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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

Competition binding experiments with RT and NC to PPT (blue) and 591 (green) duplexes. The duplexes (20 nM) were prebound to 500 nM RT (A) and to 1000 nM NC (B). FA values for the protein-free duplexes were approximately 0.05 and 0.08 for the PPT and 591 duplexes, respectively.
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Figure 4: Competition binding experiments with RT and NC to PPT (blue) and 591 (green) duplexes. The duplexes (20 nM) were prebound to 500 nM RT (A) and to 1000 nM NC (B). FA values for the protein-free duplexes were approximately 0.05 and 0.08 for the PPT and 591 duplexes, respectively.

Mentions: To investigate the possibility that NC might be inhibiting mispriming by blocking RT from binding to the 3′-terminus of the primer (78), we performed competition experiments (Figure 4). Either RT or NC was prebound to the PPT or 591 duplexes and then increasing concentrations of the competing protein were added. Binding was evaluated by measuring FA. When NC was the competing protein, FA of both the PPT and 591 duplexes was unchanged over a broad range of NC concentration. This indicates that NC was unable to displace RT that was bound to the nucleic acid hybrids (Figure 4A). In contrast, when RT was the competing protein, FA of both duplexes was increased with increasing concentrations of RT (Figure 4B). These data show that RT competed effectively for binding to RNA–DNA hybrids prebound to NC in a sequence-independent fashion, i.e. the results were the same with the PPT and non-PPT duplexes. Collectively, these findings rule out a direct binding competition model to account for the ability of NC to specifically inhibit mispriming of non-PPT primers. The results are also in agreement with the measured dissociation constants (Table 2; Figure S3).Figure 4.


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)

Competition binding experiments with RT and NC to PPT (blue) and 591 (green) duplexes. The duplexes (20 nM) were prebound to 500 nM RT (A) and to 1000 nM NC (B). FA values for the protein-free duplexes were approximately 0.05 and 0.08 for the PPT and 591 duplexes, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665208&req=5

Figure 4: Competition binding experiments with RT and NC to PPT (blue) and 591 (green) duplexes. The duplexes (20 nM) were prebound to 500 nM RT (A) and to 1000 nM NC (B). FA values for the protein-free duplexes were approximately 0.05 and 0.08 for the PPT and 591 duplexes, respectively.
Mentions: To investigate the possibility that NC might be inhibiting mispriming by blocking RT from binding to the 3′-terminus of the primer (78), we performed competition experiments (Figure 4). Either RT or NC was prebound to the PPT or 591 duplexes and then increasing concentrations of the competing protein were added. Binding was evaluated by measuring FA. When NC was the competing protein, FA of both the PPT and 591 duplexes was unchanged over a broad range of NC concentration. This indicates that NC was unable to displace RT that was bound to the nucleic acid hybrids (Figure 4A). In contrast, when RT was the competing protein, FA of both duplexes was increased with increasing concentrations of RT (Figure 4B). These data show that RT competed effectively for binding to RNA–DNA hybrids prebound to NC in a sequence-independent fashion, i.e. the results were the same with the PPT and non-PPT duplexes. Collectively, these findings rule out a direct binding competition model to account for the ability of NC to specifically inhibit mispriming of non-PPT primers. The results are also in agreement with the measured dissociation constants (Table 2; Figure S3).Figure 4.

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