Portland State University. Department of Biology
Robert L. Milletette
Term of Graduation
Date of Publication
Master of Arts (M.A.) in Biology
Herpes simplex virus, Mutation (Biology), Glycoproteins
1 online resource (viii, 155 pages)
The purpose of this study was to construct and analyze four recombinant herpes simplex virus type 1 (HSV-1) viruses having mutations in the YYl and/or Sp1 binding sites of the glycoprotein D (gD) gene promoter and a control virus containing a wild type (wt) gD promoter. The YYl and Spl binding sites in the HSV-1-gD gene promoter have been shown in earlier in vitro experiments to be essential for virus-induced gD gene expression with Sp1 playing the major role. The first part of my project required the construction of four different plasmids containing a HSV-1 sequence that included the gD gene with promoter and additional flanking sequences. The gD promoters in theses constructs were either wt or contained the mutations in the YYl and/or Spl binding sites. The second step was to introduce the mutations into the virus. This was achieved by cotransfection of the linearized plasmids and infectious HSV-1FgDβ DNA, which contains a β-galactosidase gene in place of the gD gene, into a complementing cell line. The recombinant viruses were selected on the basis of ~-galactosidase (blue-white) screening. The structure was verified by PCR with subsequent restriction and sequence analysis of the PCR product. The growth rates and yields of the recombinant viruses were determined by infection assays and one-step growth curves on Vero cells. The results of this analysis showed that the mutations of the YYl and Spl binding sites of the gD promoter had only a slight effect on the growth yield and growth rate of HSV-1. Analysis of gD mRNA will be needed to detennine the effects of the mutations on gD expression.
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Ludwig, Sabine, "Construction and Analysis of Mutant HSV-1 Viruses Having Mutations in the YY1 And/or Sp1 Binding Sites of the Glycoprotein D (gD) Gene Promoter" (1998). Dissertations and Theses. Paper 6497.