Dataset Information

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 ?m). However, our LC-MS/MS study revealed that the non-canonical dUTP concentration (2.9 ?m) 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.

SUBMITTER: Kennedy EM

PROVIDER: S-EPMC3137078 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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

Kennedy Edward M EM Daddacha Waaqo W Slater Rebecca R Gavegnano Christina C Fromentin Emilie E Schinazi Raymond F RF Kim Baek B

The Journal of biological chemistry 20110331 28

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 un ...[more]

PMID: 21454906

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Dataset Information

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

Publications

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

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