Abundant Non-canonical dUTP Found in Primary Human Macrophages Drives Its Frequent Incorporation by HIV-1 Reverse Transcriptase

• Published in: The Journal of biological chemistry 2011-07, Vol.286 (28), p.25047-25055
• Main Authors: Kennedy, Edward M., Daddacha, Waaqo, Slater, Rebecca, Gavegnano, Christina, Fromentin, Emilie, Schinazi, Raymond F., Kim, Baek
• Format: Article
• Language: English
• Subjects: Cells, Cultured; DNA and Chromosomes; DNA Polymerase; DNA, Viral - biosynthesis; Enzyme Kinetics; HIV Reverse Transcriptase - metabolism; HIV-1 - enzymology; Humans; Kinetics; Macrophages - metabolism; Mutagenesis Mechanisms; Nucleotide; Proviruses - metabolism; Reverse Transcription; Reverse Transcription - physiology; Simian Immunodeficiency Virus - enzymology; Uridine Triphosphate - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.234047

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.