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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.

Wu T, Heilman-Miller SL, Levin JG - Nucleic Acids Res. (2007)

Bottom Line: Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect.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.

View Article: PubMed Central - PubMed

Affiliation: Section on Viral Gene Regulation, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

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.

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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.
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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.

Mentions: To test this hypothesis, our strategy was to make mutations that either stabilize or destabilize the local structure of the acceptor RNAs and then to measure the effect of the mutations on minus-strand transfer. The mutations we chose were ones that retain the original RNA 50 fold (Figure 2A), thereby allowing a direct comparison of mutant and wild-type activities. Figure 3A illustrates these changes and also gives the predicted overall ΔG values for each mutant. The RNA 50 structure was destabilized in two ways: (i) by creating a mismatch (G46U); and (ii) by changing a G-C bp to a G-U wobble pair (C49U). We also made a more stable mutant of RNA 50 by changing the G-G mismatch to a G-U wobble pair (G48U). Since the RNA 50 mutants have the same fold as the wild-type (WT) RNA (data not shown), an increase or decrease in predicted ΔG values can be attributed to increased or decreased thermostability induced by the changes in local structure. Thus, mutants RNA 50G46U and RNA 50C49U (destabilizing mutations) have lower predicted ΔG values than WT RNA, while RNA 50G48U (stabilizing mutation) has a higher predicted ΔG value than WT (Figure 3A). Note that we did not make mutations in the upper stem containing the last 4 bases (nt 28–31) that are annealed to the 11 base sequence in DNA 50, since mFOLD analysis (77,78) revealed that most of the potential mutations in this region would result in structures that differed from RNA 50 in their overall fold.Figure 3.


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.

Wu T, Heilman-Miller SL, Levin JG - Nucleic Acids Res. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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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.
Mentions: To test this hypothesis, our strategy was to make mutations that either stabilize or destabilize the local structure of the acceptor RNAs and then to measure the effect of the mutations on minus-strand transfer. The mutations we chose were ones that retain the original RNA 50 fold (Figure 2A), thereby allowing a direct comparison of mutant and wild-type activities. Figure 3A illustrates these changes and also gives the predicted overall ΔG values for each mutant. The RNA 50 structure was destabilized in two ways: (i) by creating a mismatch (G46U); and (ii) by changing a G-C bp to a G-U wobble pair (C49U). We also made a more stable mutant of RNA 50 by changing the G-G mismatch to a G-U wobble pair (G48U). Since the RNA 50 mutants have the same fold as the wild-type (WT) RNA (data not shown), an increase or decrease in predicted ΔG values can be attributed to increased or decreased thermostability induced by the changes in local structure. Thus, mutants RNA 50G46U and RNA 50C49U (destabilizing mutations) have lower predicted ΔG values than WT RNA, while RNA 50G48U (stabilizing mutation) has a higher predicted ΔG value than WT (Figure 3A). Note that we did not make mutations in the upper stem containing the last 4 bases (nt 28–31) that are annealed to the 11 base sequence in DNA 50, since mFOLD analysis (77,78) revealed that most of the potential mutations in this region would result in structures that differed from RNA 50 in their overall fold.Figure 3.

Bottom Line: Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect.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.

View Article: PubMed Central - PubMed

Affiliation: Section on Viral Gene Regulation, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

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.

Show MeSH
Related in: MedlinePlus