Characterization of HIV-1 reverse transcriptase drug resistance connection subdomain mutation N348I

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Connection subdomain mutations are a recently discovered class of reverse transcriptase (RT) drug resistance mutations which are positioned at some distance from previously described resistance mutations. The work presented in this thesis investigates drug resistance mechanisms conferred by HIV-1 RT connection subdomain mutation N348I. N348I was chosen as a representative connection subdomain mutation due to its clinical relevance to resistance against drugs belonging to both classes of HIV-1 RT inhibitors: the NRTIs and NNRTIs. N348I is the first, and currently only, clinically relevant HIV-1 RT single amino acid substitution mutation known to confer cross-resistance to drugs from both classes of RT inhibitors. I describe here a mechanism of HIV-1 RT N348I in vitro resistance against inhibition by the NNRTI nevirapine (NVP), where I show the mutant enzyme exhibits a decreased affinity towards inhibitor binding. Using pre-steady state kinetics techniques, I further show in detail how the N348I mutation on either of the heterodimer enzyme's subunits affects enzymatic activities. Interestingly, the mutation on either subunit decreases the rate of catalytic turnover for nucleotide incorporation reactions. The N348I mutant enzyme also displays an altered RNAse H activity, and I demonstrate that the N348I mutation on the p51 subunit provides the major contribution towards this altered activity. I also investigated RT N348I in vitro susceptibility to ddATP, the active form of the NRTI prodrug didanosine (ddI). Multiple mechanisms of NRTI resistance were studied, but significant levels of in vitro resistance to ddATP were not detected despite the fact that the N34I mutant virus has been shown to be resistant to ddI. Here I found that the N348I mutation does not negatively impact the steady-state kinetics of single nucleotide incorporation reactions, or the affinity for nucleotide substrate.