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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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The 2-Ap substituted cTAR (A) and NC(11–55) (B) sequences. The cTAR sequence from the Lai strain of HIV-1 was selectively modified either at position 9, 17, 21, 28, 35, 45, 49, 53 or 55 with 2-Ap.
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gkt164-F1: The 2-Ap substituted cTAR (A) and NC(11–55) (B) sequences. The cTAR sequence from the Lai strain of HIV-1 was selectively modified either at position 9, 17, 21, 28, 35, 45, 49, 53 or 55 with 2-Ap.

Mentions: The nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) is a small basic protein resulting from the protease-mediated cleavage of the Gag polyprotein precursor. NCp7 is formed by two highly conserved CCHC zinc-finger motifs connected by a short flexible linker and flanked by poorly folded N- and C-terminal basic domains (1,2) (Figure 1). NCp7 is critical during the early and late steps of the HIV-1 viral life cycle (3–11). Most NCp7 functions rely on its RNA- and DNA-chaperone properties that direct the rearrangement of nucleic acids into their most thermodynamically stable structure (12–15). These chaperone properties are thought to play a critical role in reverse transcription (5,16,17). For instance, during the first strand-transfer of reverse transcription, NCp7 favours the transfer of the newly synthesized minus strong-stop DNA [(−) ssDNA] to the 3′-end of the viral RNA genome, by promoting the base pairing of the complementary repeat (R) sequences at the 3′-ends of the (−)ssDNA and genomic RNA, respectively (18). In vitro, the stable stem–loop (SL) of the transactivation response element (TAR) sequence within the R domain of the genomic RNA does not readily anneal with the complementary cTAR sequence of the (−)ssDNA in the absence of NCp7 (18–21). Addition of NCp7 strongly increases the rate and extent of the cTAR/TAR annealing reaction (18–23) and switches the reaction pathway to direct the hybridization of these sequences through the end of their double-stranded (ds) stems (20,21). The main chaperone properties involved in promoting the first strand transfer have been characterized as two interconnected components: a destabilization activity and a hybridization-promoting activity. Promotion of the annealing of complementary sequences requires an efficient aggregation of nucleic acids (24). This is achieved by the numerous basic residues of NCp7 that decrease the electrostatic repulsion between complementary oligonucleotides by neutralizing the negatively charged phosphate groups (5,21,25). The annealing facilitator property of NCp7 is shared with many other basic proteins that are also able to promote the cTAR/TAR DNA hybridization (26–28). On the contrary, the ability to destabilize the cTAR sequence appears to be a hallmark of NCp7. NCp7 activates the transient opening (fraying) of the lower half of the cTAR stem (29–34) constituted by a series of short ds-segments separated by a bulge and two mismatches. This activation follows a non–two-state mechanism, which results in complex spatial and temporal fluctuations of the cTAR ends that greatly facilitate cTAR/TAR hybridization. This activity has been assigned to the conserved hydrophobic platform formed by the Val13, Phe16, Thr24, Ala25, Trp37, Gln45 and Met46 residues at the surface of the folded zinc-finger (ZF) domains (1,2,35). More specifically, the Trp37 residue plays a critical role through stacking with G residues (17,36–43). This hydrophobic platform has also been found critical for binding (35,38). Nevertheless, the precise molecular mechanisms by which NCp7 destabilizes cTAR remain poorly described. Here, we selectively modified the 57-nt cTAR stem–loop sequence of the Lai strain with 2-aminopurine (2-Ap) at different positions to investigate by fluorescence spectroscopy the structural and dynamic modifications that occur within cTAR in response to NCp7 binding. In contrast to most techniques that monitor only global structural changes, the exquisite sensitivity of 2-Ap fluorescence to its local environment allows region-specific characterization of the structural modifications within the cTAR hairpin sequence. The 2-Ap has been previously used to site-specifically characterize the NCp7-induced changes in the dynamics of short single-stranded sequences (44,45), as well as in the loop of the primer-binding site sequence of HIV-1 (46). These studies showed that binding of the intact ZF domain was responsible for a decrease of the DNA base stacking and for a strong restriction of the base mobility on a picosecond–nanosecond timescale. By selectively replacing different adenine positions of the cTAR sequence by 2-Ap (Figure 1), we aimed to picture the preferential destabilization sites and to gain insight into the molecular mechanism by which the NCp7 ZFs destabilize the cTAR stem–loop. Although NC(11–55) was found to bind all over the sequence, it preferentially restricted the dynamics and destabilized the bases of the penultimate ds-segment sandwiched between two C•A mismatches in the lower part of the cTAR stem. These NC(11–55)-induced changes are highly specific, as they were not observed when NC(11–55) was substituted by the SSHS–NC(11–55) mutant where the Cys residues in the ZFs were substituted by Ser residues to prevent the binding of zinc and the folding of the ZFs. These structural changes in this part of cTAR play likely a critical role in the ability of NCp7 to promote cTAR/TAR annealing through a specific zipping pathway.Figure 1.


