The pyrophosphate analogue foscarnet traps the pre-translocational state of HIV-1 reverse transcriptase in a Brownian ratchet model of polymerase translocation
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.
- SourceAvailable from: Mikhail Kashlev
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- "   , in the T7 RNAP , and in the HIV-1 reverse transcriptase . Furthermore, pyrophosphate shifts the translocation equilibrium toward the pre-translocated state in T7 RNAP , and pyrophosphate analogues shift the translocation equilibrium toward the pre-translocated state in HIV reverse transcriptase ; the effect of pyrophosphate or a pyrophosphate analogue on the translocation equilibrium of multi-subunit RNAPs has not yet been addressed. "
ABSTRACT: DNA template and RNA/DNA hybrid movement through RNA polymerase (RNAP) is referred to as "translocation". Because nucleic acid movement is coupled to NTP loading, pyrophosphate release, and conformational changes, the precise ordering of events during bond addition is consequential. Moreover, based on several lines of experimental evidence, translocation, pyrophosphate release or an associated conformational change may determine the transcription elongation rate. In this review we discuss various models of translocation, the data supporting the hypothesis that translocation rate determines transcription elongation rate and also data that may be inconsistent with this point of view. A model of the nucleotide addition cycle accommodating available experimental data is proposed. On the basis of this model, the molecular mechanisms regulating translocation and potential routes for NTP entry are discussed.Biochimica et Biophysica Acta 05/2010; 1799(5-6):389-401. DOI:10.1016/j.bbagrm.2010.01.007 · 4.66 Impact Factor
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ABSTRACT: Human herpesvirus 6 (HHV-6), which is closely related to human cytomegalovirus, is sensitive to foscarnet (PFA). Up to now, the resistance of HHV-6 to PFA has not been investigated. The goal of the study was to isolate and characterize PFA-resistant HHV-6 mutants in order to determine the mechanisms of resistance to the drug. PFA-resistant viruses, isolated in MT4 cell culture under increasing concentrations of PFA, were characterized phenotypically and genotypically. The mutations identified in the HHV-6 DNA polymerase gene were evaluated in a functional assay using recombinant mutated forms of the enzyme, and their effect on protein structure was analysed in a three-dimensional model derived from available structures of DNA polymerases. Two mutants were selected and were 8- and 15-fold more resistant to PFA than the wild-type strain. Four amino acid changes were detected in the HHV-6 DNA polymerase in association with PFA resistance: T435R, H507Y, C525S, located in the deltaC conserved domain, and F292S. Either alone or in combination, these substitutions significantly decreased the inhibitory effect of PFA at the level of the polymerase, as measured by the incorporation of radiolabelled nucleotides in a DNA elongation assay. In the three-dimensional model of HHV-6 DNA polymerase structure the four changes were not located within the putative catalytic site, but they might induce either a disturbance of local conformation or a restricted access of PFA to its target site. This first characterization of HHV-6 resistance to PFA highlights the role of distinct DNA polymerase gene mutations.Antiviral therapy 02/2007; 12(6):877-88. · 3.14 Impact Factor
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ABSTRACT: Binding of the next complementary dNTP by the binary complex containing HIV-1 reverse transcriptase (RT) and primer-template induces conformational changes that have been implicated in catalytic function of RT. We have used DNase I footprinting, gel electrophoretic mobility shift, and exonuclease protection assays to characterize the interactions between HIV-1 RT and chain-terminated primer-template in the absence and presence of various ligands. Distinguishable stable complexes were formed in the presence of foscarnet (an analog of pyrophosphate), the dNTP complementary to the first (+1) templating nucleotide or the dNTP complementary to the second (+2) templating nucleotide. The position of HIV-1 RT on the primer-template in each of these complexes is different. RT is located upstream in the foscarnet complex, relative to the +1 complex, and downstream in the +2 complex. These results suggest that HIV-1 RT can translocate along the primer-template in the absence of phosphodiester bond formation. The ability to form a specific foscarnet complex might explain the inhibitory properties of this compound. The ability to recognize the second templating nucleotide has implications for nucleotide misincorporation.Journal of Molecular Biology 05/2007; 369(1):41-54. DOI:10.1016/j.jmb.2007.03.006 · 4.33 Impact Factor