HIV-1 reverse transcriptase plus-strand initiation exhibits preferential sensitivity to non-nucleoside reverse transcriptase inhibitors in vitro

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are highly specific and potent allosteric inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. NNRTIs inhibit reverse transcription in a substrate length-dependent manner in biochemical assays and in cell-based HIV-1 replication assays, suggesting a stochastic inhibitory mechanism. Surprisingly, we observed that NNRTIs potently inhibited plus-strand initiation in vitro under conditions in which little or no inhibition of minus-strand DNA synthesis was observed. In assays that recapitulated the initiation of plus-strand DNA synthesis, greater inhibition was observed with an RNA PPT primer than with a DNA primer of corresponding sequence and with wild-type reverse transcriptase but not with NNRTI-resistant enzymes. Structural elements that dictate sensitivity to NNRTIs were revealed using modified plus-strand initiation substrates. The data presented here suggest that specific inhibition of plus-strand initiation may be an important mechanism by which NNRTIs block HIV-1 replication.