The valine-to-threonine 75 substitution in human immunodeficiency virus type 1 reverse transcriptase and its relation with stavudine resistance

The amino acid change V75T in human immunodeficiency virus type 1 reverse t ranscriptase confers a low level of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T) resistance in vivo and in vitro, Valine 75 is located at t he basis of the fingers subdomain of reverse transcriptase between the temp late contact point and the nucleotide-binding pocket. V75T reverse transcri ptase discriminates 3.6-fold d4T 5'-triphosphate relative to dTTP, as judge d by pre-steady state kinetics of incorporation of a single nucleotide into DNA, In addition, V75T increases the DNA polymerization rate up to 5-fold by facilitating translocation along nucleic acid single-stranded templates. V75T also increases the reverse transcriptase-mediated repair of the d4TMP -terminated DNA by pyrophosphate but not by ATP. The V75T/Y146F double subs titution partially suppressed both increases in rate of polymerization and pyrophosphorolysis, indicating that the hydroxyl group of Thr-75 interacts with that of Tyr-146. V75T recombinant virus was 3-4-fold d4T-resistant and 3-fold resistant to phosphonoformic acid relative to wild type, confirming that the pyrophosphate traffic is affected in V75T reverse transcriptase. Thus, in addition to nucleotide selectivity V75T defines a type of amino ac id change conferring resistance to nucleoside analogues that links transloc ation rate to the traffic of pyrophosphate at the reverse transcriptase act ive site.