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 ϳ60 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.

GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long-lived activated state

“…An enigmatic feature of SAMHD1 activity in resting immune cells is the presence of elevated dUTP levels in an otherwise depleted dNTP pool background (26,27). Given our findings that dUTP is an excellent substrate, there must be undiscovered regulatory mechanisms that allow dUTP levels to persist or accumulate after the other dNTPs have been depleted.…”

Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

“…This is due to the higher discrepancy between dNTP and rNTP levels in macrophages (4). Also, previously published studies have shown that the presence of rNMPs in a DNA template causes DNA polymerases to pause during DNA synthesis (7,23). Thus, we tested whether the presence of rNMPs in DNA templates also has the same pausing impact on HIV-1 RT-mediated DNA synthesis.…”

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

“…Finally, if the viral cDNA is uracilated to a sufficiently high density (12,25), it seems inevitable that the host nuclear UNG would excise the uracils, leading to its degradation once it entered the nucleus. There is little or no experimental data available to establish the importance of any of the above possible outcomes in human cells.…”

“…Not surprisingly, dUTPase and UNG are coordinately regulated throughout the cell cycle of dividing cells, with the highest expression levels during the G1 and S phases (9,10). However, in quiescent cells such as macrophages and neurons, dUTP can accumulate to high levels, and mRNA expression of both enzymes is barely detectable (11)(12)(13).…”

“…Biochemical evidence for this innate immunity pathway is suggested by the observation that dUTPase-deficient mutants of nonprimate lentiviruses cannot infect nondividing cells (where host cell dUTPase expression is low and dUTP levels are high) but are fully competent for infection of dividing cells (18)(19)(20)(21). Interestingly, HIV-1 does not encode a dUTPase yet is still able to infect human macrophages (albeit with reduced efficiency), even though the dUTP:dTTP ratio in these cells is as high as 60:1 (12).…”