Human immunodeficiency virus type 1 reverse transcriptase: Drug resistance and structure-function studies

Abstract

The thesis project examines: (a) various properties of a drug resistant mutant RT which contains an E89G mutation in one or both subunits of the RT heterodimer and (b) the role of the fingers subdomain in RT functional activities. To study the contribution of E89G mutation in p66 and p51 subunits of RT heterodimer to nucleoside analog resistance, divalent cation preference, and steady-state kinetic properties, chimeric, wild-type and mutant RT heterodimers were generated. E89G mutant RT displayed higher K{dollar}\rm\sb{lcub}i{rcub}{dollar} (for ddTTP and PFA), K{dollar}\rm\sb{lcub}m{rcub}{dollar} and V{dollar}\rm\sb{lcub}max{rcub}{dollar} values compared to wild-type RT as well as the loss of Mg{dollar}\sp{lcub}2+{rcub}{dollar} preference. The results from the chimeric RTs allowed us to map the increase in K{dollar}\rm\sb{lcub}i{rcub}{dollar} and K{dollar}\rm\sb{lcub}m{rcub}{dollar} values, and the loss of Mg{dollar}\sp{lcub}2+{rcub}{dollar} preference to E89G mutation in the p66 subunit. The increased V{dollar}\rm\sb{lcub}max{rcub}{dollar} appears to be a result of mutations in both p51 and p66 subunit suggesting that the p51 has indirect contribution to RT catalysis. Further, we observed that E89G RT displayed resistance to delavirdine, nevirapine and TIBO. This is the first single mutation in HIV-1 RT shown to confer resistance to both classes of RT drugs.;To determine the role of the {dollar}\beta3{lcub}-{rcub}\beta4{dollar} region of the fingers subdomain in RT enzymatic activities, we have made various deletion and insertion mutations. Our deletion mutants are more active than wild-type RT by two orders of magnitude on heteropolymeric template in the presence of NP-40 but inactive on homopolymeric templates and exhibited high-level resistance to ddNTPs. In contrast, {dollar}\beta3{lcub}-{rcub}\beta4{dollar} insertion mutants were active on both homopolymeric and heteropolymeric templates and exhibited wild-type level sensitivity to ddNTPs. Their kinetic constants on poly(rA).oligo (dT) were similar to those of wild-type RT. Interestingly, the insertion mutants demonstrated significantly higher processivity than wild-type RT and were still capable of producing strand transfer products. These data suggest that the {dollar}\beta3{lcub}-{rcub}\beta4{dollar} hairpin of the fingers subdomain plays an important role in template-primer interaction, processive DNA synthesis and sensitivity to dNTP analogs.