Doctoral Dissertations

Date of Award

12-1984

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Richard J. Courtney

Committee Members

Jeffrey Becker, David Brian, Leaf Huang

Abstract

Using an inhibitor of DNA synthesis as well as a temperature sensitive, DNA negative mutant of herpes simplex virus type 1 (HSV-I), the role of viral DNA synthesis in the expression of the virus-specific glycoproteins was examined. Analysis by immunoblotting employing monospecific antisera revealed that the gC glycoprotein was not synthesized when viral DNA synthesis was inhibited. In contrast, the gB glycoprotein was detectable in significant, albeit somewhat reduced, amounts. These data suggest that gC is a "true late" protein of HSV-I. In addition, temperature-shift-up experiments suggested some viral gene product(s) is needed continuously to achieve wild type levels of gC synthesis.

The distribution of the gB and gC glycoproteins was examined after cell fractionation of HSV-1 infected cells. It was determined that the high-mannose precursors were the predominant components of the nuclear fraction. In addition, various culture conditions were examined for their effect on the intracellular distribution of HSV-1 glycoproteins. In the presence of an inhibitor of asparagine-(N-) linked glycosylation, marked preferential localization of the nonglycosylated precursors to the nuclear fraction was observed. Furthermore, it was found that in certain cell lines incubated at reduced temperature, the nonglycosylated forms of HSV-1 glycoproteins were detectable in the absence of inhibitors of glycosylation. The post-translational addition of high-mannose core sugars to the nonglycosylated forms was demonstrated in infected cultures treated with an inhibitor of protein synthesis at a time when significant amounts of the nonglycosylated protein was present.

In order to determine if there were host cell limitations of biosynthetic components, a differential extraction procedure was employed to separate and identify monosaccharide-dolichol from oligosaccharide-1ipid and glycoproteins. Measurement of [3H]mannose incorporation into mannosyl phosphoryl-dolichol revealed no significant difference between infected and uninfected cells. In contrast, there was a marked reduction of mannose incorporation into oligosaccharide lipid in HSV-1 infected Vero cells while incorporation into glycoproteins was two-fold greater in the same cells. These results imply there is an accelerated turnover of oligosaccharide-lipid in infected cells which may be due in part to extensive HSV-induced glycoprotein synthesis.

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