Mutational Analysis of Tyr-501 of HIV-1 Reverse Transcriptase: EFFECTS ON RIBONUCLEASE H ACTIVITY AND INHIBITION OF THIS ACTIVITY BY N-ACYLHYDRAZONES

Dominique Arion,Nicolas Sluis-Cremer,Kyung-Lyum Min,Michael E Abram,Ronald S Fletcher,Michael A Parniak

doi:10.1074/jbc.m110254200

Dominique Arion, Nicolas Sluis-Cremer + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110254200

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Abstract

N-(4-tert-Butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone (BBNH) is a potent inhibitor of the ribonuclease H (RNase H) activity of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Molecular modeling predicted that BBNH binds to the HIV-1 RT RNase H active site via two major interactions, coordination to the metal ion cofactor (Mg(2+) or Mn(2+)) in the enzyme active site and aromatic ring-stacking interaction between the naphthyl ring of BBNH and amino acid Tyr-501. The latter residue equivalent is conserved in virtually all RNases H, suggesting the need for an aromatic or pi-stacking interaction in this region. To assess the importance of Tyr-501 in the binding of BBNH for the inhibition of RT RNase H activity, we used site-specific mutagenesis to generate RT with a variety of substitutions at this position. Most substitutions resulted virtually in a complete loss of RNase H activity. However, three mutants, Y501F, Y501W, and Y501R, possessed RNase H activities comparable with wild-type enzyme. Whereas BBNH inhibited Y501F RT RNase H activity with potency equivalent to wild-type RT, the Y501W mutant showed a 6-fold resistance to inhibition by BBNH, and the Y501R mutant was completely resistant to inhibition by BBNH. The replication "fitness" of HIV molecular clones with the Y501W or Y510R mutation was significantly compromised compared with wild-type virus. Importantly, BBNH was an effective inhibitor of the DNA polymerase activity of all Y501X mutants tested. Our results highlight the importance of Tyr-501 in RT RNase H activity and in N-acylhydrazone inhibitor binding and suggest that drugs that target critical residues in HIV-1 proteins may be a useful approach in new antiviral development.

Highlights

N-(4-tert-Butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone (BBNH) is a potent inhibitor of the ribonuclease H (RNase H) activity of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT)
Our hypothesis was that binding to a site at or near the nucleoside RT inhibitorbinding pocket (NNIBP) in the polymerase domain of RT results in the inhibition of the DNA polymerase activity of the enzyme, whereas binding to the second site in the RNase H domain provides inhibition of the RNase H activity of the enzyme
Molecular modeling showed that BBNH can be readily docked into the RT RNase H domain in a manner consistent with previously reported data (Fig. 2A)

Summary

EXPERIMENTAL PROCEDURES

Materials—BBNH and N-(4-tert-butylbenzoyl)-2-hydroxy-1-salicylylhydrazone (BBSH) were synthesized by standard methods [14]. 1H NMR and elemental analyses of these compounds were entirely consistent with the expected chemical structures. [3H]TTP, [␥-32P]ATP, and the homopolymeric template/primer poly(rA)-oligo(dT) were purchased from Amersham Biosciences, Inc. Molecular Modeling—A model for the interaction of BBNH with HIV-1 RT was constructed using the x-ray crystallographic coordinates for HIV-1 RT complexed with nevirapine [15] (Protein Data Bank accession number 3HVT) This structure was chosen because of the presence of magnesium atoms bound in the RNase H active site. Assays for the inhibition of RT DNA polymerase activity by BBNH were carried out as described previously [11]. For assay of BBNH or BBSH inhibition of RT RNase H activity, WT or mutant RT (30 nM) was preincubated with BBNH or BBSH (dissolved in Me2SO) in 50 mM Tris-HCl, pH 7.9, 60 mM KCl, 2.5 mM MgCl2, 0.2 mg/ml of ovalbumin in a final volume of 50 ␮l for 10 min. Aliquots of cell-free supernatants were reserved for the assessment of viral p24 levels

RESULTS

45 Ϯ 6 inactive

DISCUSSION