Sequence variation plays a significant role in the pathogenesis and persistence of infections and is a major obstacle in the development of vaccines as well as therapies against lethal diseases caused by retroviruses. It is the goal of the investigator to further elucidate the basic molecular mechanisms of retroviral variation. More specifically, she plans to focus on the mechanisms of retroviral recombination. It is the investigator's hypothesis that certain nucleic acid sequences are more prone to recombination than others and that at least some of these potential recombination hotspots correlate with pause and termination sites for reverse transcriptase. In order to test this hypothesis, she will: 1) determine whether hotspots for mutation correlate with hotspots for retroviral recombination; 2) determine the pause and termination sites for reverse transcriptase in vivo; and 3) identify naturally occurring retroviral sequences which serve as hotspots for recombination. The basic in vivo system will utilize retroviral vectors and helper cells that are designed to isolate one cycle of viral replication. Recombinants will be analyzed by a variety of methods including restriction enzyme mapping, base mutation screening, and DNA sequencing. For the pause and termination assay, unintegrated viral DNA will be analyzed by polymerase chain reaction (PCR). Study of these basic mechanisms will contribute to understanding the role of retroviral strand transfer.
