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Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

Godet J, Kenfack C, Przybilla F, Richert L, Duportail G, Mély Y - Nucleic Acids Res. (2013)

Bottom Line: NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem.Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC.Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, Université de Strasbourg, 67401 Illkirch, France.

ABSTRACT
The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

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Sequence logo of cTAR. Sequence logo resulting from 587 aligned cTAR sequences showed the globally strong conservation of the cTAR residues. Residues flanking the G10–C47 base pairs seemed to be among the least conserved.
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gkt164-F7: Sequence logo of cTAR. Sequence logo resulting from 587 aligned cTAR sequences showed the globally strong conservation of the cTAR residues. Residues flanking the G10–C47 base pairs seemed to be among the least conserved.

Mentions: If G10 and G50 residues are critically involved in NCp7 recognition sites needed for efficient strand transfer, it is likely that these positions must be conserved. To check whether these positions corresponded to conserved motifs within the HIV subtypes, we aligned the cTAR sequence presented in Figure 1 to the LANL HIV subtype reference database (http://www.hiv.lanl.gov). The 587 sequences retrieved from the QuickAlign procedure were used to calculate a position weight matrix from which a sequence logo was derived (Figure 7). This sequence logo is drawn such that the overall height of the stack indicates the information content at each position (derived from the Shannon entropy), whereas the height of the base symbols within the stack indicates their relative frequency (51,73,74). The information content, which can reach a maximal theoretical value of 2 bits for nucleic acids, is usually interpreted as an indicator of evolutionary conservation. The cTAR sequence seems to be well conserved, including the G10 and G50 residues. In contrast, residues A9 and T11 flanking G10 were associated to low information content as compared with the other residues of the cTAR ends, suggesting that the nature of the bases at these positions is of limited importance. As a result of this low base conservation, A9 was replaced by a T in one-third of the sequences. In addition, the C48 residue was frequently replaced by an A or a T. Therefore, the A9–C48 mismatch is barely as probable as the canonical A–T base pair at this position (Figure 8B). However, a C11–A46 mismatch was also frequent, indicating that the position of the distal CA mismatch seems to be exchangeable on both sides of the G10–C47 base pair (data not shown). Similarly, a C8–A49 mismatch was also frequently found when the C6–A51 mismatch was absent. It resulted that albeit in different positions, CA mismatches were largely conserved, in close proximity of the conserved C7–G50 and G10–C47 base pairs, highlighting that their presence is required to finely tune the thermodynamic stability of the two terminal ds-segments of cTAR (75).Figure 7.


Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

Godet J, Kenfack C, Przybilla F, Richert L, Duportail G, Mély Y - Nucleic Acids Res. (2013)

Sequence logo of cTAR. Sequence logo resulting from 587 aligned cTAR sequences showed the globally strong conservation of the cTAR residues. Residues flanking the G10–C47 base pairs seemed to be among the least conserved.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643577&req=5

gkt164-F7: Sequence logo of cTAR. Sequence logo resulting from 587 aligned cTAR sequences showed the globally strong conservation of the cTAR residues. Residues flanking the G10–C47 base pairs seemed to be among the least conserved.
Mentions: If G10 and G50 residues are critically involved in NCp7 recognition sites needed for efficient strand transfer, it is likely that these positions must be conserved. To check whether these positions corresponded to conserved motifs within the HIV subtypes, we aligned the cTAR sequence presented in Figure 1 to the LANL HIV subtype reference database (http://www.hiv.lanl.gov). The 587 sequences retrieved from the QuickAlign procedure were used to calculate a position weight matrix from which a sequence logo was derived (Figure 7). This sequence logo is drawn such that the overall height of the stack indicates the information content at each position (derived from the Shannon entropy), whereas the height of the base symbols within the stack indicates their relative frequency (51,73,74). The information content, which can reach a maximal theoretical value of 2 bits for nucleic acids, is usually interpreted as an indicator of evolutionary conservation. The cTAR sequence seems to be well conserved, including the G10 and G50 residues. In contrast, residues A9 and T11 flanking G10 were associated to low information content as compared with the other residues of the cTAR ends, suggesting that the nature of the bases at these positions is of limited importance. As a result of this low base conservation, A9 was replaced by a T in one-third of the sequences. In addition, the C48 residue was frequently replaced by an A or a T. Therefore, the A9–C48 mismatch is barely as probable as the canonical A–T base pair at this position (Figure 8B). However, a C11–A46 mismatch was also frequent, indicating that the position of the distal CA mismatch seems to be exchangeable on both sides of the G10–C47 base pair (data not shown). Similarly, a C8–A49 mismatch was also frequently found when the C6–A51 mismatch was absent. It resulted that albeit in different positions, CA mismatches were largely conserved, in close proximity of the conserved C7–G50 and G10–C47 base pairs, highlighting that their presence is required to finely tune the thermodynamic stability of the two terminal ds-segments of cTAR (75).Figure 7.

Bottom Line: NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem.Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC.Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, Université de Strasbourg, 67401 Illkirch, France.

ABSTRACT
The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

Show MeSH
Related in: MedlinePlus