Date of Award

1992

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Ronald C. Montelaro

Abstract

Biochemical, immunological, and structural characterizations of the structural proteins of simian immunodeficiency virus (SIV) were performed. Various electrophoretic and chromatographic analyses of native and metabolically radiolabeled virus preparations coupled with immunological procedures such as Western blotting and radioimmunoprecipitation were utilized to identify SIV/DeltaB670-encoded polypeptides. Two-dimensional tryptic peptide mapping of each putative viral protein was employed to confirm that each of these components were unique and not cleavage products of precursor polyproteins. Site-directed serological studies using synthetic peptides analogous to regions of the SU and TM protein predicted to have high antigenic potential were performed. These studies resulted in the identification of several group-specific and type-specific antigenic determinants of SIV envelope proteins and demonstrated serological similarities between SIV and several other lentiviruses. The lentivirus lytic peptide (LLP), a segment within the carboxyl terminus of the TM protein which has high amphipathic potential and unusually high positive charge density, was identified during secondary structural modeling studies. Since the LLP appeared to share structural properties with natural cytolytic peptides such as magainins, cecropins, melittin, etc., synthetic peptides homologous to the LLP of HIV-1 and SIV were employed in standard assays designed to measure the ability of cytolytic peptides to inhibit prokaryotic or eukaryotic cells, and were found to share functional characteristics with natural cytolytic peptides. Results from membrane permeability studies and $\sp{51}$Cr-release assays suggested that the LLPs inhibit cells via membrane perturbation. The LLPs also share structural characteristics with calmodulin (CaM)-binding proteins. CaM is the primary sensor of Ca$\sp{2+}$ within eukaryotic cells. Since Ca$\sp{2+}$ is the primary signal for activation of many enzyme systems, CaM plays an essential role in the activation of many cellular enzymatic processes. Using gel mobility shift assays and standard phosphodiesterase competition assays, we have shown that LLPs bind CaM with high affinity and can inhibit CaM-mediated activation of CaM-dependent enzymes. LLP-mediated interference with enzyme activation and/or alteration of critical plasma membrane properties may explain many of the unusual manifestations of HIV-1-mediated disease and may represent a novel mechanism of pathogenesis.

Pages

210

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