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Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein.

Post K, Kankia B, Gopalakrishnan S, Yang V, Cramer E, Saladores P, Gorelick RJ, Guo J, Musier-Forsyth K, Levin JG - Nucleic Acids Res. (2009)

Bottom Line: NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected.Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency.Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3' polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5'-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

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Related in: MedlinePlus

Effect of HIV-1 NC on plus-strand initiation with four RNA primers. The 194R (PPT+5), 587R, 591R and 589R primers were extended by HIV-1 RT in the absence or presence of HIV-1 NC. (A) Gel analysis. FL DNA products synthesized during primer extension after incubation at 37°C for 30 min in the absence (No) (lanes 1, 7, 13, 19) or presence of increasing concentrations of HIV-1 NC as follows: 14 nt/NC (0.17 µM), lanes 2, 8, 14, 20; 7 nt/NC (0.34 µM), lanes 3, 9, 15, 21; 3.5 nt/NC (0.7 µM), lanes 4, 10, 16, 22; 1.75 nt/NC (1.4 µM), lanes 5, 11, 17, 23; 0.88 nt/NC (2.7 µM), lanes 6, 12, 18, 24. The positions of the primer (P) and the FL DNA products formed by 587R (55 nt), 591R (40 nt) and 589R (85 nt) are shown on the right and for 194R (80 nt), on the left. The bracketed bands are RNase H cleavage products. The sizes of the DNA products were verified with appropriate markers. (B) Bar graphs showing the percentage of total radioactivity in a given lane present as the FL 32P-labeled DNA (% FL DNA) as a function of NC concentration. The numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Note that the inset in the bar graph for 587R shows the values for % FL DNA on an expanded scale. Symbols: 194R (PPT+5), open bars; 587R, filled bars; 591R, hatched bars; and 589R, stippled bars.
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Figure 2: Effect of HIV-1 NC on plus-strand initiation with four RNA primers. The 194R (PPT+5), 587R, 591R and 589R primers were extended by HIV-1 RT in the absence or presence of HIV-1 NC. (A) Gel analysis. FL DNA products synthesized during primer extension after incubation at 37°C for 30 min in the absence (No) (lanes 1, 7, 13, 19) or presence of increasing concentrations of HIV-1 NC as follows: 14 nt/NC (0.17 µM), lanes 2, 8, 14, 20; 7 nt/NC (0.34 µM), lanes 3, 9, 15, 21; 3.5 nt/NC (0.7 µM), lanes 4, 10, 16, 22; 1.75 nt/NC (1.4 µM), lanes 5, 11, 17, 23; 0.88 nt/NC (2.7 µM), lanes 6, 12, 18, 24. The positions of the primer (P) and the FL DNA products formed by 587R (55 nt), 591R (40 nt) and 589R (85 nt) are shown on the right and for 194R (80 nt), on the left. The bracketed bands are RNase H cleavage products. The sizes of the DNA products were verified with appropriate markers. (B) Bar graphs showing the percentage of total radioactivity in a given lane present as the FL 32P-labeled DNA (% FL DNA) as a function of NC concentration. The numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Note that the inset in the bar graph for 587R shows the values for % FL DNA on an expanded scale. Symbols: 194R (PPT+5), open bars; 587R, filled bars; 591R, hatched bars; and 589R, stippled bars.

Mentions: Each RNA was labeled at its 5′-end with 32P and was then hybridized to a complementary 100-nt DNA template (581D for 194R, 587R and 591R; 582D for 589R). Primer extension was assayed as described in Materials and Methods section and the results are shown in Figure 2. The only gel products detected are extended DNAs that retain the 5′ 32P label, i.e. DNAs still attached to the intact primer, and small labeled RNAs produced by RNase H cleavage of the labeled RNA primer in the RNA–DNA hybrid (bands located beneath the primer band [P]) (Figure 2A). As predicted from the sequence of the templates, the sizes of the FL DNA extension products (including the RNA sequence) for each non-PPT primer were: 587R, 55 nt; 591R, 40 nt; and 589R, 85 nt. For 194R, the FL product was 80 nt. Unannealed primers in reactions without RT and NC migrated as essentially a single band (Figure S1).Figure 2.


Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein.

