A pyrophosphatase activity associated with purified HIV-1 particles

Biochimie. 2012 Dec;94(12):2498-507. doi: 10.1016/j.biochi.2012.06.025. Epub 2012 Jul 3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Anti-HIV Agents / metabolism
  • Anti-HIV Agents / pharmacology
  • DNA, Viral / genetics
  • DNA, Viral / metabolism
  • Dideoxynucleotides / metabolism
  • Diphosphates / metabolism
  • Drug Resistance, Viral / drug effects
  • Drug Resistance, Viral / genetics
  • HIV Reverse Transcriptase / antagonists & inhibitors
  • HIV Reverse Transcriptase / genetics
  • HIV Reverse Transcriptase / metabolism*
  • HIV-1 / drug effects
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • Kinetics
  • Mutation
  • Pyrophosphatases / genetics
  • Pyrophosphatases / metabolism*
  • Stavudine / metabolism
  • Stavudine / pharmacology
  • Substrate Specificity
  • Thymine Nucleotides / metabolism
  • Virion / drug effects
  • Virion / enzymology*
  • Virion / genetics
  • Zidovudine / analogs & derivatives
  • Zidovudine / metabolism
  • Zidovudine / pharmacology

Substances

  • Anti-HIV Agents
  • DNA, Viral
  • Dideoxynucleotides
  • Diphosphates
  • Thymine Nucleotides
  • strong-stop DNA
  • Zidovudine
  • diphosphoric acid
  • zidovudine triphosphate
  • Adenosine Triphosphate
  • Stavudine
  • HIV Reverse Transcriptase
  • Pyrophosphatases