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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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Arrhenius analysis of the cTAR/TAR annealing reaction in the presence of NC(11–55) or (SSHS)2NC(11–55). The temperature dependence of the cTAR/TAR annealing reaction was determined by reacting 10 nM of the doubly labelled TMR-5′-cTAR-3′-Fl species together with 200 nM of TAR in the presence of NC(11–55) (closed triangle) or (SSHS)2NC(11–55) (squares correspond to the fast component and circles to the slow one) added at a ratio of one peptide per 5 nt. The retrieved thermodynamic parameters are reported in Table 2. In the presence of (SSHS)2NC(11–55), the fraction afast of the fast component (upper panel open square) increased with temperature.
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gkt164-F6: Arrhenius analysis of the cTAR/TAR annealing reaction in the presence of NC(11–55) or (SSHS)2NC(11–55). The temperature dependence of the cTAR/TAR annealing reaction was determined by reacting 10 nM of the doubly labelled TMR-5′-cTAR-3′-Fl species together with 200 nM of TAR in the presence of NC(11–55) (closed triangle) or (SSHS)2NC(11–55) (squares correspond to the fast component and circles to the slow one) added at a ratio of one peptide per 5 nt. The retrieved thermodynamic parameters are reported in Table 2. In the presence of (SSHS)2NC(11–55), the fraction afast of the fast component (upper panel open square) increased with temperature.

Mentions: To evaluate whether the NC(11–55)-induced remodelling of cTAR could facilitate the cTAR/TAR annealing reaction, we compared the cTAR/TAR annealing kinetics in the presence of NC(11–55) and (SSHS)2NC(11–55). We reacted 10 nM of doubly labelled 5′-TMR-cTAR-3′Fl together with TAR in pseudo-first order conditions, in the presence of either of the two peptides at a ratio of 5 nt per peptide and monitored the hybridization reaction in real-time through the Fl fluorescence restoration occurring as the ds-DNA/RNA duplex forms. As their initial part was too fast to be monitored, the kinetic curves in the presence of NC(11–55) could be fitted with a single kinetic rate constant of 2.5 ± 0.2 × 104 M−1s−1, close to the 1.8 ± 0.2 × 104 M−1s−1 value found for the NC(12–55)-promoted annealing reaction of the cTAR and TAR Mal strain sequences (20). In contrast, the kinetic curves observed in the presence of (SSHS)2NC(11–55) were clearly bi-exponential with rate constants of 1.9 ± 0.2 ×1 04 M−1s−1 and 1.5 ± 0.4 × 103 M−1s−1 for the fast and slow component, respectively (Table 2). Thus, the slow component with (SSHS)2NC(11–55) was at least one order of magnitude slower than that observed with NC(11–55). Thermodynamic parameters of the reactions associated with the two NC mutants were determined (Table 2). The transition state enthalpy was estimated from the temperature dependence of the annealing reaction (Figure 6) according to:(1)Figure 6.


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)

Arrhenius analysis of the cTAR/TAR annealing reaction in the presence of NC(11–55) or (SSHS)2NC(11–55). The temperature dependence of the cTAR/TAR annealing reaction was determined by reacting 10 nM of the doubly labelled TMR-5′-cTAR-3′-Fl species together with 200 nM of TAR in the presence of NC(11–55) (closed triangle) or (SSHS)2NC(11–55) (squares correspond to the fast component and circles to the slow one) added at a ratio of one peptide per 5 nt. The retrieved thermodynamic parameters are reported in Table 2. In the presence of (SSHS)2NC(11–55), the fraction afast of the fast component (upper panel open square) increased with temperature.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt164-F6: Arrhenius analysis of the cTAR/TAR annealing reaction in the presence of NC(11–55) or (SSHS)2NC(11–55). The temperature dependence of the cTAR/TAR annealing reaction was determined by reacting 10 nM of the doubly labelled TMR-5′-cTAR-3′-Fl species together with 200 nM of TAR in the presence of NC(11–55) (closed triangle) or (SSHS)2NC(11–55) (squares correspond to the fast component and circles to the slow one) added at a ratio of one peptide per 5 nt. The retrieved thermodynamic parameters are reported in Table 2. In the presence of (SSHS)2NC(11–55), the fraction afast of the fast component (upper panel open square) increased with temperature.
Mentions: To evaluate whether the NC(11–55)-induced remodelling of cTAR could facilitate the cTAR/TAR annealing reaction, we compared the cTAR/TAR annealing kinetics in the presence of NC(11–55) and (SSHS)2NC(11–55). We reacted 10 nM of doubly labelled 5′-TMR-cTAR-3′Fl together with TAR in pseudo-first order conditions, in the presence of either of the two peptides at a ratio of 5 nt per peptide and monitored the hybridization reaction in real-time through the Fl fluorescence restoration occurring as the ds-DNA/RNA duplex forms. As their initial part was too fast to be monitored, the kinetic curves in the presence of NC(11–55) could be fitted with a single kinetic rate constant of 2.5 ± 0.2 × 104 M−1s−1, close to the 1.8 ± 0.2 × 104 M−1s−1 value found for the NC(12–55)-promoted annealing reaction of the cTAR and TAR Mal strain sequences (20). In contrast, the kinetic curves observed in the presence of (SSHS)2NC(11–55) were clearly bi-exponential with rate constants of 1.9 ± 0.2 ×1 04 M−1s−1 and 1.5 ± 0.4 × 103 M−1s−1 for the fast and slow component, respectively (Table 2). Thus, the slow component with (SSHS)2NC(11–55) was at least one order of magnitude slower than that observed with NC(11–55). Thermodynamic parameters of the reactions associated with the two NC mutants were determined (Table 2). The transition state enthalpy was estimated from the temperature dependence of the annealing reaction (Figure 6) according to:(1)Figure 6.

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