The pyrophosphate analogue foscarnet traps the pre-translocational state of HIV-1 reverse transcriptase in a Brownian ratchet model of polymerase translocation

J Biol Chem. 2007 Feb 2;282(5):3337-46. doi: 10.1074/jbc.M607710200. Epub 2006 Nov 30.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Primers
  • DNA Replication / drug effects
  • DNA, Viral / drug effects
  • DNA, Viral / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Foscarnet / pharmacology*
  • HIV Reverse Transcriptase / antagonists & inhibitors*
  • HIV Reverse Transcriptase / genetics
  • Kinetics
  • Models, Biological
  • Protein Transport
  • Recombinant Proteins / antagonists & inhibitors
  • Reverse Transcriptase Inhibitors / pharmacology*
  • Templates, Genetic


  • DNA Primers
  • DNA, Viral
  • Recombinant Proteins
  • Reverse Transcriptase Inhibitors
  • Foscarnet
  • HIV Reverse Transcriptase
  • DNA-Directed DNA Polymerase