Our work demonstrates that the cytoplasmic domain of gC is necessary for stable membrane anchoring of this glycoprotein. In this application, I propose to extend these studies to identify what constitutes a good membrane anchor for gC. The major hypothesis to be tested is that the basic amino acids normally found in cytoplasmic domains are critical for anchoring. This will be tested by using oligonucleotide directed mutagenesis to construct a new series of mutant gC genes that encode glycoproteins with specifically tailored cytoplasmic domains. The sequences of these cytoplasmic domains will be chosen to evaluate the effects of charge and amino acid constitution. The mutant glycoproteins will be expressed by construction of recombinant viruses. Additionally, to accelerate the characterization of the mutant glycoproteins, an in vitro expression system will be developed. The mutant genes will be constructed and expressed so that they can be transcribed in vitro from a plasmid template using an upstream phage promoter. The in vitro transcripts will be translated in vitro in a system containing microsomes capable of carrying out the postranslational processing steps that are normally done in the rough endoplasmic reticulum. Although additional data will have to be collected from other systems, the conclusions drawn from the gC system should have general applicability. A second area of research concerns the membrane configuration of HSV-1 gB. Other investigators have proposed that gB has three transmembrane domains. I propose a model of gB membrane topogenesis in which specific topogenic functions are associated with individual transmembrane domains of gB. Both the gB structural model and the model of gB topogenesis will be tested by construction of a series of gB deletion mutants in which one or more of proposed transmembrane domains are deleted. The mutant glycoproteins will be expressed and their membrane configurations evaluated using the in vitro expression system. Finally, the protein product of one of the major HSV-1 genetic loci involved in cell fusion, the syn1 locus, will be identified and characterized. Hybridization selection will be used to purify syn1 mRNA, which will be translated in vitro in a microsomal system to preliminary identify the syn1 protein. If necessary, mRNA mapping will be done to positively identify the syn1 mRNA and reading frame. Antibodies to the syn1 protein will be produced by expressing fragments of the gene in E. coli. These antibodies will be used to characterize expression of the protein in infected cells and to determine its membrane topology.
| Mo, C; Holland, T C (1997) Determination of the transmembrane topology of herpes simplex virus type 1 glycoprotein K. J Biol Chem 272:33305-11 |
| Dolter, K E; Ramaswamy, R; Holland, T C (1994) Syncytial mutations in the herpes simplex virus type 1 gK (UL53) gene occur in two distinct domains. J Virol 68:8277-81 |
| Dolter, K E; King, S R; Holland, T C (1993) Incorporation of CD4 into virions by a recombinant herpes simplex virus. J Virol 67:189-95 |
| Skoff, A M; Holland, T C (1993) The effect of cytoplasmic domain mutations on membrane anchoring and glycoprotein processing of herpes simplex virus type 1 glycoprotein C. Virology 196:804-16 |
| Ramaswamy, R; Holland, T C (1992) In vitro characterization of the HSV-1 UL53 gene product. Virology 186:579-87 |
| Martin, L B; Montgomery, P C; Holland, T C (1992) Soluble glycoprotein D blocks herpes simplex virus type 1 infection of rat eyes. J Virol 66:5183-9 |
