Yves Mély

Destabilization of the HIV-1 complementary sequence of TAR by the nucleocapsid protein through activation of conformational fluctuations.

The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two obligatory strand transfer reactions required for the synthesis of a complete proviral DNA by reverse transcriptase. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response region (TAR) sequence at the 3 end of the genomic RNA and the complementary sequence cTAR at the 3 terminus of minus strong-stop DNA, the early product of reverse transcription. In order to determine the dynamics of NCp7-mediated nucleic acid destabilization, we investigated by time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly labeled cTAR sequence with NC(12-55) containing NCp7 CCHC zinc fingers and flanking basic amino acid residues. From the chemical rates and the activation energy associated with the conformational fluctuations observed in the absence of NC, it is concluded that such fluctuations are associated with the opening and closing of the double-stranded terminal segments of cTAR. The destabilizing activity of NC(12-55) occurs mainly through a major increase of the opening rate constant of cTAR. Moreover, NC appears to augment the number of pathways between the open and closed states of cTAR, suggesting that it initiates melting of base-pairs at different locations within the terminal segments of cTAR. This activity of NC on the dynamics of cTAR secondary structure is thought to be critical for the formation of the cTAR-TAR complex, which is essential for the specificity and extent of proviral DNA synthesis by reverse transcriptase.

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.

How the HIV-1 Nucleocapsid Protein Binds and Destabilises the (−)Primer Binding Site During Reverse Transcription

The human immunodeficiency virus type 1 nucleocapsid protein (NCp7) plays an important role in the second strand transfer during reverse transcription. It promotes annealing of the 18-nucleotide complementary DNA primer-binding site (PBS) sequences at the 3 ends of (-)DNA and (+)DNA. NMR studies show that NCp7(12-55) and NCp7(1-55) interact at the 5 end of the loop of DeltaP(-)PBS, a (-)PBS derivative without the 3 protruding sequence, in a slow-exchange equilibrium. This interaction is mediated through the binding of the hydrophobic plateau (Val13, Phe16, Thr24, Ala25, Trp37, and Met46) on the zinc finger domain of both peptides to the 5-CTG-7 sequence of DeltaP(-)PBS. The stacking of the Trp37 aromatic ring with the G7 residue likely constitutes the determinant factor of the interaction. Although NCp7(12-55) does not melt the DeltaP(-)PBS stem-loop structure, it opens the loop and weakens the C5.G11 base pair next to the loop. Moreover, NCp7(12-55) was also found to bind but with lower affinity to the 10-CGG-12 sequence in an intermediate-exchange equilibrium on the NMR time scale. The loop modifications may favour a kissing interaction with the complementary (+)PBS loop. Moreover, the weakening of the upper base pair of the stem likely promotes the melting of the stem that is required to convert the kissing complex into the final (+/-)PBS extended duplex.

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.

Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine

The nucleocapsid protein (NC) plays an important role in HIV-1, mainly through interactions with the genomic RNA and its DNA copies. Though the structures of several complexes of NC with oligonucleotides (ODNs) are known, detailed information on the ODN dynamics in the complexes is missing. To address this, we investigated the steady state and time-resolved fluorescence properties of 2-aminopurine (2Ap), a fluorescent adenine analog introduced at positions 2 and 5 of AACGCC and AATGCC sequences. In the absence of NC, 2Ap fluorescence was strongly quenched in the flexible ODNs, mainly through picosecond to nanosecond dynamic quenching by its neighboring bases. NC strongly restricted the ODN flexibility and 2Ap local mobility, impeding the collisions of 2Ap with its neighbors and thus, reducing its dynamic quenching. Phe16→Ala and Trp37→Leu mutations largely decreased the ability of NC to affect the local dynamics of 2Ap at positions 2 and 5, respectively, while a fingerless NC was totally ineffective. The restriction of 2Ap local mobility was thus associated with the NC hydrophobic platform at the top of the folded fingers. Since this platform supports the NC chaperone properties, the restriction of the local mobility of the bases is likely a mechanistic component of these properties.

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.

Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein.

Due to its highly conserved zinc fingers and its nucleic acid chaperone properties which are critical for HIV-1 replication, the nucleocapsid protein (NC) constitutes a major target in AIDS therapy. Different families of molecules targeting NC zinc fingers and/or inhibiting the binding of NC with its target nucleic acids have been developed. However, their limited specificity and their cellular toxicity prompted us to develop a screening assay to target molecules able to inhibit NC chaperone properties, and more specifically the initial NC-promoted destabilization of the nucleic acid secondary structure. Since this destabilization is critically dependent on the properly folded fingers, the developed assay is thought to be highly specific. The assay was based on the use of cTAR DNA, a stem-loop sequence complementary to the transactivation response element, doubly labelled at its 5 and 3 ends by a rhodamine 6G fluorophore and a fluorescence quencher, respectively. Addition of NC(12-55), a peptide corresponding to the zinc finger domain of NC, to this doubly-labelled cTAR, led to a partial melting of the cTAR stem, which increases the distance between the two labels and thus, restores the rhodamine 6G fluorescence. Thus, positive hits were detected through the decrease of rhodamine 6G fluorescence. An in-house chemical library of 4800 molecules was screened and five compounds with IC(50) values in the micromolar range have been selected. The hits were shown by mass spectrometry and fluorescence anisotropy titration to prevent binding of NC(12-55) to cTAR through direct interaction with the NC folded fingers, but without promoting zinc ejection. These non-zinc ejecting NC binders are a new series of anti-NC molecules that could be used to rationally design molecules with potential anti-viral activities.

