Site-specific incorporation of nucleoside analogs by HIV-1 reverse transcriptase and the template grip mutant P157S - Template interactions influencesubstrate recognition at the polymerase active site

Studies of drug-resistant reverse transcriptases (RTs) reveal the roles of specific structural elements and amino acids in polymerase function. To cha racterize better the effects of RT/template interactions on dNTP substrate recognition, we examined the sensitivity of human immunodeficiency virus ty pe 1 (HIV-1) RT containing a new mutation in a "template grip" residue (P15 7S) to the 5'-triphosphates of (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) , (-)-beta-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), and 3'-azido-3'-de oxythymidine (AZT). A primer extension assay was used to monitor quantitati vely drug monophosphate incorporation opposite each of multiple target site s. Wild-type and P157S RTs had similar catalytic activities and processivit ies on heteropolymeric RNA and DNA templates. When averaged over multiple t emplate sites, P157S RT was 2-7-fold resistant to the 5'-triphosphates of 3 TC, FTC, and AZT, Each drug triphosphate inhibited polymerization more effi ciently on the DNA template compared with an RNA template of identical sequ ence. Moreover, chain termination by 3TC and FTC was strongly influenced by template sequence context. Incorporation of FTC and 3TC monophosphate vari ed up to 10-fold opposite 7 different G residues in the DNA template, and t he P157S mutation altered this site specificity. In summary, these data ide ntify Pro(157) as an important residue affecting nucleoside analog resistan ce and suggest that interactions between RT and the template strand influen ce dNTP substrate recognition at the RT active site. Our findings are discu ssed within the context of the HIV-1 RT structure.