EFFECTS OF MUTATIONS IN THE POLYMERASE DOMAIN ON THE POLYMERASE, RNASE-H AND STRAND TRANSFER ACTIVITIES OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE

Based on structural analyses and on the behavior of mutants, we sugges t that the polymerase domain of HIV-1 reverse transcriptase (RT) plays a critical role in holding and appropriately positioning the template -primer both at the polymerase active site and at the RNase H active s ite. For RT to successfully copy the viral RNA genome, RNase H must cl eave the RNA with absolute precision. We believe that a combination of the structure of the template-primer and its precise positioning are responsible for the specific cleavages RNase H makes. We have proposed that resistance of HIV-1 RT to nucleoside analogs involves a subtle r epositioning of the template-primer. This hypothesis is based on both structural and biochemical analyses. Mutations that confer resistance to nucleoside analogs do not cluster at the polymerase active site; ho wever, they are in pos itions where they could alter the interaction b etween RT and the template-primer. If, as we:have hypothesized, the po lymerase domain is primarily responsible for positioning the template- primer and RNase H cleavage depends on this positioning, it should be possible to use RNase H cleavage to monitor at least some of the major changes in the position of the template-primer. We have used three as says (polymerase, RNase H, and strand transfer) to investigate the eff ects of mutations in the polymerase domain, including mutations that c onfer resistance to nucleotide analogs, on HIV-1 RT. All three assays involve RNA sequences derived from the viral genome. The data show tha t alterations in the polymerase domain, in particular, mutations that are in positions that would be expected to alter the interaction of RT with the template-primer, can alter both the efficiency and specifici ty of RNase H cleavage. These results are discussed in light of the st ructure of HIV-1 RT. (C) 1998 Academic Press Limited.