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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

Schematic diagram of the RNA primers used in this study. The gray rectangle represents nt 8994–9138 from the 3′-end of the HIV-1 NL4-3 RNA genome (numbering according to GenBank accession number: AF324493) (70). The RNA primers (each 20 nt) are shown beneath the gray rectangle and the tick marks are placed according to the position of the first base in the primer sequence in the viral RNA genome. Note that in the case of the PPT, we used a primer containing the PPT plus the five downstream bases, so that all primers would be the same size. The five additional bases are removed by RNase H to generate the actual PPT primer (9). Symbols: 589R, stippled; 194R (PPT+5), open; 587R, solid; and 591R, hatched. The table below the diagram indicates the nt positions and the sequence (5′ to 3′ direction) of each primer.
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Figure 1: Schematic diagram of the RNA primers used in this study. The gray rectangle represents nt 8994–9138 from the 3′-end of the HIV-1 NL4-3 RNA genome (numbering according to GenBank accession number: AF324493) (70). The RNA primers (each 20 nt) are shown beneath the gray rectangle and the tick marks are placed according to the position of the first base in the primer sequence in the viral RNA genome. Note that in the case of the PPT, we used a primer containing the PPT plus the five downstream bases, so that all primers would be the same size. The five additional bases are removed by RNase H to generate the actual PPT primer (9). Symbols: 589R, stippled; 194R (PPT+5), open; 587R, solid; and 591R, hatched. The table below the diagram indicates the nt positions and the sequence (5′ to 3′ direction) of each primer.

Mentions: From available evidence we know that the exclusive use of the PPT is achieved, at least in part, because the PPT duplex, in contrast to all other RNA–DNA hybrids, is resistant to RNase H degradation during reverse transcription (6–8). Several factors are responsible for this unusual property. For example, the PPT has a unique sequence, including six Gs at its 3′-end (Figure 1) that are required for proper RNase H cleavage at the PPT-U3 junction and extension by HIV-1 RT (9) [for further details on mutational analysis of retroviral PPT sequences, see refs. (7,8) and references therein].Figure 1.


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)

Schematic diagram of the RNA primers used in this study. The gray rectangle represents nt 8994–9138 from the 3′-end of the HIV-1 NL4-3 RNA genome (numbering according to GenBank accession number: AF324493) (70). The RNA primers (each 20 nt) are shown beneath the gray rectangle and the tick marks are placed according to the position of the first base in the primer sequence in the viral RNA genome. Note that in the case of the PPT, we used a primer containing the PPT plus the five downstream bases, so that all primers would be the same size. The five additional bases are removed by RNase H to generate the actual PPT primer (9). Symbols: 589R, stippled; 194R (PPT+5), open; 587R, solid; and 591R, hatched. The table below the diagram indicates the nt positions and the sequence (5′ to 3′ direction) of each primer.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Schematic diagram of the RNA primers used in this study. The gray rectangle represents nt 8994–9138 from the 3′-end of the HIV-1 NL4-3 RNA genome (numbering according to GenBank accession number: AF324493) (70). The RNA primers (each 20 nt) are shown beneath the gray rectangle and the tick marks are placed according to the position of the first base in the primer sequence in the viral RNA genome. Note that in the case of the PPT, we used a primer containing the PPT plus the five downstream bases, so that all primers would be the same size. The five additional bases are removed by RNase H to generate the actual PPT primer (9). Symbols: 589R, stippled; 194R (PPT+5), open; 587R, solid; and 591R, hatched. The table below the diagram indicates the nt positions and the sequence (5′ to 3′ direction) of each primer.
Mentions: From available evidence we know that the exclusive use of the PPT is achieved, at least in part, because the PPT duplex, in contrast to all other RNA–DNA hybrids, is resistant to RNase H degradation during reverse transcription (6–8). Several factors are responsible for this unusual property. For example, the PPT has a unique sequence, including six Gs at its 3′-end (Figure 1) that are required for proper RNase H cleavage at the PPT-U3 junction and extension by HIV-1 RT (9) [for further details on mutational analysis of retroviral PPT sequences, see refs. (7,8) and references therein].Figure 1.

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