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 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.

FIGURE 1.

Comparison of dUTP and TTP concentrations of human primary macrophages and activated PBMCs. A, the dUTP concentrations of macrophages and activated PBMCs, which were determined by LC-MS/MS (summarized in Table 1), are plotted with gray bars. The black bars represent TTP concentrations of human primary macrophages and activated CD4⁺ T cells previously determined by LC-MS/MS (9). The -fold differences between dTTP and dUTP in each cell type are shown. B, the dUTP/TTP concentration ratio difference between the two cell types calculated from the LC-MS/MS analysis data.

FIGURE 2.

dUTP incorporation by HIV-1 RT with nucleotide substrate pools found in primary human macrophages and PBMCs. An RT extension reaction coupled with a UNG2 digestion assay is shown in A. A 5′-end ³²P-labeled 23-mer DNA primer annealed to the 40-mer RNA template was extended in vitro by HXB2 HIV-1 RT with the reconstituted dNTP concentrations found in macrophages or PBMCs in the presence and absence of dUTP concentration found in those cell types, and then these reactions were quenched with EDTA and subjected to E. coli UNG2 digestion, followed by 95 °C incubation and analyzed by urea denaturing PAGE. The cleavage products induced by the dUTP incorporation are marked with an asterisk. P, primer. B, total fully extended products (FE) after heat treatment were determined, and the -fold difference between the fully extended product levels of the reactions under the two cell type conditions is shown.

FIGURE 3.

TTP and dUTP incorporation rates of HIV-1 RT in macrophage conditions. A, single nucleotide extension reaction time-courses using the same template-primer (P) used in Fig. 2 were performed in vitro with the reconstituted TTP and dUTP concentrations found in macrophages. E, extended product. B, product formation for TTP (black line), dUTP (blue line), and both TTP and dUTP (green line) were plotted. C, the rates of the product formation (B) were determined with a double exponential equation as described under “Experimental Procedures.” A 20 nm final concentration of HIV-1 RT was used to initiate the reaction in which small aliquots were obtained over the time course indicated. The experiments were repeated in triplicate.

FIGURE 4.

dUTP incorporation by other lentiviral and non-lentiviral RT proteins. UNG-2 digestion assays were carried out as described in Fig. 2 for SIV_agm, MuLV, and FV RTs. Uracil digestion products . are marked with an asterisk. FE, fully extended product; P, primer.

FIGURE 5.

Effect of ddU treatment on HIV-1 replication in primary human macrophages and activated CD4⁺ T cells. Primary human macrophage were pretreated with ddU and then transduced with a single round vesicular stomatitis virus glycoprotein pseudotyped HIV-1 vector (D3HIV). A, the normalized vector transduction efficiency in macrophages at the indicated ddU concentrations from n = 5 independent human donors. B, FACS analysis for the macrophages in A for GFP-expressing cells. C, the normalized vector transduction efficiency in human primary activated CD4⁺ T cells at the indicated ddU concentrations from three independent human donors. D, 2LTR circle quantitative PCR plotted by ddU concentration from four independent human donors. E, ddU toxicity assessed by the live and dead cell assay for primary human lung fibroblasts (MRC5), human microglial cell line (CHME5), human primary activated CD4⁺ T cells, and primary human macrophage.