Selective Excision of Chain-terminating Nucleotides by HIV-1 Reverse Transcriptase with Phosphonoformate as Substrate

Abstract

A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP i ) analogs on PP i -and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP i -and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT⅐template/primer complex was hindered by the presence of a chain terminator at the 3-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZTtriphosphate in the presence of PP i or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

“…To account for the non-competitive mode of inhibition it has been suggested that PFA may likewise bind to the pre-translocated complex (8). High concentrations of PFA appear to excise incorporated AZT-monophosphate, which provides some support for this notion (44). However, it remains to be seen whether the pre-translocational state is accessible during active DNA synthesis.…”

Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

“…[11][12][13][14]31 Comparison of crystal structures of ternary complexes 20,32 with those of binary complexes 33,34 show an open-toclosed transformation. Electrophoretic mobility shift experiments with the pyrophosphate (PPi) product analogue, phosphonoformate (foscarnet), show that a stable complex can also be formed with this compound, [35][36][37] suggesting that the enzyme can adopt a closed conformation in the presence of either dNTP substrate or PPi product. Crystal structures of polymerase•P/T•PPi complexes have recently been reported for T7 RNA polymerase, 38 the Y-family DNA polymerase Dpo4, 39 and the X-family DNA polymerase pol-λ.…”

Foscarnet salvage therapy efficacy is associated with the presence of thymidine-associated mutations (TAMs) in HIV-infected patients

“…4,5 The relationship observed here between the virological response to foscarnet in vivo and the number of TAMs is compatible with a recent study showing that foscarnet interferes with phosphorolysis reactions in vitro. 6 By acting on RT-catalysed nucleotide excision, foscarnet would enhance the effect of AZT, particularly on AZT-resistant viruses harbouring TAMs. Thus, TAMs can be beneficial in two ways: (i) by inducing hypersusceptibility to foscarnet, and (ii) by preventing the selection of foscarnet resistance mutations.…”

“…5 Being a PP i analog, foscarnet acts as a substrate for phosphorolysis and is able to block the excision of chain-terminating nucleotides catalysed by HIV RT. 6 Excision of chain-terminating nucleotides is the main mechanism of resistance to thymidine analogs (AZT and d4T). TAMs increase the capacity of RT to remove thymidine analogs from growing DNA chains.…”