Comparative Enzymatic Study of HIV-1 Reverse Transcriptase Resistant to 2‘,3‘-Dideoxynucleotide Analogs Using the Single-Nucleotide Incorporation Assay

Abstract

Employing the single-nucleotide incorporation assay using a heteropolymeric RNA template and DNA primers, we defined enzymatic profiles of recombinant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing a set of five mutations [A62V, V75I, F77L, F116Y, and Q151M] which confers resistance to multiple 2‘,3‘-dideoxynucleosides (ddNs) on HIV-1. RTs containing other drug-resistance-associated mutations were also examined. The K_m for dNTPs, the k_cat, and the k_cat/K_m ratios of mutant RTs were all comparable to those of wild-type RT (RT_wt). The processive primer extension activity of mutant RTs was also comparable to that of RT_wt as examined in the presence of saturating concentrations of dNTPs and heparin. Determination of the K_i values toward 5‘-triphosphates (TP) of various ddNs [3‘-azido-2‘,3‘-dideoxythymidine (AZT), 2‘,3‘-didehydro-2‘,3‘-dideoxythymidine (D4T), 2‘,3‘-dideoxycytidine (ddC), (−)-β-l-2‘,3‘-dideoxy-3‘-thiacytidine (3TC), (−)-β-l-2‘,3‘-dideoxy-5-fluorocytidine (FddC), 2‘,3‘-dideoxyadenosine (ddA), and 2‘-β-fluoro-2‘,3‘-dideoxyadenosine (FddA)] and 9-(2-phosphonylmethoxyethyl)adenine diphosphate (PMEApp) revealed that RT_{A62V/V75I/F77L/F116Y/Q151M} was insensitive to ddATP, AZTTP, D4TTP, FddATP, and ddCTP, but was sensitive to PMEApp, 3TCTP, and FddCTP. RT_K65R was less sensitive to ddATP, FddATP, PMEApp, ddCTP, and 3TCTP, while RT_M184V was less sensitive only to 3TCTP and ddCTP. The determination of K_i(ddNTP)/K_m(dNTP) ratios showed that AZTTP, D4TTP, and ddCTP are, as substrates, as efficient for RT_wt as their corresponding dNTPs, that ddATP, PMEApp, and 3TCTP are moderately efficient substrates for RT_wt, and that FddATP is the least efficient substrate among ddNTPs examined. 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 discrimination of ddNTPs from natural substrates.