Abstract
The pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet) inhibits the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1); however, the mechanisms of drug action and resistance remain elusive. Here we studied the effects of the translocational status of HIV-1 RT on drug binding and inhibition of DNA synthesis. We identified "hot spots" for inhibition during active elongation. Site-specific footprinting analyses revealed that the corresponding complexes exist predominantly in the pre-translocational state. The sensitivity to PFA is significantly reduced with sequences that show a bias toward the post-translocational state. Binding studies showed that PFA stabilizes selectively the complex in the pre-translocated configuration. These findings are consistent with a Brownian ratchet model of polymerase translocation. The enzyme can rapidly shuttle between pre- and post-translocated states. The bound inhibitor acts like a pawl of a ratchet and prevents the forward motion of HIV-1 RT, whereas the bound nucleotide binds to the post-translocated complex and prevents the reverse motion. The proposed mechanisms of RT translocation and drug action are consistent with the PFA-resistant phenotypes. We show that certain sequences and the PFA-resistant E89K mutant diminishes the stability of the pre-translocated complex. In these cases, the enzyme is seen at multiple positions around the 3' end of the primer, which provides a novel mechanism for resistance. These findings validate the pre-translocated complex as a target for the development of novel, perhaps less toxic and more potent inhibitors that block HIV-1 RT translocation.
Highlights
Transcriptase inhibitors (NRTIs) are major components in drug regimens that are currently used in the clinic
Steady-state kinetics with retroviral reverse transcriptase (RT) enzymes, and DNA polymerases of herpes simplex virus or cytomegalovirus suggest a non-competitive mode of inhibition of DNA synthesis with respect to the deoxynucleoside triphosphate substrate [4, 16, 17]
The substrate was derived from the polypurine tract (PPT) of human immunodeficiency virus type 1 (HIV-1) as plate, and 0.08 pmol of active site RT
Summary
Transcriptase inhibitors (NRTIs) are major components in drug regimens that are currently used in the clinic. The enzyme must translocate a single template position further downstream to clear the nucleotide binding site (N-site) This motion relative to the nucleic acid substrate brings the 3Ј end of the primer to the priming or product site (P-site) [24, 25]. High concentrations of PFA appear to excise incorporated AZT-monophosphate, which provides some support for this notion [44] It remains to be seen whether the pre-translocational state is accessible during active DNA synthesis. PFA-mediated inhibition of DNA synthesis is linked to primer/template sequences that facilitate the formation of the pre-translocational state. These findings suggest that pre- and post-translocated complexes equilibrate immediately following each nucleotide incorporation event. The results of this study validate the pretranslocated complex as a target for drug discovery and development efforts
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