Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

Abstract

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (-) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop structure at the 5' end of the transactivation response element (TAR) in a 70-nt RNA acceptor (RNA 70) appears to be the major nucleation pathway. Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect. In this case, the efficiencies of both annealing and strand transfer reactions are similar. However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer. Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing. This suggests that Mg2+ competes with NC for binding to the nucleic acid substrates. Collectively, our findings provide new insights into the mechanism of NC-dependent and -independent minus-strand transfer.

Figure 1.

Schematic diagram illustrating the strand transfer system used for this study. (A) RNA 70. The diagram shows annealing of the 50 complementary bases in the RNA 70 acceptor and the (−) SSDNA, DNA 50, which is labeled at its 5′ end with ³²P. The 20 nt sequence from U3 serves as the template for RT-catalyzed DNA extension. (B) RNA 50. The only difference between (A) and (B) is that the RNA 50 acceptor contains only 30 nt complementary to DNA 50. With both acceptors, the final product is a 70-nt labeled DNA (dashed lines at the bottom of the figure). For RNA 70, the ‘TAR’ sequence is 50 nt; 9 nt at the 3′ end of full-length TAR are missing (28). RNA 50 ‘TAR’ consists of the 5′ half of TAR (28). White rectangles, DNA 50; gray rectangles, acceptor RNA. The stars denote the ³²P label. The diagram is not drawn to scale.

Figure 2.

Influence of acceptor RNA secondary structure on minus-strand transfer. (A) Secondary structures of RNA 70 and RNA 50 acceptor RNAs, based on mFold analysis and extensive RNase mapping studies (28). The 20-nt U3 sequences are indicated. (Note that mFold predicts that two bases from U3, C19 and U20, are part of the stem-loop structure at the 5′ end of RNA 70). The predicted ΔG values are shown beneath the structures. The potential nucleation site at the 5′ end of each RNA is boxed. The arrows point to residues in RNA 70 and RNA 50 (gray shading) that were mutated. (B) Secondary structure of DNA 50, based on mFold analysis and enzymatic mapping studies (28). The predicted ΔG value is shown on the right. The arrows indicate residues C17 and C18 (gray shading) that were mutated. Note the 11-nt single-stranded sequence at the 3′ end of the DNA. (C) NC-mediated minus-strand transfer. DNA 50 and RNA 70 or RNA 50 were present in reactions at a 1:1 ratio of (−) SSDNA to acceptor RNA, each with a final concentration of 10 nM. These nucleic acid concentrations were used in all of the experiments described below. Incubation was for 60 min in the absence or presence of HIV-1 NC (0.88 nt/NC), as described under the Materials and Methods section, and was followed by PAGE and PhosphorImager analysis. The bar graph shows the percentage (%) of minus-strand transfer product synthesized in each reaction. Plus NC, closed bars; minus NC, open bars.

Figure 3.

Effect of HIV-1 NC on minus-strand transfer with RNA 50 and RNA 50 mutants. (A) RNA 50 and mutants. The predicted ΔG values shown for RNA 50 and mutants represent the values for overall thermodynamic stability. The relevant sequences in each RNA are boxed. (B) Gel analysis. DNA 50 was incubated with acceptor RNA 50 and mutants for 60 min in the absence (No) (lanes 1, 6, 11, 16) or presence of increasing concentrations of HIV-1 NC, as follows: lanes 2, 7, 12, 17, 7 nt/NC (0.14 μM); lanes 3, 8, 13, 18, 3.5 nt/NC (0.3 μM); lanes 4, 9, 14, 19, 1.75 nt/NC (0.6 μM); lanes 5, 10, 15, 20, 0.88 nt/NC (1.2 μM). (C) Bar graphs showing the percentage (%) of minus-strand transfer product synthesized as a function of NC concentration. Note that the numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Symbols: RNA 50, open bars; RNA 50G46U, hatched bars; RNA 50C49U, cross-hatched bars and RNA 50G48U, gray bars.

Figure 4.

Kinetics of minus-strand transfer with RNA 70 and RNA 70 mutants. Reaction mixtures containing ³²P-labeled DNA 50 and acceptor RNA 70 (A), RNA 70U28C (B), RNA 70U30C (C) and RNA 70U28,30C (D), were incubated from 1 to 60 min without NC or with two different NC concentrations (3.5 nt/NC [0.3 µM] and 0.88 nt/NC [1.4 µM]). The percentage (%) of transfer product synthesized was plotted against time of incubation. Symbols: no NC, open circles; 3.5 nt/NC, closed circles; 0.88 nt/NC, closed inverted triangles.

Figure 5.

Kinetics of minus-strand transfer with an RNA 70 TAR loop mutant. Reaction mixtures containing the acceptor RNA 70 loop mutant and ³²P-labeled DNA 50 (A) or ³²P-labeled DNA 50C17,18T (a compensatory mutant) (B) were incubated from 1 to 60 min at 37°C without NC or with two different NC concentrations (3.5 nt/NC [0.3 µM] and 0.88 nt/NC [1.4 µM]). The percentage (%) of strand transfer product synthesized was plotted as a function of time of incubation. The symbols are described in Figure 4 legend.

Figure 6.

Annealing of RNA 70 and mutants to DNA 50. ³²P-labeled DNA 50 was incubated with RNA 70 (A), RNA 70G53,54A (B), RNA 70U28C (C) and RNA 70U28,30C (D) from 0.5 to 30 min at 37°C without NC or with two different NC concentrations (3.5 nt/NC [0.3 µM] and 0.88 nt/NC [1.4 µM]). The percentage (%) of DNA 50 annealed was plotted against time of incubation. Symbols are the same as those given in Figure 4 legend.

Figure 7.

Effect of Mg²⁺ on annealing of RNA 70 and RNA 70 mutants to DNA 50. ³²P-labeled DNA 50 was incubated with RNA 70 (lanes 1–8), RNA 70G53,54A (lanes 9–16) or RNA 70U28,30C (lanes 17–24) for 30 min at 37°C under conditions indicated below. Bar graphs show the percentage (%) of DNA 50 annealed for each reaction. Symbols: open bars, no NC and no Mg²⁺ (lanes 1, 9, 17); striped bars, 7 mM Mg²⁺ alone (lanes 2, 10, 18); gray bars, 0.88 nt/NC (1.4 µM) and no Mg²⁺ (lanes 3, 11, 19); black bars, 0.88 nt/NC and increasing concentrations of Mg²⁺as follows: 0.25 mM Mg²⁺ (lanes 4, 12, 20); 0.5 mM Mg²⁺ (lanes 5, 13, 21); 1.75 mM Mg²⁺ (lanes 6, 14, 22); 3.5 mM Mg²⁺ (lanes 7, 15, 23); 7 mM Mg²⁺ (lanes 8, 16, 24). Note that the order of addition of NC and MgCl₂ had no effect on the extent of annealing (data not shown).