Philippe Fossé

Flexible Nature and Specific Functions of the HIV-1 Nucleocapsid Protein

One salient feature of reverse transcription in retroviruses, notably in the human immunodeficiency virus type 1, is that it requires the homologous nucleocapsid (NC) protein acting as a chaperoning partner of the genomic RNA template and the reverse transcriptase, from the initiation to the completion of viral DNA synthesis. This short review on the NC protein of human immunodeficiency virus type 1 aims at briefly presenting the flexible nature of NC protein, how it interacts with nucleic acids via its invariant zinc fingers and flanking basic residues, and the possible mechanisms that account for its multiple functions in the early steps of virus replication, notably in the obligatory strand transfer reactions during viral DNA synthesis by the reverse transcriptase enzyme.

Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5′ thymine interacts with residues of the N-terminal zinc knuckle and the 3′ guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA–protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids.

Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein

Annealing of the TAR RNA hairpin to the cTAR DNA hairpin is required for the minus-strand transfer step of HIV-1 reverse transcription. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. To gain insight into the mechanism of NC-mediated TAR RNA–DNA annealing, we used structural probes (nucleases and potassium permanganate), gel retardation assays, fluorescence anisotropy and cTAR mutants under conditions allowing strand transfer. In the absence of NC, cTAR DNA-TAR RNA annealing depends on nucleation through the apical loops. We show that the annealing intermediate of the kissing pathway is a loop–loop kissing complex involving six base-pairs and that the apical stems are not destabilized by this loop–loop interaction. Our data support a dynamic structure of the cTAR hairpin in the absence of NC, involving equilibrium between both the closed conformation and the partially open ‘Y’ conformation. This study is the first to show that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively. NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the ‘Y’ conformation exhibiting at least 12 unpaired nucleotides in its lower part.

Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin

First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop–loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loops is stable, but that the lower stem of mini-cTAR is unstable. The residues of the internal loop undergo slow motions at the NMR time-scale that are consistent with conformational exchange phenomena. In contrast, residues of the apical loop undergo fast motions. The lower stem is destabilized by the slow interconversion processes in the internal loop, and thus the internal loop is responsible for asymmetric destabilization of mini-cTAR. These findings are consistent with the functions of cTAR in first strand transfer: its apical loop is suitably exposed to interact with the apical loop of TAR RNA and its lower stem is significantly destabilized to facilitate the subsequent action of the nucleocapsid protein which promotes the annealing reaction.

Intrinsic Nucleic Acid Dynamics Modulates HIV-1 Nucleocapsid Protein Binding to Its Targets

HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using 13C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome.

Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers.

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.

Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding

Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase’s DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning.

Dynamic interactions of the HIV-1 Tat with nucleic acids are critical for Tat activity in reverse transcription

The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44–61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44–61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer. To further understand the structure–activity relationships of this highly conserved domain, we investigated by electrophoresis and fluorescence approaches the binding and annealing properties of various Tat(44–61) mutants. Our data showed that the Tyr47 and basic residues of the Tat(44–61) domain were instrumental for binding to cTAR through stacking and electrostatic interactions, respectively, and promoting its annealing with dTAR. Furthermore, the annealing efficiency of the mutants clearly correlates with their ability to rapidly associate and dissociate the complementary oligonucleotides and to promote RTion. Thus, transient and dynamic nucleic acid interactions likely constitute a key mechanistic component of annealers and the role of Tat in the late steps of RTion. Finally, our data suggest that Lys50 and Lys51 acetylation regulates Tat activity in RTion.

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59.