Abundant Non-Canonical dUTP Found in Primary Human Macrophages Drives Its Frequent Incorporation by Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Terminally differentiated/non-dividing macrophages contain extremely low cellular dNTP concentrations (20-40 nM), compared to 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, while 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 demonstrated that the non-canonical dUTP was abundant relative to TTP, and efficiently incorporated during HIV-1 reverse transcription, particularly in non-dividing macrophages.