The role of lentiviral reverse transcriptase in viral replication and pathogenesis: Identification and characterization of the high-fidelity V148I reverse transcriptase mutant

The HIV/AIDS epidemic has been spreading across the globe over the past three decades. Researchers have been trying to understand the course of disease and develop HIV vaccines and antiretroviral agents. It has become increasingly apparent that the large quasispecies found within individuals and the high degree of diversity found between individuals makes it difficult to develop broadly effective vaccines or maintain long-term susceptibility to drugs. The virally encoded DNA polymerase, reverse transcriptase (RT), is one of the main driving forces for the fast genomic hypermutation seen during the replication of lentiviruses (e.g. SIV and HIV-1).; In this thesis the role of RT in viral replication and pathogenesis are discussed. By tracking RT fidelity throughout the course of SIV infection in a pig-tailed macaque, an RT with increased fidelity was identified at late stages of disease progression when the immune system in the animal was depleted. This RT was the first high-fidelity RT isolated in vivo during the course of natural infection. Among six mutations that appeared within the DNA polymerase domain of the RT in this infected animal, one mutation, V148I, was identified as a high fidelity mutation. This thesis presents biochemical evidence for a conserved role of the V148 residue in SIV and HIV-1 RTs. Mutations in this residue affect the fidelity of purified RTs by altering the ability of the enzyme to bind incoming dNTPs. Interestingly, an HIV-1 pseudotyped virus containing the V148I RT mutant has a decreased viral mutation rate as expected from our biochemical data.; In addition to the role of V148I in RT fidelity, the effects of the V148I mutation on viral tropism have also been investigated. The decreased dNTP binding exhibited by V148I mutants significantly reduces the ability of pseudotyped viruses to infect macrophages. This may be the first association between mutations in the viral RT and HIV-1 cell-type specificity. Altogether, the data presented here proposes roles for the V148I mutation, and possibly other RT mutants, in maintaining viral fitness and cellular tropism at various stages throughout the course of infection.