Semester

Summer

Date of Graduation

2000

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Vinay Pathak.

Abstract

Retroviruses exhibit high mutation rates. High mutation and recombination rates increase variation within a viral population and result in production of drug-resistant mutants and/or mutants that can escape a host immune response. Mutations are introduced into the viral genome by error-prone reverse transcriptase (RT), a virally encoded enzyme that converts single-stranded viral RNA into double-stranded DNA. In the current study we are trying to understand what structural determinants of RTs are important for fidelity, frequency of template switching, and drug-resistance. First, we developed an in vivo assay and performed mutational analysis of manne leukemia virus (MLV) RT to identify structural elements important for template switching. Based on obtained results, we proposed a dynamic copy-choice model in which both the rate of DNA polymerization and the rate of degradation of the RNA template influence the frequency of RT template switching. Second, we employed a previously described in vivo fidelity assay to determine whether a minor groove binding helix of the thumb domain and primer grip of MLV RT are important for in vivo fidelity of reverse transcription. Because the thumb domain of MLV RT has not been crystallized, we utilized homology alignment and molecular modeling to identify the minor groove binding helix of the thumb domain of MLV RT. Mutations in the minor groove binding helix residues R301 and F309 decreased RT fidelity by up to 2.8-fold, suggesting that this region plays an important role in accuracy of DNA synthesis. Finally, we attempted to elucidate a mechanism of drug-resistance to the antiretroviral nucleoside analog 2',3'-dideoxy-3 '-thiacytidine (3TC), an inhibitor of wild-type human immunodeficiency virus type 1 (HIV-1) RT. We tested our hypothesis that a valine residue at the 223 position in YVDD motif of the MLV RT leads to a natural high level of resistance of MLV to 3TC in a manner similar to that proposed for the YVDD mutant of HIV-1 RT. The results indicated that the wild-type, V223M, V2231, V223A, and V223S mutants of MLV RT were all highly resistant to 3TC, suggesting that determinants outside the YVDD motif of MLV RT confer a high level of resistance to 3TC.

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