AEGiS-11IAC: Comparative enzymatic study of HIV reverse transcriptase resistant to multiple 2',3'-dideoxynucleoside 5'-triphosphates.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Comparative enzymatic study of HIV reverse transcriptase resistant to multiple 2',3'-dideoxynucleoside 5'-triphosphates.

Int Conf AIDS 1996 Jul 7-12; 11:212 (abstract no. Th.A.151)
Ueno T, Mitsuya H; Japan Energy Co., Toda-shi, Saitama 335, Japan. Fax: +81-48-443-1605.

OBJECTIVE: To define enzymatic profiles of recombinat HIV-1 reverse transcriptase (RT) which contains a set of five mutations (A62V, V75I, F77L, F116Y, and Q151M) identified in HIV-1 variants which show resistance to multiple 2',3'-dideoxynucleosides (ddNs).

METHODS: The single nucleotide incorporation assay using heteropolymeric RNA template and DNA primers was employed. RTQ151M, RTA62V/V75I/F77L/F116Y/Q151M, and RTs containing other mutations, such as K65R, L74V, M184V, or T215Y were examined.

RESULTS: The Km values for dNTPs, the kcat values, the kcat/Km ratio, and the processivity of mutant RTs examined were all comparable to those of wild-type RT (RTwt). Determination of the Ki values toward 5'-triphosphates (TP) of various ddNs [AZT, D4T, ddC, 3TC, (-)-beta-L-2',3'-dideoxy-5-fluorocytidine ((-)FddC), ddA, 2'-beta-fluoro-2',3'-dideoxyadenosine (FddA)] and PMEA diphosphate (PMEApp) revealed that RTA62V/V75I/F77L/F116Y/Q151M was highly insensitive to ddATP (different in Ki compared to RTwt by 17-fold) and AZTTP (21-fold), and less sensitive to D4TTP, FddATP, and ddCTP (5- to 10-fold), but was sensitive to PMEApp, 3TCTP, and (-)FddCTP (less than 3-fold). RTk65R was less sensitive to ddATP, FddATP, PMEApp, ddCTP and 3TCTP (5- to 13-fold), although it was sensitive to AZTTP and D4TTP (less than or equal to 3-fold), while RTM184v was less sensitive only to 3TCTP (72-fold) and ddCTP (7-fold). RTL74V was modestly less sensitive to ddATP (3.2-fold) and FddATP (3.7-fold), while RTT215Y was sensitive to all ddNTPs examined (less than 3-fold).

CONCLUSIONS: The observed cross-resistance of HIV-1 RT to various ddNTPs should reflect the alteration of RT's substrate recognition and should provide insights into the molecular mechanism of RT discriminating ddNTPs from natural substrates.

Keywords: AEGIS, RNA-Directed DNA Polymerase, Dideoxynucleosides, HIV-1 Reverse Transcriptase, Reverse Transcriptase Inhibitors, HIV-1, Lamivudine, Zidovudine, Zalcitabine, Anti-HIV Agents, Templates, Genetic, DNA Primers, Thymine Nucleotides, Deoxyadenine Nucleotides, Deoxycytosine Nucleotides, Polyphosphates, Dideoxyadenosine, 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxy-5-fluorocytidine 5'-triphosphate, 2',3'-dideoxyadenosine triphosphate, triphosphoric acid, genetics, ICA11

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ThA151