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

Department of Microbiology & Immunology, McGill University, Montréal, Québec H3A 2B4, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2007; 282(5):3337-46. DOI: 10.1074/jbc.M607710200
Source: PubMed

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.

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    • "[11] [28] [70] [71], in the T7 RNAP [20], and in the HIV-1 reverse transcriptase [72]. Furthermore, pyrophosphate shifts the translocation equilibrium toward the pre-translocated state in T7 RNAP [20], and pyrophosphate analogues shift the translocation equilibrium toward the pre-translocated state in HIV reverse transcriptase [37]; the effect of pyrophosphate or a pyrophosphate analogue on the translocation equilibrium of multi-subunit RNAPs has not yet been addressed. "
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