Mechanistic and Kinetic Differences between Reverse Transcriptases of Vpx Coding and Non-coding Lentiviruses

Abstract

Among lentiviruses, HIV Type 2 (HIV-2) and many simian immunodeficiency virus (SIV) strains replicate rapidly in non-dividing macrophages, whereas HIV Type 1 (HIV-1) replication in this cell type is kinetically delayed. The efficient replication capability of HIV-2/SIV in non-dividing cells is induced by a unique, virally encoded accessory protein, Vpx, which proteasomally degrades the host antiviral restriction factor, SAM domain- and HD domain-containing protein 1 (SAMHD1). SAMHD1 is a dNTPase and kinetically suppresses the reverse transcription step of HIV-1 in macrophages by hydrolyzing and depleting cellular dNTPs. In contrast, Vpx, which is encoded by HIV-2/SIV, kinetically accelerates reverse transcription by counteracting SAMHD1 and then elevating cellular dNTP concentration in non-dividing cells. Here, we conducted the pre-steady-state kinetic analysis of reverse transcriptases (RTs) from two Vpx non-coding and two Vpx coding lentiviruses. At all three sites of the template tested, the two RTs of the Vpx non-coding viruses (HIV-1) displayed higher kpol values than the RTs of the Vpx coding HIV-2/SIV, whereas there was no significant difference in the Kd values of these two groups of RTs. When we employed viral RNA templates that induce RT pausing by their secondary structures, the HIV-1 RTs showed more efficient DNA synthesis through pause sites than the HIV-2/SIV RTs, particularly at low dNTP concentrations found in macrophages. This kinetic study suggests that RTs of the Vpx non-coding HIV-1 may have evolved to execute a faster kpol step, which includes the conformational changes and incorporation chemistry, to counteract the limited dNTP concentration found in non-dividing cells and still promote efficient viral reverse transcription.

Keywords: DNA replication; SAMHD1; Vpx; dNTP; enzyme kinetics; lentivirus; macrophage; reverse transcription.

FIGURE 1.

dNTP concentration-dependent DNA synthesis of RT enzymes from Vpx coding and non-coding lentiviruses. A, template (T) and primer (P) used in this study. 5′ ³²P-labeled 17-mer DNA primer was annealed to 40-mer RNA template. The three sites (*) used for pre-steady-state analysis are indicated. B, the T/P was extended by four purified RT enzymes from either Vpx non-coding or Vpx coding lentiviruses under the conditions described under “Experimental Procedures” at different dNTP concentrations (lanes 1–5: 1 μm, 500 nm, 200 nm, 100, nm, 50 nm). The RTs used from Vpx non-coding viruses were HIV-1 Cy (subtype A) and HIV-1 Ug (subtype D), and the RTs used from Vpx coding viruses were HIV-2 Rod and SIV 9063-2. RT activity used in this assay generated ∼50% primer extension at the high dNTP concentration found in activated CD4⁺ T cells (T and lane 1) as determined by the quantitation of the 40-bp fully extended product (F and ←). The three sites analyzed for the pre-steady-state kinetic study are also marked with *. (+): 50 μm dNTP positive control; (−): no dNTP control. T: dNTP concentration found in activated CD4⁺ T cells; M: dNTP concentration found in macrophages; X: dNTP concentration found in macrophages treated with Vpx (16). F: fully extended products; P: primer and unextended substrate.

FIGURE 2.

Active concentration determination and the pre-steady-state dATP incorporation kinetics of RT enzymes from Vpx non-coding and coding lentiviruses at Site 1 of the 40-mer RNA template. A, pre-steady-state burst kinetics of incorporation of dATP onto the T/P described under “Experimental Procedures” by the four RT enzymes. The solid line represents a fit to a burst equation. Burst experiments were repeated 2–3 times for each enzyme, and a representative curve for each enzyme is shown. Percentages of the active concentrations for HIV-1 Cy, HIV-1 Ug, HIV-2 Rod, and SIV 9063-2 are 40, 73, 55, and 84%, respectively. B, pre-steady-state incorporation rates of the four RT proteins at varying dATP concentration (1–100 μm) at Site 1 of the T/P described in the legend for Fig. 1 were plotted. The fit to the data gave the following K_d (dNTP binding constant) and k_pol (maximum incorporation rate) values, respectively: HIV-1 Cy, 30.9 μm and 67.2 s⁻¹; HIV-1 Ug, 28.4 μm and 86.6 s⁻¹; HIV-2 Rod, 30.4 μm and 29.6 s⁻¹; and SIV 9063-2, 40.0 μm and 22.3 s⁻¹ (see Table 1 for detail). Experiments were repeated 3–7 times for the four enzymes at Site 1, and the average is shown with error bars representing S.E.

FIGURE 3.

k_pol, K_d, and k_pol/K_d comparison of the four RT enzymes at three different sites on the 40-mer RNA template. A–C, the maximum incorporation rates (k_pol) (A), the dNTP binding affinity (K_d) (B), and the incorporation efficiency (k_pol/K_d) (C) of RT protein from Vpx non-coding (black bars) and Vpx coding (open bars) lentiviruses at the three different sites described in the legend for Fig. 1 were determined 3–7 times, and the average values are shown with error bars representing S.E. -Fold changes are indicated by brackets above the bars, and statistical significance from an unpaired Student's t test is indicated as: NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

dNTP concentration-dependent RNA-dependent DNA synthesis of the four RT enzymes with two long viral RNA templates. A and B, schematic showing 5′ ³²P-labeled 20-mer primer (P) annealed to HIV-1 Pol (A, top) or TAR (B, top) RNA template. Template structure was based on Mfold prediction for lowest free energy (36). The predicted bottoms of each stem-loop structure in these RNA templates were marked by *. The 5′ ³²P-labeled primers annealed to the Pol or TAR RNAs (A and B) were extended by an equal active concentration of the four purified RT proteins at five different dNTP concentrations (lanes 1–5: 50, 10, 1, 0.25, 0.1 μm), which are close to the dNTP concentrations found in activated T cells (__), macrophages (M), and macrophages treated with Vpx (X), respectively. * indicates pause sites produced by kinetic delays of dNTP incorporations at lower dNTP concentrations near the bottom of each stem-loop structure predicted in the RNA templates. (+): 100 μm dNTP positive control; (−): no dNTP control. F: fully extended products; P: primer and unextended substrate.