Mutagenesis of Human Immunodeficiency Virus Reverse Transcriptase p51 Subunit Defines Residues Contributing to Vinylogous Urea Inhibition of Ribonuclease H Activity

Suhman Chung,Jennifer T Miller,Barry C Johnson,Stephen H Hughes,Stuart F.J Le Grice

doi:10.1074/jbc.m111.314781

Abstract

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, whereas protein footprinting suggested a contribution from α-helix I residues Cys-280 and Lys-281. To more thoroughly characterize the vinylogous urea binding site, horizontal alanine scanning mutagenesis between p51 residues Lys-275 and Thr-286 (comprising α-helix I and portions of the neighboring αH/αI and αI/αJ connecting loops) was combined with a limited vertical scan of Cys-280. A contribution from Cys-280 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 IC(50) for p51 mutant T286A coupled with a 2-8-fold increased IC(50) 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.

Highlights

Binding of vinylogous urea-derived inhibitors to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was examined by scanning mutagenesis
Construction and Analysis of Selectively Mutated p66/p51 RT Heterodimers—To more precisely define p51 thumb residues contributing to DNTP binding, we performed alanine scanning mutagenesis between Lys-275 and Thr-286, which constitutes ␣-helix I and a portion of the adjacent ␣H/␣I and ␣I/␣J connecting loops (Fig. 1b)
Our previous communication illustrated that unlike active site inhibitors, which can be displaced from the ribonuclease H (RNase H) active site by nucleic acid, vinylogous ureas are still effective in the presence of substrate [11]

Summary

Background

Binding of vinylogous urea-derived inhibitors to HIV-1 RT was examined by scanning mutagenesis. An ϳ19-fold reduced IC50 for p51 mutant T286A coupled with a 2– 8-fold increased IC50 when intervening residues were substituted supports our original proposal of p51 ␣-helix I as the vinylogous urea binding site In contrast to these allosteric inhibitors, mutant enzymes retained equivalent sensitivity to the natural product ␣-hydroxytropolone inhibitor manicol, which x-ray crystallography has demonstrated functions by chelating divalent metal at the p66 RNase H active site. High throughput screening of NCI, National Institutes of Health libraries of chemical entities identified the vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4, 5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as moderately potent HIV-1 and HIV-2 RNase H inhibitors [11, 20] Despite their lower activity compared with ␣-hydroxytropolones, these compounds had the advantage of retaining activity against the enzyme-substrate complex [11]. Together with observations from selectively deleted p66/p51 heterodimers, our data suggest an indirect contribution from inhibitor binding to the p66 thumb subdomain is unlikely

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION