Semester

Fall

Date of Graduation

2003

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Vinay K. Pathak.

Abstract

Initiation of reverse transcription in retroviruses occurs on a specific place in viral genome, called primer-binding site (PBS). Cellular tRNA is annealed to the complementary PBS and is used as a primer. We constructed murine leukemia virus (MLV)-based vectors containing two PBSs, one in normal position and another in the middle of the vector. Green fluorescence protein (GFP) gene was inserted between the two PBSs. The internal PBS was derived from a spleen necrosis virus (SNV), which uses the same tRNA-Pro as a primer. We have shown that the internal PBS was functional for initiation of reverse transcription. Replication of vectors containing two functional PBSs is expected to result in deletion of sequences between the two PBSs if initiations on these two sites occur simultaneously. The deletion was detected in 20--30% of infected cells by the GFP-negative phenotype and by direct Southern blotting analysis of DNA of infected cells. This indicated that a single virion has the capacity to initiate reverse transcription more than once. In order to fund the efficiency of initiation of a single PBS we constructed a mathematical model, which provided a relationship between several parameters of retroviral replication and the outcome of reverse transcription of vectors with two PBSs. Using this model we found that efficiency of initiation at MLV and SNV PBS is 35--50%. Next, we constructed human immunodeficiency virus type 1 (HIV-1)-based vectors with two PBSs by inserting human immunodeficiency virus type 2 (HIV-2) PBS in the middle. Again, the internal PBS was found functional and simultaneous initiations were occurring in 22--24% of all infections. This system was used to analyze the effects of mutations within HIV-2 PBS region on the efficiency of initiation. By using wild type HIV-1 PBS (present in the same vector) as internal control we were able to get accurate quantitative measurements of initiation efficiency at mutant HIV-2 PBS. Our results indicated that U5-IR stem of HIV-2 plays a very important role in initiation. Also, additional interactions between tRNA and viral genome, proposed for HIV-1, were found to be important for HIV-2 initiation, indicating that these two viruses use similar mechanisms to achieve specific and efficient initiation of reverse transcription.

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