Hugues de Rocquigny

NCp7 Activates HIV-1Lai RNA Dimerization by Converting a Transient Loop-Loop Complex into a Stable Dimer

Nucleocapsid protein 7 (NCp7), the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, was shown to strongly potentiate the dimerization of the retroviral genomic RNA. This process involves the interaction of two retroviral RNA monomer subunits near their 5′-ends. A region located upstream from the splice donor site was recently identified as being responsible for the formation of dimeric HIV-1 RNA. This region appeared to be confined within a stem-loop structure, with an autocomplementary sequence in the loop. In an in vitro study of spontaneous dimer formation, we reported that the 77-402 RNA transcript forms two distinct dimers differing in their thermostability: D37 and D55. We identified D37 as a “kissing” complex structure, formed via a loop-loop interaction between the two monomers, and D55 as a double stranded structure involving all nucleotides of the stem-loop via canonical base pairing. In this report, we have characterized the role of NCp7 in the HIV-1Lai RNA dimerization process by using in vitro dimerization assays with RNA transcripts of different lengths and dimer thermal dissociation. Our results show that the nucleocapsid protein NCp7 activates RNA dimerization very likely through interaction with the kissing complex and converts it into a stable dimer. Furthermore, this NCp7-promoted conversion only occurs if the 240-280 stem-loop structure is present in HIV-1Lai RNA molecules and contains the autocomplementary G257CGCGC262 sequence. This study suggests that, under physiological conditions, an NCp7-mediated RNA conformational change is involved in the maturation of the HIV-1 RNA dimer.

Evidence of Interactions between the Nucleocapsid Protein NCp7 and the Reverse Transcriptase of HIV-1

The human immunodeficiency virus (HIV-1) nucleocapsid protein NCp7 containing two CX 2CX 4HX 4C-type zinc fingers was proposed to be involved in reverse transcriptase (RT)-catalyzed proviral DNA synthesis through promotion of tRNA3 Lys annealing to the RNA primer binding site, improvement of DNA strand transfers, and enhancement of RT processivity. The NCp7 structural characteristics are crucial because mutations altering the finger domain conformation led to noninfectious viruses characterized by defects in provirus integration. These findings prompted us to study a putative RT/NCp7 protein-protein interaction. Binding assays using far Western analysis or RT immobilized on beads clearly showed the formation of a complex between NCp7 and RT. The affinity of NCp7 for p66/p51RT was 0.60 μm with a 1:1 stoechiometry. This interaction was confirmed by chemical cross-linking and co-immunoprecipitation of the two proteins in a viral environment. Competition experiments using different NCp7 mutants showed that alteration of the finger structure disrupted RT recognition, giving insights into the loss of infectivity of corresponding HIV-1 mutants. Together with structural data on RT, these results suggest that the role of NCp7 could be to enhance RT processivity through stabilization of a p51-induced active form of the p66 subunit and open the way for designing new antiviral agents.

Targeting the viral nucleocapsid protein in anti-HIV-1 therapy.

The nucleocapsid protein (NC) plays seminal roles in HIV replication, thus representing a major drug target. NC functions rely on its two zinc-fingers and flanking basic residues. Zinc ejectors inhibit NC functions, but with limited specificity. New classes of molecules competing with NC or its viral nucleic acid and enzyme partners are reviewed here.

Properties, Functions, and Drug Targeting of the Multifunctional Nucleocapsid Protein of the Human Immunodeficiency Virus

Publisher Summary This chapter reviews the structure and functions of the human immunodeficiency virus type 1 (HIV‐1) nucleocapsid (NC) protein and discusses the rational for a simple, rapid screening of anti‐NC drugs aimed at inhibiting virion production. Retroviral nucleocapsid (NC) proteins are small proteins generated by the cleavage of the Gag structural polyprotein by the viral protease and are characterized by one or two copies of a highly conserved CCHC zinc finger (ZF) flanked by basic residues. Retroviral NC proteins are nucleic acid–binding proteins with potent RNA‐chaperoning properties, enabling important structural rearrangements that are required for genomic RNA replication and its packaging during virion assembly. The three‐dimensional (3D) conformation of HIV‐1 NC shows that the central domain folds into a hydrophobic plateau flanked by disordered basic sequences. The NC hydrophobic plateau pilots the selection, dimerization, and packaging of the genomic RNA during the virion assembly process, which ensures the formation of a mature functional inner capsid structure. The chapter describes a new one‐step screening assay, which allows for the rapid in vitro identification of anti‐NC compounds aimed at binding to the hydrophobic plateau, thus, inhibiting NC during the early and late steps of HIV‐1 replication.

