title: Abundant non-canonical dUTP found in primary human macrophages drives its frequent incorporation by HIV-1 reverse transcriptase.
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.
authors: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors: Edward M
authors: EM
authors: Kennedy
authors: Waaqo
authors: W
authors: Daddacha
authors: Rebecca
authors: R
authors: Slater
authors: Christina
authors: C
authors: Gavegnano
authors: Emilie
authors: E
authors: Fromentin
authors: Raymond F
authors: RF
authors: Schinazi
authors: Baek
authors: B
authors: Kim
mesh_terms: D002478:Cells, Cultured; D004279:DNA, Viral / Q000096:biosynthesis*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008264:Macrophages / Q000378:metabolism*; D011533:Proviruses / Q000378:metabolism*; D048348:Reverse Transcription / Q000502:physiology*; D015302:Simian Immunodeficiency Virus / Q000201:enzymology; D014544:Uridine Triphosphate / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D014544:Uridine Triphosphate