The pyrophosphate analogue foscarnet traps the pre-translocational state of HIV-1 reverse transcriptase in a Brownian ratchet model of polymerase translocation.
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.
Marchand
Bruno
B
Department of Microbiology & Immunology, McGill University, Montréal, Québec H3A 2B4, Canada.
Tchesnokov
Egor P
EP
Götte
Matthias
M
D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000187:drug effects / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D017245:Foscarnet / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000235:genetics; D007700:Kinetics; D008954:Models, Biological; D021381:Protein Transport; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013698:Templates, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D017245:Foscarnet; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase