Date of Award

1992

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Abstract

A detailed analysis of polypeptides located in the envelope of vaccinia virus (strain IHD-W) was performed with the goal of characterizing the physical properties and function of specific envelope polypeptides. Envelope polypeptides were identified by: (1) detergent extraction of virions followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), (2) reaction of virions with a surface-reactive biotinylating reagent, (3) reaction of immunoblots of viral polypeptides with an antiserum raised against NP-40 solubilized envelope components. The most prominent envelope polypeptide detected using all three procedures migrated with an apparent MW or 35K and was chosen as the subject for more detailed analysis aimed at elucidating its structure and the nature of its interaction with the envelope.;Polypeptides recognized as antigens were identified by probing Western blots of vaccinia virus and virus infected cells with sera from infected mice. The major antigen identified in this manner corresponded to the 35K envelope polypeptide described above. Other prominent virion-associated antigens migrated at 28K and 62K.;The gene encoding the 35K envelope polypeptide (designated Ag35) was located within HindIII fragment H of vaccinia virus by hybridization arrested translation coupled with immunoprecipitation. S1 nuclease protection analysis identified a viral transcript within this region. DNA sequence analysis identified an open reading frame (ORF) corresponding to this transcript which encoded a polypeptide of only 22,300 daltons, much smaller than is evident by SDS-PAGE. To confirm that the ORF identified did indeed encode Ag35, the gene was expressed in E. coli using the lac promoter of pUC19; the expressed polypeptide migrated in a similar manner to authentic Ag35.;Two possible functions of Ag35 were suggested from studies using monospecific anti-Ag35 antibodies. First, antibody to Ag35 neutralized virus infection in vitro indicating that this polypeptide may play a role in virus-cell interaction. In addition, evidence for an association of this polypeptide with the lipids of the envelope of vaccinia during several stages of biogenesis was obtained by immune-electron microscopy. The association of this polypeptide with the early stages of the de novo assembly of the vaccinia envelope provides a useful model for the study of envelope biogenesis.

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