Title

Use of monoclonal antibodies for the analysis of the major capsid protein of simian virus 40

Date of Award

1988

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Walter A. Scott, Committee Chair

Abstract

I have chosen to study structure/function relationships for the major capsid protein of simian virus 40 (SV40 VP1) using a panel of monoclonal antibodies which react with this protein. My studies have shown that a subpopulation of SV40 chromatin contains VP1. This subfraction lacks the nuclease-hypersensitive site detected near the replication origin in a large proportion of the SV40 chromatin molecules isolated from infected cells. Sequence-specific binding of VP1 to isolated SV40 DNA fragments was not detected. Analysis of VP1 isoforms in the nucleoprotein complexes revealed changes on the ratio of modified to unmodified VP1 during the maturation process. The presence of VP1 in nucleoprotein complexes from temperature sensitive mutants unable to assemble virions suggests that these mutations in VP1 affect a level of capsid organization beyond the VP1/chromatin-association step. These results support the hypothesis that VP1 interactions with the viral chromatin play a major role in the gradual encapsidation of the SV40 virus.Monoclonal antibodies recognizing both continuous and discontinuous epitopes were identified. A continuous epitope was mapped near the carboxyl terminus of the VP1 sequence. A synthetic peptide corresponding to this epitope competed with SV40 virus for the binding to antibodies. Antibodies which recognize each type of epitope were able to neutralize SV40 infections. Monoclonal antibodies to some discontinuous epitopes neutralized infectivity more efficiently. A complex pattern of competition for binding to VP1 was observed between the antibodies, suggesting a series of overlapping antigenic determinants. The ability of each monoclonal antibody to interact with the nucleoprotein complexes as well as with the virus suggests that the VP1 protein structure is not dramatically altered during virion encapsidation. Prediction of secondary structure and surface-exposed regions from primary sequence using published computer algorithms agrees with a structure in which the continuous epitope protrudes from the surface of the protein. My results support the hypothesis that a protruding domain is present in the carboxyl terminus of VP1 which contains antigenic sequences that are involved in the infectivity of the virus.

Keywords

Biology, Molecular

Link to Full Text

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