ALTERATIONS TO THE PRIMER GRIP OF P66 HIV-1 REVERSE-TRANSCRIPTASE ANDTHEIR CONSEQUENCES FOR TEMPLATE-PRIMER UTILIZATION

Alanine scanning mutagenesis was undertaken to evaluate the structural significance of Met(230)-His(235) Of the 66 kDa subunit of p66/p51 hu man immunodeficiency virus reverse transcriptase (HIV-1 RT). Together with Glu(224)-Trp(229), these residues provide the framework of the p6 6 ''primer grip'', whose proposed role is maintaining the primer termi nus in an orientation appropriate for nucleophilic attack on an incomi ng dNTP. Of these residues, altering Leu(234) results in a p66 subunit incapable of associating into heterodimer. The remaining selectively mutated enzymes were successfully reconstituted and purified to homoge neity for evaluation of RT-associated activities. We show here that al terations to any residue within the p66-Trp(229)-Met(230)-Gly(231)-Tyr (232)-quartet alter functions associated with both the DNA polymerase and ribonuclease H (RNase H) domains. Detailed analysis of mutant p66( Y232A)/p51 with an intact or a model ''precleaved'' RNA-DNA hybrid sug gests an altered RNase H phenotype could result from relocation of tem plate-primer in the nucleic acid binding cleft. As a consequence, temp late nucleotide -8 is positioned in the immediate vicinity of the RNas e H catalytic center rather than nucleotide -17.