TY - JOUR
T1 - Abundant non-canonical dUTP found in primary human macrophages drives its frequent incorporation by HIV-1 reverse transcriptase
AU - Kennedy, Edward M.
AU - Daddacha, Waaqo
AU - Slater, Rebecca
AU - Gavegnano, Christina
AU - Fromentin, Emilie
AU - Schinazi, Raymond F.
AU - Kim, Baek
PY - 2011/7/15
Y1 - 2011/7/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=79960124315&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79960124315&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111.234047
DO - 10.1074/jbc.M111.234047
M3 - Article
C2 - 21454906
AN - SCOPUS:79960124315
SN - 0021-9258
VL - 286
SP - 25047
EP - 25055
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 28
ER -