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

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

Retroviral nucleocapsid proteins and DNA strand transfers

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RNA Structural Requirements for Nucleocapsid Protein-Mediated Extended Dimer Formation

Retroviruses package two copies of their genomic RNA (gRNA) as non-covalently linked dimers. Many studies suggest that the retroviral nucleocapsid protein (NC) plays an important role in gRNA dimerization. The upper part of the L3 RNA stem-loop in the 5′ leader of the avian leukosis virus (ALV) is converted to the extended dimer by ALV NC. The L3 hairpin contains three stems and two internal loops. To investigate the roles of internal loops and stems in the NC-mediated extended dimer formation, we performed site-directed mutagenesis, gel electrophoresis, and analysis of thermostability of dimeric RNAs. We showed that the internal loops are necessary for efficient extended dimer formation. Destabilization of the lower stem of L3 is necessary for RNA dimerization, although it is not involved in the linkage structure of the extended dimer. We found that NCs from ALV, human immunodeficiency virus type 1 (HIV-1), and Moloney murine leukemia virus (M-MuLV) cannot promote the formation of the extended dimer when the apical stem contains ten consecutive base pairs. Five base pairs correspond to the maximum length for efficient L3 dimerization induced by the three NCs. L3 dimerization was less efficient with M-MuLV NC than with ALV NC and HIV-1 NC

In vitro characterization of a base pairing interaction between the primer binding site and the minimal packaging signal of avian leukosis virus genomic RNA

The 5′ leader region of avian sarcoma-leukosis viruses (ASLVs) folds into a series of RNA secondary structures which are involved in key steps in the viral replication cycle such as reverse transcription, dimerization and packaging of genomic RNA. The O3 stem and three stem–loops (O3SLa, O3SLb and O3SLc) form the minimal packaging signal that is located downstream of the primer binding site (PBS). The U5–PBS region contributes to packaging via a mechanism that remains unknown. In this in vitro study, we have investigated the possibility of interactions between the R–U5–PBS region and the minimal packaging signal using chemical and enzymatic probing, antisense oligonucleotides and site-directed mutagenesis. We have identified a base pairing interaction between the PBS sequence and the terminal loop of O3SLa. It was found that the PBS/O3SLa interaction was intramolecular since it occurred not only in dimeric RNA but also in monomeric RNA. This interaction probably corresponds to a pseudoknot interaction. The PBS/O3SLa interaction may be formed in vivo since the sequences are highly conserved in ASLV strains. The PBS/O3SLa interaction may regulate the processes of primer tRNA annealing, packaging and initiation of Gag translation through its involvement in leader tertiary structure. Interestingly, we found that in other retroviruses the PBS sequence can also base pair with a terminal loop of the stem–loops involved in RNA packaging

Characterization of loose and tight dimer forms of avian leukosis virus RNA.

International audienceRetroviral genomes consist of two identical RNA molecules joined non-covalently near their 5'-ends. Recently, we showed that an imperfect autocomplementary sequence, located in the L3 domain, plays an essential role in avian sarcoma-leukosis virus (ASLV) RNA dimerization in vitro. This sequence can adopt a stem-loop structure and is involved in ASLV replication. Here, we found that in the absence of nucleocapsid protein, RNA transcripts of avian leukosis virus (ALV) were able to form two types of dimers in vitro that differ in their stability: a loose dimer, formed at a physiological temperature, and a tight dimer, formed at a high temperature. A mutational analysis was performed to define the features of these dimers. The results of this analysis unambiguously confirm that the two L3 stem-loops interact directly in both types of dimers. A loop-loop interaction is the main linkage in the loose dimer. In contrast, in the tight dimer, the stem and the loop of the L3 hairpin form an extended duplex. Surprisingly, we also found that the dimerization properties defined for our ALV strain (type SR-A) differ from those found in other ASLV strains

Identification of acylation products in SHAPE Chemistry

International audienceSHAPE chemistry (selective 2’-hydroxyl acylation analyzed by primer extension) has been developed to specifically target flexible nucleotides (often unpaired nucleotides) independently to their purine or pyrimidine nature for RNA secondary structure determination. However, to the best of our knowledge, the structure of 2’-O-acylation products has never been confirmed by NMR or X-ray data. We have realized the acylation reactions between cNMP and NMIA under SHAPE chemistry conditions and identified the acylation products using standard NMR spectroscopy and LC-MS/MS experiments. For cAMP and cGMP, the major acylation product is the 2’-O-acylated compound (> 99 %). A trace amount of N-acylated cAMP has also been identified by LC-UV-MS². While for cCMP, the isolated acylation products are composed of 96 % of 2’-O-acylated, 4 % of N,O-diacylated, and trace amount of N-acylated compounds. In addition, the characterization of the major 2’-O-acylated compound by NMR showed slight differences in the conformation of the acylated sugar between the three cyclic nucleotides. This interesting result should be useful to explain some unexpected reactivity of the SHAPE chemistry

RNA SHAPE chemistry with aromatic acylating reagents

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