Abundant non-canonical dUTP found in primary human macrophages drives its frequent incorporation by HIV-1 reverse transcriptase

Edward M. Kennedy, Waaqo Daddacha, Rebecca Slater, Christina Gavegnano, Emilie Fromentin, Raymond F. Schinazi, Baek Kim

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Terminally differentiated/non-dividing macrophages contain extremely low cellular dNTP concentrations (20-40 nM), compared with activated CD4 + T cells (2-5 μM). However, our LC-MS/MS study revealed that the non-canonical dUTP concentration (2.9 μM) is ∼60 times higher than TTP in macrophages, whereas the concentrations of dUTP and TTP in dividing human primary lymphocytes are very similar. Specifically, we evaluated the contribution of HIV-1 reverse transcriptase to proviral DNA uracilation under the physiological conditions found in HIV-1 target cells. Indeed, biochemical simulation of HIV-1 reverse transcription demonstrates that HIV-1 RT efficiently incorporates dUTP in the macrophage nucleotide pools but not in the T cell nucleotide pools. Measurement of both pre-steady state and steady state kinetic parameters of dUTP incorporation reveals minimal selectivity of HIV-1 RT for TTP over dUTP, implying that the cellular dUTP/TTP ratio determines the frequency of HIV-1 RT-mediated dUTP incorporation. The RT of another lentivirus, simian immunodeficiency virus, also displays efficient dUTP incorporation in the dNTP/dUTP pools found in macrophages but not in T cells. Finally, 2′,3′-dideoxyuridine was inhibitory to HIV-1 proviral DNA synthesis in macrophages but not in T cells. The data presented demonstrates that the non-canonical dUTP was abundant relative to TTP, and efficiently incorporated during HIV-1 reverse transcription, particularly in non-dividing macrophages.

Original languageEnglish (US)
Pages (from-to)25047-25055
Number of pages9
JournalJournal of Biological Chemistry
Volume286
Issue number28
DOIs
StatePublished - Jul 15 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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