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)

The 2-Ap substituted cTAR (A) and NC(11–55) (B) sequences. The cTAR sequence from the Lai strain of HIV-1 was selectively modified either at position 9, 17, 21, 28, 35, 45, 49, 53 or 55 with 2-Ap.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt164-F1: The 2-Ap substituted cTAR (A) and NC(11–55) (B) sequences. The cTAR sequence from the Lai strain of HIV-1 was selectively modified either at position 9, 17, 21, 28, 35, 45, 49, 53 or 55 with 2-Ap.
Mentions: The nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) is a small basic protein resulting from the protease-mediated cleavage of the Gag polyprotein precursor. NCp7 is formed by two highly conserved CCHC zinc-finger motifs connected by a short flexible linker and flanked by poorly folded N- and C-terminal basic domains (1,2) (Figure 1). NCp7 is critical during the early and late steps of the HIV-1 viral life cycle (3–11). Most NCp7 functions rely on its RNA- and DNA-chaperone properties that direct the rearrangement of nucleic acids into their most thermodynamically stable structure (12–15). These chaperone properties are thought to play a critical role in reverse transcription (5,16,17). For instance, during the first strand-transfer of reverse transcription, NCp7 favours the transfer of the newly synthesized minus strong-stop DNA [(−) ssDNA] to the 3′-end of the viral RNA genome, by promoting the base pairing of the complementary repeat (R) sequences at the 3′-ends of the (−)ssDNA and genomic RNA, respectively (18). In vitro, the stable stem–loop (SL) of the transactivation response element (TAR) sequence within the R domain of the genomic RNA does not readily anneal with the complementary cTAR sequence of the (−)ssDNA in the absence of NCp7 (18–21). Addition of NCp7 strongly increases the rate and extent of the cTAR/TAR annealing reaction (18–23) and switches the reaction pathway to direct the hybridization of these sequences through the end of their double-stranded (ds) stems (20,21). The main chaperone properties involved in promoting the first strand transfer have been characterized as two interconnected components: a destabilization activity and a hybridization-promoting activity. Promotion of the annealing of complementary sequences requires an efficient aggregation of nucleic acids (24). This is achieved by the numerous basic residues of NCp7 that decrease the electrostatic repulsion between complementary oligonucleotides by neutralizing the negatively charged phosphate groups (5,21,25). The annealing facilitator property of NCp7 is shared with many other basic proteins that are also able to promote the cTAR/TAR DNA hybridization (26–28). On the contrary, the ability to destabilize the cTAR sequence appears to be a hallmark of NCp7. NCp7 activates the transient opening (fraying) of the lower half of the cTAR stem (29–34) constituted by a series of short ds-segments separated by a bulge and two mismatches. This activation follows a non–two-state mechanism, which results in complex spatial and temporal fluctuations of the cTAR ends that greatly facilitate cTAR/TAR hybridization. This activity has been assigned to the conserved hydrophobic platform formed by the Val13, Phe16, Thr24, Ala25, Trp37, Gln45 and Met46 residues at the surface of the folded zinc-finger (ZF) domains (1,2,35). More specifically, the Trp37 residue plays a critical role through stacking with G residues (17,36–43). This hydrophobic platform has also been found critical for binding (35,38). Nevertheless, the precise molecular mechanisms by which NCp7 destabilizes cTAR remain poorly described. Here, we selectively modified the 57-nt cTAR stem–loop sequence of the Lai strain with 2-aminopurine (2-Ap) at different positions to investigate by fluorescence spectroscopy the structural and dynamic modifications that occur within cTAR in response to NCp7 binding. In contrast to most techniques that monitor only global structural changes, the exquisite sensitivity of 2-Ap fluorescence to its local environment allows region-specific characterization of the structural modifications within the cTAR hairpin sequence. The 2-Ap has been previously used to site-specifically characterize the NCp7-induced changes in the dynamics of short single-stranded sequences (44,45), as well as in the loop of the primer-binding site sequence of HIV-1 (46). These studies showed that binding of the intact ZF domain was responsible for a decrease of the DNA base stacking and for a strong restriction of the base mobility on a picosecond–nanosecond timescale. By selectively replacing different adenine positions of the cTAR sequence by 2-Ap (Figure 1), we aimed to picture the preferential destabilization sites and to gain insight into the molecular mechanism by which the NCp7 ZFs destabilize the cTAR stem–loop. Although NC(11–55) was found to bind all over the sequence, it preferentially restricted the dynamics and destabilized the bases of the penultimate ds-segment sandwiched between two C•A mismatches in the lower part of the cTAR stem. These NC(11–55)-induced changes are highly specific, as they were not observed when NC(11–55) was substituted by the SSHS–NC(11–55) mutant where the Cys residues in the ZFs were substituted by Ser residues to prevent the binding of zinc and the folding of the ZFs. These structural changes in this part of cTAR play likely a critical role in the ability of NCp7 to promote cTAR/TAR annealing through a specific zipping pathway.Figure 1.

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