Diversification and evolution of HIV-1 enables the virus to persist despite a vigorous immune response and the administration of potent antiretroviral therapy. Globally, viral diversification has led to distinct phylogenetic clades and subtypes of recognized concern to vaccine development. While much attention has been devoted to the study of viral sequence changes resulting from sequential single base substitutions, the contribution of recombination to HIV-1 diversification and evolution is less well understood. Previous studies quantifying retroviral recombination or examining factors which affect recombination have been performed either in vitro using purified viral components, or with transformed fibroblastic cells that are not targets of infection in vivo. We have developed methods which permit rapid and quantitative study of recombination during infection of essentially any human cell type, including primary T cells and macrophages. Preliminary studies reveal (1) that by examining HIV-1 recombination in the relevant target cell population, HIV-1 is revealed to be more recombinogenic than previously appreciated, (2) that HIV-1 recombination rates are substantially higher in some cell types than in others, (3) that differentiation of monocytic cells leads to a 3 fold increase in recombination rates, and (4) that HIV-1 recombination can be influenced by mutations within the reverse transcriptase enzyme which alter enzyme activity and confer clinical resistance to antiretroviral drugs. Based on these preliminary studies, we hypothesize that the rate of recombination during infection is linked to the differentiation, activation and/or proliferation of the infected cell. We also hypothesize that recombination rates are directly linked to the kinetics of reverse transcription as determined by both the infected cell and by mutations in RT, which affect enzyme function. We will test these hypotheses through the following specific aims: 1. Determine the effects of myeloid differentiation on recombination rates. 2. Determine the effects of T cell activation and proliferation on recombination. 3. Determine the relationship between the kinetics of reverse transcription and recombination within aims 1, 2 and 4. 4. Determine the role in recombination of mutations which influence RT activity and drug resistance. ? ?
