Date of Award

1993

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

Konstantin G. Kousoulas

Abstract

This research focuses on the phenotypic and genotypic characteristics of a temperature sensitive mutant of herpes simplex virus type 1, HSV-1 (tsZ47). This virus was previously reported to carry two different mutations, which caused it to be temperature sensitive for viral replication and transport of glycoproteins to the infected cell surface. However, altered glycoprotein transport to the cell surface was not observed in this study. In addition, cells transformed with unique long gene 28 (UL28), but not glycoprotein B (gB), complemented tsZ47 at the nonpermissive temperature indicating that a single mutation could restore the wild type phenotype to the mutant virus. A UL28-null virus (gC$\Delta$7B) was not capable of complementing the tsZ47 mutation, indicating that the mutation was within the UL28 gene and there was no other mutation elsewhere. Surprisingly ts1203, containing a mutation in the UL28 gene region, was able to rescue the wildtype phenotype. This suggests intragenic (allelic) complementation. Marker rescue experiments were used to determine the region(s) of the genome which contained a mutation. A panel of overlapping cloned DNA fragments from the wild type virus were co-transfected with infectious viral DNA into Vero cells. A single area of 1333 base pairs within the UL28 gene region rescued the wildtype phenotype. Transfer of this single region into an otherwise wild type viral genome resulted in a virus that was phenotypically identical to tsZ47. This confirmed that a single mutation was responsible for the observed phenotype of tsZ47. Sequence analysis of this region revealed a single mutation, C to T, that substituted $\sp{531}$Tryptophan for $\sp{531}$Arginine. Computer analyses were used to predict the effects of this mutation. DNA from tsZ47 infected cells is present in long concatemers rather than being cleaved to genomic length. In addition, electron microscopic analysis revealed that DNA was not being packaged into the nucleocapsids. It can be concluded that HSV-1 (tsZ47) does not have a defect in glycoprotein transport and contains only a single mutation in the UL28 gene. This mutation affects the ability of the virus to effectively cleave and package DNA.

Pages

122

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