Inhibition of human immunodeficiency virus type 1 reverse transcriptase by 3'-blocked oligonucleotide primers.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (EC 2.7.7.49) with a high specific activity has been purified from the overexpressing Escherichia coli strain DH5 alpha [pJS3.7]. Steady-state kinetics of DNA synthesis catalysed by RT were analysed on polyriboadenylate 20-mer of (3'-5')deoxythymidylate [poly(rA).(dT)20] and polyribouridylate 20-mer of (3'-5')-deoxyadenylate [poly(rU).(dA)20] homopolymeric template-primers. Km values of 40 and 140 nM (3'-OH ends) and kcat values of 4 and 0.14 sec-1 were determined for the two different substrates. Oligonucleotide primers (dA)20 and (dT)20 were elongated in a terminal transferase-catalysed reaction (EC 2.7.7.31) with ddATP, 3'-dATP (cordycepin), 2',3'-epoxy-ATP and arabino-ATP; and ddTTP, 3'-azido-TTP, 3'-dUTP, 3'-F-dTTP and rUTP, respectively. The resulting oligonucleotides were hybridized to their complementary templates and the inhibitory potential of these compounds towards DNA synthesis started from unchanged primers was measured. Oligonucleotides with unextendable 3'-groups were shown to act as strong inhibitors of DNA synthesis catalysed by HIV-1 RT. In particular, poly(rA).(dT)20-[ddTMP] and poly(rU).(dA)20-[3'-dAMP] were potent competitive inhibitors, displaying Ki values of about 6 and 12 nM, respectively. Also 3'-azido-, and 3'-fluoro-terminated oligonucleotides showed competitive inhibition with inhibition constants in the range of 20-35 nM. In contrast, 2',3'-epoxy-terminated (dA)21 displayed a mixed-type inhibition with a Ki value of 67 nM. Arabino-terminated (dA)21 was found to be an uncompetitive inhibitor of HIV-1 RT with an inhibition constant of 318 nM. Arabino-terminated primers did not act as strict chain terminators because they could be elongated by HIV-1 RT. This study provides information on the structure-activity relationship of modified 3'-termini of primer molecules which might be exploited as inhibitors of HIV in the future.