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 heteropolymeri c RNA template and DNA primers, we defined enzymatic profiles of recom binant human immunodeficiency virus type 1 (HIV-I) reverse transcripta se (RT) containing a set of five mutations [A62V, V75I, F77L, F116Y, a nd Q151M] which confers resistance to multiple 2',3'-dideoxynucleoside s (ddNs) on HIV-1. RTs containing other drug-resistance-associated mut ations were also examined. The K-m for dNTPs, the k(cat), and the k(ca t)/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 saturat ing concentrations of dNTPs and heparin. Determination of the Ki value s toward 5'-triphosphates (TP) of various ddNs [3'-azido-2',3'-dideoxy thymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (D4T), 2',3'-d ideoxycytidine (ddC), (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), (-)-beta-L-2',3'-dideoxy-5-fluorocytidine (FddC), 2',3'-dideoxyadenosi ne (ddA), and 2'-beta-fluoro-2',3'-dideoxyadenosine (FddA)1 and 9-(2-p hosphonylmethoxyethyl)adenine diphosphate (PMEApp) revealed that RT(A6 2V/V75I/F77/L/F116Y/Q151M) was insensitive to ddATP, AZTTP, D4TTP. Fdd ATP, and ddCTP, but was sensitive to PMEApr,, 3TCTP, and FddCTP. RT(K6 5R) was less sensitive to ddATP, FddATP, PMEApp, ddCTP, and 3TCTP, whi le RT(M184V) was less sensitive only to 3TCTP and ddCTP. The determina tion of K-i(ddNTP)/Km((dNTP)) ratios showed that AZTTP, D4TTP, and ddC TP are, as substrates, as efficient for RT(wt) as their corresponding dNTPs. that substrate among ddNTPs examined. The observed cross-resist ance of HIV-1 RT to various ddNTPs should reflect the alteration of RT 's substrate recognition and should provide insights into the molecula r mechanis, of RT descrimination of ddNTPs from natural substances.