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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 zinc coordinating activity of NC on the kinetics of 591R primer extension. Reactions were performed with RT in the presence or absence of WT NC, SSHS NC (32), or zinc-less NC (69). The data were plotted as % FL DNA versus Time (min). Symbols: minus NC, squares; WT NC, triangles; SSHS NC, open circles, dashed line; zinc-less NC, diamonds. Note that the minus NC and WT NC curves are the same as those shown in Figure 5 for reactions with WT RT, but the scale on the y-axis is expanded here.
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Figure 6: Effect of zinc coordinating activity of NC on the kinetics of 591R primer extension. Reactions were performed with RT in the presence or absence of WT NC, SSHS NC (32), or zinc-less NC (69). The data were plotted as % FL DNA versus Time (min). Symbols: minus NC, squares; WT NC, triangles; SSHS NC, open circles, dashed line; zinc-less NC, diamonds. Note that the minus NC and WT NC curves are the same as those shown in Figure 5 for reactions with WT RT, but the scale on the y-axis is expanded here.

Mentions: It was also of interest to determine whether NC's ability to coordinate zinc is required for inhibition of mispriming. Since NC's destabilization activity is associated with the zinc finger domains [for references before 2005, see ref. (36); (59,62)], this question is of great importance for elucidating the mechanism of NC activity in our system. To address this issue, we tested two NC proteins that do not have zinc finger structures: (i) the SSHS mutant, which has all six Cys residues changed to Ser (32) and (ii) chemically synthesized WT NC that was never exposed to zinc (zinc-less NC) (69) (Figure 6). The time course of 591R priming activity was assessed with the two NC variants and was compared with activity in the absence and presence of WT NC. Only WT RT was used in these experiments. The data for reactions with and without WT NC are taken from Figure 5, except that in this case, the values for percentage FL DNA are displayed on an expanded y-axis.Figure 6.


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 zinc coordinating activity of NC on the kinetics of 591R primer extension. Reactions were performed with RT in the presence or absence of WT NC, SSHS NC (32), or zinc-less NC (69). The data were plotted as % FL DNA versus Time (min). Symbols: minus NC, squares; WT NC, triangles; SSHS NC, open circles, dashed line; zinc-less NC, diamonds. Note that the minus NC and WT NC curves are the same as those shown in Figure 5 for reactions with WT RT, but the scale on the y-axis is expanded here.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Effect of zinc coordinating activity of NC on the kinetics of 591R primer extension. Reactions were performed with RT in the presence or absence of WT NC, SSHS NC (32), or zinc-less NC (69). The data were plotted as % FL DNA versus Time (min). Symbols: minus NC, squares; WT NC, triangles; SSHS NC, open circles, dashed line; zinc-less NC, diamonds. Note that the minus NC and WT NC curves are the same as those shown in Figure 5 for reactions with WT RT, but the scale on the y-axis is expanded here.
Mentions: It was also of interest to determine whether NC's ability to coordinate zinc is required for inhibition of mispriming. Since NC's destabilization activity is associated with the zinc finger domains [for references before 2005, see ref. (36); (59,62)], this question is of great importance for elucidating the mechanism of NC activity in our system. To address this issue, we tested two NC proteins that do not have zinc finger structures: (i) the SSHS mutant, which has all six Cys residues changed to Ser (32) and (ii) chemically synthesized WT NC that was never exposed to zinc (zinc-less NC) (69) (Figure 6). The time course of 591R priming activity was assessed with the two NC variants and was compared with activity in the absence and presence of WT NC. Only WT RT was used in these experiments. The data for reactions with and without WT NC are taken from Figure 5, except that in this case, the values for percentage FL DNA are displayed on an expanded y-axis.Figure 6.

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