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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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Site-selective monitoring of the thermal melting of the 2-Ap-substituted cTAR sequences. (A and B) Melting curves are monitored through the 2-Ap fluorescence emission. Two types of transitions were observed. (A) Upward melting curves for 2-Ap9 (closed squares), 2-Ap17 (open circles), 2-Ap21 (open squares), 2-Ap45 (open triangles) and 2-Ap49 (closed circles). (B) Downward melting curves for 2-Ap28 (black squares), 2-Ap53 (open circles) and 2-Ap55 (closed triangles). (C) Local melting temperatures recorded for the various 2-Ap positions. The melting temperatures clearly show a lower stability of the lower part of cTAR stem (light grey bars) as compared with the upper cTAR part (dark grey bars). The last ds-segment probed by the 2-Ap55 residue seemed poorly stable. No melting transition was observed for 2-Ap35, (see Supplementary Figure S1) indicating that 2-Ap in the internal loop experienced interactions with its neighbours which are similar to those in single-stranded DNA.
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gkt164-F3: Site-selective monitoring of the thermal melting of the 2-Ap-substituted cTAR sequences. (A and B) Melting curves are monitored through the 2-Ap fluorescence emission. Two types of transitions were observed. (A) Upward melting curves for 2-Ap9 (closed squares), 2-Ap17 (open circles), 2-Ap21 (open squares), 2-Ap45 (open triangles) and 2-Ap49 (closed circles). (B) Downward melting curves for 2-Ap28 (black squares), 2-Ap53 (open circles) and 2-Ap55 (closed triangles). (C) Local melting temperatures recorded for the various 2-Ap positions. The melting temperatures clearly show a lower stability of the lower part of cTAR stem (light grey bars) as compared with the upper cTAR part (dark grey bars). The last ds-segment probed by the 2-Ap55 residue seemed poorly stable. No melting transition was observed for 2-Ap35, (see Supplementary Figure S1) indicating that 2-Ap in the internal loop experienced interactions with its neighbours which are similar to those in single-stranded DNA.

Mentions: To further characterize the local 2-Ap environment in the various labelled cTAR sequences, thermal melting curves were recorded using the 2-Ap fluorescence signal. These curves allow evidencing local conformations and dynamics of 2-Ap and its close neighbours (59). On melting, two types of transitions were observed. When the 2-Ap residue was located in ds-segments (positions 17, 21, 45 and 49), its fluorescence emission strongly increased during duplex melting (Figure 3A). This was ascribed to the progressive loss of base stacking as the temperature increases. The 2-Ap9 presented a similar transition pattern, suggesting that 2-Ap9 is likely base paired with C48. On the contrary, downward transitions were observed for 2-Ap53 and 2-Ap55 (Figure 3B), evidencing an extra-helical conformation of G54 when the last two ds-segments are formed (60). Indeed, such an extra-helical conformation is thought to limit the dynamical quenching of G54 with both 2-Ap53 and 2-Ap55, explaining a higher QY for the folded state than for the melted form. Moreover, the low melting temperature associated with the 2-Ap55 residue indicated that the last ds-segment is the least stable one. In addition, the broad melting transition observed with this residue further suggested a high propensity of the last ds-segment to transiently melt at room temperature, in line with previous reports (29,31). More surprisingly, 2-Ap28 also underwent a downward melting transition (Figure 3B), probably as the consequence of the disappearance of the structural constraints within the apical loop when the upper ds-segment melted. Taken together, the range of melting temperatures of the different ds-segments of cTAR clearly supported a non–two-state melting transition (61,62) where ds-segments melted independently and where the upper part of the stem–loop seemed more stable than the lower part (Figure 3C).Figure 3.


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)

Site-selective monitoring of the thermal melting of the 2-Ap-substituted cTAR sequences. (A and B) Melting curves are monitored through the 2-Ap fluorescence emission. Two types of transitions were observed. (A) Upward melting curves for 2-Ap9 (closed squares), 2-Ap17 (open circles), 2-Ap21 (open squares), 2-Ap45 (open triangles) and 2-Ap49 (closed circles). (B) Downward melting curves for 2-Ap28 (black squares), 2-Ap53 (open circles) and 2-Ap55 (closed triangles). (C) Local melting temperatures recorded for the various 2-Ap positions. The melting temperatures clearly show a lower stability of the lower part of cTAR stem (light grey bars) as compared with the upper cTAR part (dark grey bars). The last ds-segment probed by the 2-Ap55 residue seemed poorly stable. No melting transition was observed for 2-Ap35, (see Supplementary Figure S1) indicating that 2-Ap in the internal loop experienced interactions with its neighbours which are similar to those in single-stranded DNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643577&req=5

gkt164-F3: Site-selective monitoring of the thermal melting of the 2-Ap-substituted cTAR sequences. (A and B) Melting curves are monitored through the 2-Ap fluorescence emission. Two types of transitions were observed. (A) Upward melting curves for 2-Ap9 (closed squares), 2-Ap17 (open circles), 2-Ap21 (open squares), 2-Ap45 (open triangles) and 2-Ap49 (closed circles). (B) Downward melting curves for 2-Ap28 (black squares), 2-Ap53 (open circles) and 2-Ap55 (closed triangles). (C) Local melting temperatures recorded for the various 2-Ap positions. The melting temperatures clearly show a lower stability of the lower part of cTAR stem (light grey bars) as compared with the upper cTAR part (dark grey bars). The last ds-segment probed by the 2-Ap55 residue seemed poorly stable. No melting transition was observed for 2-Ap35, (see Supplementary Figure S1) indicating that 2-Ap in the internal loop experienced interactions with its neighbours which are similar to those in single-stranded DNA.
Mentions: To further characterize the local 2-Ap environment in the various labelled cTAR sequences, thermal melting curves were recorded using the 2-Ap fluorescence signal. These curves allow evidencing local conformations and dynamics of 2-Ap and its close neighbours (59). On melting, two types of transitions were observed. When the 2-Ap residue was located in ds-segments (positions 17, 21, 45 and 49), its fluorescence emission strongly increased during duplex melting (Figure 3A). This was ascribed to the progressive loss of base stacking as the temperature increases. The 2-Ap9 presented a similar transition pattern, suggesting that 2-Ap9 is likely base paired with C48. On the contrary, downward transitions were observed for 2-Ap53 and 2-Ap55 (Figure 3B), evidencing an extra-helical conformation of G54 when the last two ds-segments are formed (60). Indeed, such an extra-helical conformation is thought to limit the dynamical quenching of G54 with both 2-Ap53 and 2-Ap55, explaining a higher QY for the folded state than for the melted form. Moreover, the low melting temperature associated with the 2-Ap55 residue indicated that the last ds-segment is the least stable one. In addition, the broad melting transition observed with this residue further suggested a high propensity of the last ds-segment to transiently melt at room temperature, in line with previous reports (29,31). More surprisingly, 2-Ap28 also underwent a downward melting transition (Figure 3B), probably as the consequence of the disappearance of the structural constraints within the apical loop when the upper ds-segment melted. Taken together, the range of melting temperatures of the different ds-segments of cTAR clearly supported a non–two-state melting transition (61,62) where ds-segments melted independently and where the upper part of the stem–loop seemed more stable than the lower part (Figure 3C).Figure 3.

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