title: A pyrophosphatase activity associated with purified HIV-1 particles.
abstract: Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3'-azido-3'-deoxythymidine (AZT) and to a lesser extent to 2'-3'-didehydro-2'-3'-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate. However, in vitro, resistant RT still uses pyrophosphate more efficiently than ATP. We performed in vitro (-) strong-stop DNA synthesis experiments, with wild-type and AZT-resistant HIV-1 RTs, in the presence of physiologically relevant pyrophosphate and/or ATP concentrations and found that in the presence of pyrophosphate, ATP and AZTTP, TAMs do not enhance in vitro (-) strong-stop DNA synthesis. We hypothesized that utilisation of ATP in vivo is driven by intrinsic low pyrophosphate concentrations within the reverse transcription complex, which could be explained by the packaging of a cellular pyrophosphatase. We showed that over-expressed flagged-pyrophosphatase was associated with HIV-1 viral-like particles. In addition, we demonstrated that when HIV-1 particles were purified in order to avoid cellular microvesicle contamination, a pyrophosphatase activity was specifically associated to them. The presence of a pyrophosphatase activity in close proximity to the reverse transcription complex is most likely advantageous to the virus, even in the absence of any drug pressure.
authors: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 15 Rue René Descartes, 67084 Strasbourg, France. celine.ducloux@gmail.com
authors: Céline
authors: C
authors: Ducloux
authors: Marylène
authors: M
authors: Mougel
authors: Valérie
authors: V
authors: Goldschmidt
authors: Ludovic
authors: L
authors: Didierlaurent
authors: Roland
authors: R
authors: Marquet
authors: Catherine
authors: C
authors: Isel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019380:Anti-HIV Agents / Q000378:metabolism / Q000494:pharmacology; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D054306:Dideoxynucleotides / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D018119:Stavudine / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism; D014771:Virion / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D015215:Zidovudine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
chemical_list: D019380:Anti-HIV Agents; D004279:DNA, Viral; D054306:Dideoxynucleotides; D011756:Diphosphates; D013942:Thymine Nucleotides; C041124:strong-stop DNA; D015215:Zidovudine; C107241:diphosphoric acid; C052873:zidovudine triphosphate; D000255:Adenosine Triphosphate; D018119:Stavudine; D054303:HIV Reverse Transcriptase; D011755:Pyrophosphatases