mutants of HSV-1 that cause extensive fusion during a productive infection have been physically mapped to the prototypic genome coordinates 0.732 to 0.753. One of these mutations in MP, as well as a second mutation that regulates the production of glycoprotein gC, has been located within the coding sequences of ICP-27. Experiments are proposed to study fusion produced by mutational changes in this region. Linker mutagenesis (XbaI and perhaps BclI) at random sites throughout the cloned DNA of this region will provide for rapid and accurate location of the linker after its transfer to KOS DNA. Base substitution mutagenesis will also be employed. Linker mutagenesis may also increase the speed with which DNA sequence analysis of the mutants may be determined. Suppressor mutations of fusion throughout the HSV-1 genome (using cloned EcoRI fragments) will also be produced and located. The production of gC by the presence and absence of ICP-27 in cis and trans will be determined. These regulatory studies, as well as those that locate target sequences in gB (a fusion-inducing gene adjacent to ICP-27) for fusion by the gene X and ICP-27 products, will employ an HSV-1/prokaryotic shuttle-vector that forms defective virus particles in the presence of a helper virus. A map of the target sequences within gB, the primary HSV-1 molecule possessing fusion activity, for syncytium formation by gene X and ICP-27 gene products will employ deletion mapping of gB. gB deletion, gene X (or ICP-27) syncytium-inducing viruses will be incubated with a shuttle vector that contains cloned gB genes with short deletions at random sites throughout the gB coding sequences and assayed for fusion during infection on nonpermissive cells. The gene X product will be obtained by expression of the HSV-1 peptides in a tripartite plasmid in E. coli. Antibodies produced against the tripartite protein will be used to precipitate the gene X product from infected cells in order to characterize its fusion-inducing role.
| Hutchinson, L; Graham, F L; Cai, W et al. (1993) Herpes simplex virus (HSV) glycoproteins B and K inhibit cell fusion induced by HSV syncytial mutants. Virology 196:514-31 |
| Cai, W H; Gu, B; Person, S (1988) Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion. J Virol 62:2596-604 |
