Semester

Summer

Date of Graduation

2000

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Vinay K. Pathak.

Abstract

Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a major contributor to variation in retroviral populations generating mutants that exhibit altered host tropisms, resistance to antiviral drugs, and the ability to escape the host's immune system. To identify structural elements of murine leukemia virus (MLV) RT important for fidelity and drug-resistance in vivo, we developed a D17-based encapsidating cell line (ANGIE P) which is designed to express the amphotropic MLV envelope. ANGIE P cell line also contains an MLV-based retroviral vector (GA-1), which encodes a wild-type bacterial beta-galactosidase gene (lacZ) and a neomycin phosphotransferase gene. Transfection of ANGIE P cells with wild type or mutated MLV gag-pol expression constructs generated GA-1 virus that was able to undergo only one cycle of viral replication upon infection of D17 cells. The infected D17 cell clones were characterized by staining with X-Gal and the frequencies of inactivating mutations in lacZ were quantified. Several structural determinants of MLV RT were identified that were important for fidelity which included position V223 of the YVDD box, several residues of the dNTP-binding site, as well as residues residing in the RNase H domain of MLV RT. A number of these MLV RT mutants resulted in statistically significant decreases in fidelity (1.2 to 2.8-fold) whereas two mutants showed a statistically significant increase in fidelity (0.8-fold) relative to wild-type MLV RT. Furthermore, these amino acid residues were observed to play critical roles in catalysis and viral replication, which was exhibited by the reductions in both viral titers as well as RT activities of these mutants. In addition to identifying structural determinants important for fidelity, we also showed that the V223 position of MLV RT may not be the only structural determinant important for resistance to the antiviral nucleoside analog, 2', 3'-dideoxy-3 '-thiacytidine (3TC). This is in contrast to results observed with human immunodeficiency virus type 1 (HIV-1) RT. These results establish a sensitive in vivo assay for identification of structural determinants important for accuracy of DNA synthesis as well as dug-resistance and indicate that several structural determinants may have an effect on the in vivo fidelity of MLV RT.

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