Abstract
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.
Highlights
In this report we demonstrate that nucleoside reverse transcriptase inhibitors (NNRTIs) potently and inhibit plus-strand initiation in vitro under conditions in which little or no inhibition of minus-strand DNA synthesis is observed, and we identify the structural features of the plus-strand initiation substrate that contribute to NNRTI sensitivity
We have explored the effects of NNRTIs on plusstrand initiation and have demonstrated the following. (i) NNRTIs selectively inhibit plus-strand initiation from an RNA PPT primer trivial possibility that the rate effect of the NNRTI on plus- under conditions in which little or no inhibition of minusstrand initiation is a consequence of this miscleavage, we strand synthesis is observed
Related substrates in which the conducted the assay using reverse transcriptase (RT) containing the RNase H active RNA PPT primer is replaced by a DNA primer of the same site mutation D443N, which is devoid of RNase H activity
Summary
Reagents—All oligonucleotides were synthesized by Integrated DNA Technologies unless otherwise noted. In some experiments a homogeneous scintillation proximity assay (SPA, Amersham Biosciences) was used to characterize the effects of NNRTIs on plus-strand initiation For this assay format, a 5Ј-biotinylated 80-nt DNA template of the same sequence of that described above annealed to the various PPT primers was utilized, and [3H]dATP was used as the radiolabel instead of [32P]dATP. Ligand Binding Assay—We used an adaptation of the scintillation proximity assay described above to measure the affinity of incoming dNTPs to RT assembled on PPT primer-template substrates For these studies, we employed dideoxy-terminated RNA and DNA primers to prevent incorporation of the incoming dNTP, and we used RT containing the D443N mutation in the RNase H activity site to preempt any degradation of the nucleic acid. Plates were allowed to rest for 8 h prior to counting in a Topcount plate reader as described above
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