Mutagenesis of the human immunodeficiency virus reverse transcriptase p51 subunit defines residues contributing to vinylogous urea inhibition of Ribonuclease H activity.

The vinylogous urea, NSC727447 was proposed to allosterically inhibit ribonuclease H (RNase H) activity of human immunodeficiency virus Type 1 reverse transcriptase (HIV-1 RT) by interacting with the thumb subdomain of its non-catalytic p51 subunit. Proximity of the p51 thumb to the p66 RNase H domain implied that inhibitor binding altered active site geometry, while protein footprinting suggested a contribution from α-helix I residues Cys280 and Lys281. To more thoroughly characterize the vinylogous urea binding site, horizontal alanine scanning mutagenesis between p51 residues Lys275 and Thr286, (comprising α-helix I and portions of the neighboring αH/αI and αI/αJ connecting loops) was combined with a limited vertical scan of Cys280. A contribution from Cys280 was strengthened by our observation that all substitutions at this position rendered selectively-mutated, reconstituted p66/p51 heterodimers ~45-fold less sensitive to inhibition. An ~19-fold reduced IC50 for p51 mutant Thr286Ala, coupled with a 2- to 8-fold increased IC50 when intervening residues were substituted, supports our original proposal of p51 α-helix I as the vinylogous urea binding site. In contrast to these allosteric inhibitors, mutant enzymes retained equivalent sensitivity to the natural product α-hydroxytropolone inhibitor manicol, which x-ray crystallography has demonstrated functions by chelating divalent metal at the p66 RNase H active site. Finally, reduced DNA strand-transfer activity, together with increased vinylogous urea sensitivity of p66/p51 heterodimers containing short p51 C-terminal deletions, suggests an additional role for the p51 C-terminus in nucleic acid binding that is compromised by inhibitor binding.