Frequent incorporation of ribonucleotides during HIV-1 reverse transcription and their attenuated repair in macrophages

Abstract

Macrophages are well known long-lived reservoirs of HIV-1. Unlike activated CD4(+) T cells, this nondividing HIV-1 target cell type contains a very low level of the deoxynucleoside triphosphates (dNTPs) required for proviral DNA synthesis whereas the ribonucleoside triphosphate (rNTP) levels remain in the millimolar range, resulting in an extremely low dNTP/rNTP ratio. Biochemical simulations demonstrate that HIV-1 reverse transcriptase (RT) efficiently incorporates ribonucleoside monophosphates (rNMPs) during DNA synthesis at this ratio, predicting frequent rNMP incorporation by the virus specifically in macrophages. Indeed, HIV-1 RT incorporates rNMPs at a remarkable rate of 1/146 nucleotides during macrophage infection. This greatly exceeds known rates for cellular replicative polymerases. In contrast, little or no rNMP incorporation is detected in CD4(+) T cells. Repair of these rNMP lesions is also substantially delayed in macrophages compared with CD4(+) T cells. Single rNMPs embedded in a DNA template are known to induce cellular DNA polymerase pausing, which mechanistically contributes to mutation synthesis. Indeed, we also observed that embedded rNMPs in a dsDNA template also induce HIV-1 RT DNA synthesis pausing. Moreover, unrepaired rNMPs incorporated into the provirus during HIV-1 reverse transcription would be generally mutagenic as was shown in Saccharomyces cerevisiae. Most importantly, the frequent incorporation of rNMPs makes them an ideal candidate for development of a new class of HIV RT inhibitors.

FIGURE 1.

Biochemical simulation of rNMP incorporation by HIV-1 RT in nucleotide pools found in human primary macrophages and CD4⁺ T cells. A–C, 5′ ³²P-17-mer DNA primer (10 nm) annealed to a 40-mer RNA template (A) was extended by HIV-1 RT (20 nm) in the presence of all four dNTPs and rNTPs found at physiological concentrations in human primary macrophages (B) or activated PBMC cells (C) (14) for 0, 2, 4, 6, 8, or 20 min. B and D, reaction products were then split and treated with 300 mm KOH or water and subjected to urea-PAGE. The KOH hydrolysis products (+PO₄) at the three C template sites (1C, 2C, and 3C) are illustrated by the arrows in B, and the first rCMP incorporation site (1C) is magnified in D. E, quantification of the KOH hydrolysis products in D is shown as the percent of its dNMP extension product counterpart for both macrophage and peripheral blood mononuclear cell concentrations at sites 1C and 2C in A. These reactions were repeated with a second template/primer set encoding different sequences with identical results in supplemental Fig. S1, and assessment of the electrophoretic mobility of the alkaline hydrolysis products resulting from monoribonucleotides in DNA is also included in supplemental Fig. S1C.

FIGURE 2.

In vivo rNMP incorporation during HIV-1 DNA synthesis in human primary macrophages and activated CD4⁺ T cells. 2LTR circles were isolated from HIV-1 vector-transduced macrophages and activated CD4⁺ T cells isolated from four independent donors. The isolated viral DNA was then treated with Jurkat nuclear extract containing potent RNase H2 activity. A, RNase H2 activity of the Jurkat nuclear extract (Ext) confirmed by specific cleavage of a dsDNA substrate containing a single rNMP whereas the buffer control (Buf) had no cleavage activity. The proviral DNA treated with nuclear extract was then subjected to 2LTR circle qPCR, after confirming the heat stability of the rNMP containing substrate and the absence of dU contamination (supplemental Fig. S2). B, qRT-PCR profiles from macrophages and T cell samples treated with buffer or nuclear extract. C, averages of the 2LTR circle DNA copy numbers and quantitative RT-PCR standard deviations for four blood donors, for activated CD4⁺ T cells and macrophages at 48 hours post infection and just for macrophages at 24 hours post infection. D, means ± S.D. (error bars) for both cell types at the 48 hr time point. The fraction of proviral DNA containing rNMPs in each of the HIV-1 target cell type was used to calculate the in vivo rNMP incorporation frequency.

FIGURE 3.

Human primary macrophage nuclear extracts exhibit reduced RNase H2 and FEN1 activity compared with CD4⁺ T cells. A, nuclear extracts from 293 FT cells, Jurkats, two donors of activated CD4⁺ T cells, and primary macrophages were normalized by total protein, and expression of RNase H2A and FEN1 were detected as described. B, depiction of RNase H2- and FEN1-specific substrates. C, nuclear extracts were diluted 1/1, 1/4, 1/80, 1/160, 1/320, and 1/640 RNase H2 activity assessed as described previously. D, nuclear extracts were diluted 1/10, 1/40, 1/80, 1/160, 1/320, and 1/640 and FEN1 activity assessed as described under “Experimental Procedures.”

FIGURE 4.

Repair of ribonucleotides embedded in dsDNA is dNTP concentration-dependent. A and B, quantification of RNase H2 (A) and FEN1 (B) activity in nuclear extracts from four donors of CD4⁺ T cells and three donors of macrophages. Extracts were normalized by total protein and diluted in enzyme specific assays as described in Fig. 3. Percent product formation was determined by densitometry using Quantity One software and normalized for total protein in each reaction per unit time. C, 5′ ³²P-labeled 80-mer DNA hairpin substrate was used as size marker (M) for the ligated repair product (**) of a 60-mer looped partial dsDNA substrate containing a 5′ rCMP annealed to a 5′ ³²P-labeled primer (*). The ribonucleotide containing substrate was incubated for 30 min with 4 μl of Jurkat nuclear extract diluted 1/20, 2 mm ATP, 1 mm dNTPs, 20 μm oligo(dT), and reaction buffer. Reactions were terminated and analyzed as described previously. D, reaction conditions similar to those described in C, except reactions contained either 100, 10, 1, 0.1, 0.01, or 0 μm dNTPs, with or without 2 mm ATP. E, capability of macrophages and activated CD4⁺ T cells to complete the entire process of rNMP repair, following RNase H2 cleavage, compared using the nuclear extracts and the substrate depicted in C which contains a nick 5′ of the rCMP. The concentrations of dNTPs found in activated CD4⁺ T cells (CD4) and macrophages (Mac) were used in these reactions and right is 10 μm. Successful rNMP removal, DNA gap filling and ligation will generate an 80-nucleotide-long repair product (**).

FIGURE 5.

Kinetic pausing of HIV-1 RT induced by rNMP-containing template. 17-mer 5′ ³²P-labeled primer was annealed to a 30-mer DNA template (left) or a DNA template containing three rNMPs (right), extended by HIV-1 RT (20 nm) at macrophage dNTP concentrations for 0, 2, 4, 6, 8, or 20 min. F is the fully extended primer, and NE represents reactions without RT. The rNMP pausing sites are marked with *.