Sensing peptide–oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein

We present a new methodology for site-specific sensing of peptide–oligonucleotide (ODN) interactions using a solvatochromic fluorescent label based on 3-hydroxychromone (3HC). This label was covalently attached to the N-terminus of a peptide corresponding to the zinc finger domain of the HIV-1 nucleocapsid protein (NC). On interaction with target ODNs, the labeled peptide shows strong changes in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from the bulk water by the ODN bases. Remarkably, this two-color response depends on the ODN sequence and correlates with the 3D structure of the corresponding complexes, suggesting that the 3HC label monitors the peptide–ODN interactions site-specifically. By measuring the two-color ratio, we were also able to determine the peptide–ODN-binding parameters and distinguish multiple binding sites in ODNs, which is rather difficult using other fluorescence methods. Moreover, this method was found to be more sensitive than the commonly used steady-state fluorescence anisotropy, especially in the case of small ODNs. The described methodology could become a new universal tool for investigating peptide–ODN interactions.

Specific implications of the HIV-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer

Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand transfer corresponds to the annealing of the (−) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(−)PBS annealing mechanism by activating a loop–loop kissing pathway that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes, we investigated the time-resolved fluorescence parameters of a (−)PBS derivative containing the fluorescent nucleoside analogue 2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(−)PBS annealing towards the loop pathway was associated to a drastic restriction of the local DNA dynamics, indicating that NCp7 can ‘freeze’ PBS conformations competent for annealing via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA.

Direct Vpr-Vpr interaction in cells monitored by two photon fluorescence correlation spectroscopy and fluorescence lifetime imaging.

BackgroundThe human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.ResultsResults show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization.ConclusionWe report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

The gag precursor contains a specific HIV-1 protease cleavage site between the NC (P7) and P1 proteins.

The predicted protease cleavage site (p7/pl; [J. Virol. 66 (1992) 1856‐1865]) within the nucleocapsid precursor protein (p15) of human immunodeficiency virus, type 1, was confirmed using an in vitro assay employing recombinant HIV‐1 protease and a chemically synthesized 72 amino acid polypeptide containing the p7 and p1 protein domains of the native gag polyprotein. The cleavage occurred between amino acid 55 (N) and amino acid 56 (F) of the polypeptide, as determined by N‐terminal sequencing. The hydrolysis was optimal at pH 6.0 and at high salt concentration. The kinetic parameters K m, k cat and K cat/K m were 99 μM (±8), 0.152 s−1 (± 0.002) and 1.56 mM−1· s−1 (± 0.11), respectively. Reconstituted as well as denatured polypeptides were cleaved at approximately the same rate, demonstrating that the conformation of the p7 protein, as a result of the Zn2+‐binding, had no significant effect on the rate of hydrolysis of the p7/pl cleavage.

Three-dimensional 1H NMR structure of the nucleocapsid protein NCp10 of Moloney murine leukemia virus

SummaryThe nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX2CysX4HisX4Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH≥5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX2CysX4HisX4Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz 1H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747±0.209 Å for backbone atoms and 1.709±0.187 Å when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr28, Trp35, Lys37, Lys41 and Lys42 residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.

HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag

ABSTRACT During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55Gag. Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G2/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55Gag-TC. This TC motif is tethered to the C terminus of Pr55Gag and does not interfere with Pr55Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55Gag-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55Gag-TC mutants confirm that the 41LXXLF domain of Gag-p6 is essential for Pr55Gag-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag-Vpr complexes are still formed when Pr55Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag-Vpr recognition and complex formation occur early during Pr55Gag assembly.