AEGiS-13IAC: Mutagenic analysis of the role of Y501 in the activity and inhibition of the ribonuclease H activity of HIV-1 reverse transcriptase.

13th International AIDS Conference

Durban, South Africa - July 9-July 14, 2000

Mutagenic analysis of the role of Y501 in the activity and inhibition of the ribonuclease H activity of HIV-1 reverse transcriptase.

Int Conf AIDS 2000 Jul 9-14; 13:(abstract no. TuOrA349)

Arion D, Sluis-Cremer N, Parniak M
D. Arion, Lady Davis Institute, McGill AIDS Centre, Jewish General Hospital, 3755 Cote Ste Catherine Road, Montreal, H3T 1E2, Quebec, Canada, Tel.: +(514) 340 8260 ext 5303, Fax: +(514) 340 7502, E-mail: d_arion@hotmail.com

BACKGROUND: HIV-1 reverse transcriptase (RT) is a multifunctional enzyme, with both DNA polymerase and RNase H activities. While numerous inhibitors of RT DNA polymerase activity have been identified, very few inhibitors of RNase H activity have been described. We were the first group to identify a potent small molecule RNase H inhibitor 'N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH)'. We have constructed a molecular model for the interaction of BBNH with RT RNase H, which shows that the inhibitor makes significant interactions with the divalent metal cofactor in the RNase H active site, and with nearby residue Y501. To test this model, we characterized the role of Y501 in the activity and inhibition of RT RNase H.

METHODS: Y501 was replaced with amino acids G, A, L, F, W, Q, S, E, H, or R. Recombinant mutant RT was characterized for RT DNA polymerase and RNase H activities, and for inhibition by BBNH.

RESULTS: The replacement of Y501 with G, L, Q, S and H resulted in elimination of RT RNase H activity. A low level of RNase H activity of the A, E and R mutants was noted at high RT concentrations, although the pattern of cleavage of the RNA/DNA duplex differed compared to wt enzyme. Replacement of Y501 by F or W had no effect on RNase H activity of RT, suggesting the requirement for an aromatic amino acid side chain at this position for optimal activity. In contrast to the Y501F mutation, the inhibitory potency of BBNH was reduced with the Y501W mutant. We propose that the increased bulk of this substitution may affect the ability of the inhibitor to contact the active site divalent metal, thereby decreasing the overall energy for binding.

CONCLUSIONS: The RNase H activity of HIV-1 RT requires an aromatic residue at position 501, as does the binding of the inhibitor BBNH. The apparent 'immutability' of Y501 suggests that contacts with this residue might be useful in the rational design of anti-RNase H therapeutics.

Keywords: AEGIS, HIV-1 Reverse Transcriptase, Ribonuclease H, Calf Thymus, DNA-Directed DNA Polymerase, HIV-1, Endoribonucleases, Hydrazones, Models, Molecular, Binding Sites, RNA, analysis
000709
TuOrA349