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Degree

Doctor of Philosophy, Case Western Reserve University, Pharmacology, 2005.

Abstract

In almost all regions of the developing world, overlapping pandemics of different subtypes or circulating recombinant forms (CRF) of HIV-1 has resulted in increased dual infections, emergence of unique recombinant forms (URFs), and subsequent selection of new stable CRFs from this CRF pool. The goal of these studies was to examine the genomic sites where recombination occurs in presence and absence of selective pressure on the virus, and to observe the frequency of recombination in presence and absence of selective pressure. To do so, a tissue culture dual infection system was used with subtypes A and D, which co-circulate in Uganda, to generate recombinant viruses in the envelope gene after approximately 15 days in culture in U87.CD4.CXCR4 cells. In addition, a cell-based single cycle assay and a cell-free in vitro reverse transcription assay were used to study intersubtype recombination in the absence of selection. Results indicate that hotspots for recombination occur in the conserved regions of the envelope gene, particularly in C1 and C3, in absence of selection when isolates from different subtypes were studied. Breakpoints were nearly absent in the variable regions. However, when two isolates of the same subtype are examined, recombination breakpoints are more dispersed over the entire gene region studied, indicating that increased sequence homology results in a higher frequency of recombination. With the tissue culture dual infection system, the majority of recombination breakpoints occurred in C1. However, there was a slight shift in breakpoints mapping to the variable region V2. This shift in breakpoints to the variable region may be indicative of a favorable selection for recombination in the variable region. These breakpoints may be better tolerated due to the relative plasticity of the genome in the variable regions. In addition, among the three assay systems used, the lowest frequency of recombination was observed in the multiple cycle system. Taken together, these results support the hypothesis that, during multiple rounds of replication, many recombinants are generated during reverse transcription, but only a small number of those recombinants have a replication capacity that allows them to become stable in the population. Thus, selective pressure influences the production of replication competent recombinants, implicating viral factors such as entry efficiency and replication capacity in evolution of HIV-1 between and within patients.

Subject Headings

Biology, Microbiology

Keywords

HIV-1; Recombination; Retrovirus; Evolution

Advisor

Eric J Arts

Pages

296p.

Document number: case1120599751
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=case1120599751

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