Post K, Kankia B, Gopalakrishnan S, Yang V, Cramer E, Saladores P, Gorelick RJ, Guo J, Musier-Forsyth K, Levin JG - Nucleic Acids Res. (2009)

Effect of HIV-1 NC on plus-strand initiation with four RNA primers. The 194R (PPT+5), 587R, 591R and 589R primers were extended by HIV-1 RT in the absence or presence of HIV-1 NC. (A) Gel analysis. FL DNA products synthesized during primer extension after incubation at 37°C for 30 min in the absence (No) (lanes 1, 7, 13, 19) or presence of increasing concentrations of HIV-1 NC as follows: 14 nt/NC (0.17 µM), lanes 2, 8, 14, 20; 7 nt/NC (0.34 µM), lanes 3, 9, 15, 21; 3.5 nt/NC (0.7 µM), lanes 4, 10, 16, 22; 1.75 nt/NC (1.4 µM), lanes 5, 11, 17, 23; 0.88 nt/NC (2.7 µM), lanes 6, 12, 18, 24. The positions of the primer (P) and the FL DNA products formed by 587R (55 nt), 591R (40 nt) and 589R (85 nt) are shown on the right and for 194R (80 nt), on the left. The bracketed bands are RNase H cleavage products. The sizes of the DNA products were verified with appropriate markers. (B) Bar graphs showing the percentage of total radioactivity in a given lane present as the FL 32P-labeled DNA (% FL DNA) as a function of NC concentration. The numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Note that the inset in the bar graph for 587R shows the values for % FL DNA on an expanded scale. Symbols: 194R (PPT+5), open bars; 587R, filled bars; 591R, hatched bars; and 589R, stippled bars.
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Related In: Results  -  Collection

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Figure 2: Effect of HIV-1 NC on plus-strand initiation with four RNA primers. The 194R (PPT+5), 587R, 591R and 589R primers were extended by HIV-1 RT in the absence or presence of HIV-1 NC. (A) Gel analysis. FL DNA products synthesized during primer extension after incubation at 37°C for 30 min in the absence (No) (lanes 1, 7, 13, 19) or presence of increasing concentrations of HIV-1 NC as follows: 14 nt/NC (0.17 µM), lanes 2, 8, 14, 20; 7 nt/NC (0.34 µM), lanes 3, 9, 15, 21; 3.5 nt/NC (0.7 µM), lanes 4, 10, 16, 22; 1.75 nt/NC (1.4 µM), lanes 5, 11, 17, 23; 0.88 nt/NC (2.7 µM), lanes 6, 12, 18, 24. The positions of the primer (P) and the FL DNA products formed by 587R (55 nt), 591R (40 nt) and 589R (85 nt) are shown on the right and for 194R (80 nt), on the left. The bracketed bands are RNase H cleavage products. The sizes of the DNA products were verified with appropriate markers. (B) Bar graphs showing the percentage of total radioactivity in a given lane present as the FL 32P-labeled DNA (% FL DNA) as a function of NC concentration. The numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Note that the inset in the bar graph for 587R shows the values for % FL DNA on an expanded scale. Symbols: 194R (PPT+5), open bars; 587R, filled bars; 591R, hatched bars; and 589R, stippled bars.
Mentions: Each RNA was labeled at its 5′-end with 32P and was then hybridized to a complementary 100-nt DNA template (581D for 194R, 587R and 591R; 582D for 589R). Primer extension was assayed as described in Materials and Methods section and the results are shown in Figure 2. The only gel products detected are extended DNAs that retain the 5′ 32P label, i.e. DNAs still attached to the intact primer, and small labeled RNAs produced by RNase H cleavage of the labeled RNA primer in the RNA–DNA hybrid (bands located beneath the primer band [P]) (Figure 2A). As predicted from the sequence of the templates, the sizes of the FL DNA extension products (including the RNA sequence) for each non-PPT primer were: 587R, 55 nt; 591R, 40 nt; and 589R, 85 nt. For 194R, the FL product was 80 nt. Unannealed primers in reactions without RT and NC migrated as essentially a single band (Figure S1).Figure 2.

Bottom Line: NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected.Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency.Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3' polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5'-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

Show MeSH
Related in: MedlinePlus