Comparative enzymatic study of HIV-1 reverse transcriptase resistant to 2',3'-dideoxynucleotide analogs using the single-nucleotide incorporation assay

Employing single-nucleotide incorporation assay using a heteropolymeric RNA template and DNA primers, we defined enzymatic profiles of recombinant human immunodeficiency virus type 1 (HIV-I) 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,,). The processive primer extension activity of mutant RTs was also comparable to that of RT,, as examined in the presence of saturating concentrations of dNTPs and heparin. Determination of the Ki 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), (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), (-)-beta-L-2',3'-dideoxy-5-fluorocytidine (FddC), 2',3'-dideoxyadenosine (ddA), and 2'-beta-fluoro-2',3'-dideoxyadenosine (FddA)1 and 9-(2-phosphonylmethoxyethyl)adenine diphosphate (PMEApp) revealed that RT(A62V/V75I/F77/L/F116Y/Q151M) was insensitive to ddATP, AZTTP, D4TTP. FddATP, and ddCTP, but was sensitive to PMEApr,, 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)/Km((dNTP)) ratios showed that AZTTP, D4TTP, and ddCTP are, as substrates, as efficient for RT(wt) as their corresponding dNTPs. that 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 mechanis, of RT descrimination of ddNTPs from natural substances.