SENSITIVITY OF WILD-TYPE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE TO DIDEOXYNUCLEOTIDES DEPENDS ON TEMPLATE LENGTH - THE SENSITIVITY OF DRUG-RESISTANT MUTANTS DOES NOT

Analysis of the three dimensional structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase CRT) complexed with double- stranded DNA indicates that while many nucleoside-resistance mutations are not at the putative dNTP binding site, several are in positions t o interact with the template-primer. Wild-type HIV-1 RT and two nucleo side-resistant variants, Leu(74) --> Val and Glu(89) --> Gly, have bee n analyzed to determine the basis of resistance. The ability of the wi ld-type enzyme to incorporate, or reject, a 2',3'-dideoxynucleoside tr iphosphate (ddNTP) is strongly affected by interactions that take plac e between the enzyme and the extended template strand 3-6 nt beyond th e polymerase active site. Inspection of a model of the enzyme with an extended template suggests that this interaction involves the fingers subdomain of the p66 subunit in the vicinity of Leu(74). These data pr ovide direct evidence that the fingers subdomain of the p66 subunit of HIV-1 RT interacts with the template strand. The wild-type enzyme is resistant to ddITP if the template extension is 3 nt or less and becom es sensitive only when the template extends more than 3 or 4 nt beyond the end of the primer strand. However, the mutant enzymes are resista nt with both short and long template extensions. Taken together with t he three-dimensional structure of HIV-1 RT in complex with double-stra nded DNA, these data suggest that resistance to the dideoxynucleotide inhibitors results from a repositioning or change in the conformation of the template-primer that alters the ability of the enzyme to select or reject an incoming dNTP.