Human immunodeficiency virus type 1 reverse transcriptase: The role of template -primer interactions in polymerase function
Fisher, Timothy Steven
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Human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) for conversion of its RNA genome into DNA. This process requires several polymerase activities inherent to RT, including RNA- and DNA-dependent DNA polymerase, ribonuclease H, strand transfer and strand displacement synthesis. While studies have demonstrated a role for RT-template-primer interactions in some of these activities, our understanding of the role of these interactions in polymerase function remains incomplete. The present work addresses the role of the thumb and fingers subdomains of HIV-1 RT in polymerase function.;We first attempted to isolate mutants of HIV-1 RT resistant to template analog RT inhibitors (TRTIs). Resistant variants (N255D and N265D) were isolated via an in situ colony screening procedure. Single mutations conferred low-level resistance to several TRTIs; when combined, both mutations greatly enhanced resistance. Resistance to TRTIs was likely due to a loss in binding affinity. Viruses carrying such mutations were defective in both viral infectivity and replication in cell culture. Interestingly, N265D was additionally found to display a template-specific defect in processive DNA synthesis. While both N255D and N265D were defective in DNA-dependent processivity, N265D was unaffected on an RNA template. This template-specific defect was also reflected in a rapid dissociation of N265D from DNA, but not RNA templates. Molecular dynamics modeling by Dr. Tom Darden (NIEHS, N.C.) suggests that template-specific loss of hydrogen bonding occurs between N265D and the template-primer. Together, these data indicate that resistance to TRTIs can arise in vitro, however they restrict viral fitness in vivo and that the thumb subdomain is, in part, responsible for RT's ability to copy RNA and DNA templates.;To determine whether the fingers subdomain plays a role in strand displacement synthesis, RTs carrying substitutions at the F61 residue were generated. Most substitutions at F61 affected strand displacement synthesis (increasing: F61L, F61Y; decreasing: F61W). Finger insertion mutants previously developed in our lab were also used to determine if the size of the fingers affects strand displacement synthesis. As the length of the insertions increased, strand displacement synthesis was enhanced, suggesting that the fingers subdomain plays a role in strand displacement synthesis. Substitutions at F61 were also found to alter polymerase processivity, fidelity and sensitivity to nucleoside analog inhibitors. Together, these results indicate that the F61 residue plays a role in several polymerase functions.