Structural determinants of HIV-1 reverse transcriptase fidelity and the influence of TAGLN2, a host factor, during HIV-1 infection

Abstract

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) performs an essential function during viral replication. It is responsible for the conversion of the single stranded viral RNA to double stranded DNA that is subsequently incorporated into the host genome. My thesis focuses primarily on understanding the process of reverse transcription in two parts.;In the first part of my thesis, we investigated the role of the Lys66 residue in two specific aspects of polymerase fidelity namely: misinsertion and mispair extension. Four Lys66 substitution mutants were tested for their efficiency in nucleotide misinsertion or mispair extension under steady-state conditions. Conservative substitutions at position 66 did not affect the efficiency of misinsertion or mispair extension, while non-conservative substitutions led to decreases in misinsertion and mispair extension efficiency ranging from 3 to 23-fold and 4 to 6-fold respectively. These differences were not due to the effects of the substitutions on the binding of RT to the t/p. Our results suggest that the Lys66 residue may have been selected during evolution of the virus because it allows HIV-1 RT the advantage of maintaining its high mutation rate.;The second part of my thesis explores the role of host protein Transgelin-2 (TAGLN2) in viral infection. Depletion of TAGLN2 by shRNA interference in virus producing cells and subsequent infection of target cells with HIV-Luc (VSV-G) led to a 2 to 3-fold reduction in HIV-1 infectivity, only under conditions where TAGLN2 was knocked down in virus producing cells. The reduction in infectivity observed could not be attributed to an inhibition of a specific step in the late and early events examined such as virus assembly, release efficiency, virion particle composition and reverse transcription.