HIV-1 nucleocapsid traps reverse transcriptase on nucleic acid substrates.

Conversion of the genomic RNA of human immunodeficiency virus (HIV) into full-length viral DNA is a complex multistep reaction catalyzed by the reverse transcriptase (RT). Numerous studies have shown that the viral nucleocapsid (NC) protein has a vital impact on various steps during reverse transcription, which is crucial for virus infection. However, the exact molecular details are poorly defined. Here, we analyzed the effect of NC on RT-catalyzed single-turnover, single-nucleotide incorporation using different nucleic acid substrates. In the presence of NC, we observed an increase in the amplitude of primer extension of up to 3-fold, whereas the transient rate of nucleotide incorporation ( k pol) dropped by up to 50-fold. To unravel the underlying molecular mechanism, we carefully analyzed the effect of NC on RT-nucleic acid substrate dissociation. The studies revealed that NC considerably enhances the stability of RT-substrate complexes by reducing the observed dissociation rate constants, which more than compensates for the observed drop in k pol. In conclusion, our data strongly support the concept that NC not only indirectly assists the reverse transcription process by its nucleic acid chaperoning activity but also positively affects the RT-catalyzed nucleotide incorporation reaction by increasing polymerase processivity presumably via a physical interaction of the two viral proteins.

Inhibition of HIV-1 replication by a bis-thiadiazolbenzene-1,2-diamine that chelates zinc ions from retroviral nucleocapsid zinc fingers.

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) nucleocapsid p7 (NCp7) protein holds two highly conserved “CCHC” zinc finger domains that are required for several phases of viral replication. Basic residues flank the zinc fingers, and both determinants are required for high-affinity binding to RNA. Several compounds were previously found to target NCp7 by reacting with the sulfhydryl group of cysteine residues from the zinc fingers. Here, we have identified an N,N′-bis(1,2,3-thiadiazol-5-yl)benzene-1,2-diamine (NV038) that efficiently blocks the replication of a wide spectrum of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) strains. Time-of-addition experiments indicate that NV038 interferes with a step of the viral replication cycle following the viral entry but preceding or coinciding with the early reverse transcription reaction, pointing toward an interaction with the nucleocapsid protein p7. In fact, in vitro, NV038 efficiently depletes zinc from NCp7, which is paralleled by the inhibition of the NCp7-induced destabilization of cTAR (complementary DNA sequence of TAR). A chemical model suggests that the two carbonyl oxygens of the esters in this compound are involved in the chelation of the Zn2+ ion. This compound thus acts via a different mechanism than the previously reported zinc ejectors, as its structural features do not allow an acyl transfer to Cys or a thiol-disulfide interchange. This new lead and the mechanistic study presented provide insight into the design of a future generation of anti-NCp7 compounds.

Site-Specific Characterization of HIV-1 Nucleocapsid Protein Binding to Oligonucleotides with Two Binding Sites†

The nucleocapsid protein (NC) of HIV-1 is a highly conserved protein essential for the virus life cycle that constitutes an attractive target for new antiviral agents. Most NC functions rely on its binding to the HIV-1 genomic RNA and its DNA copies that contain multiple and possibly interdependent binding sites. Therefore, a detailed understanding of NC binding requires a site-specific experimental approach. We have recently shown that 2-aminopurine (2Ap), a fluorescent adenine analogue, can site-selectively probe the binding of NC. Here, we introduced 2Ap at various positions of model single-stranded dodecanucleotides containing two TG motifs which constitute putative specific binding sites. Steady-state and time-resolved fluorescence experiments indicated that NC binding strongly increased the fluorescence quantum yield of 2AP by reducing the dynamic quenching of 2Ap by its close neighbors and slowing the picosecond to nanosecond conformational fluctuations of the oligonucleotides. The dodecanucleotides were found to bind two NC molecules at physiological salt concentrations, confirming the preferential binding of NC to TG motifs and an occluded binding site size for NC of five to six bases. Using the NC-induced changes in 2Ap fluorescence, we determined the microscopic affinity constants of the individual binding sites and showed that affinities can significantly differ from one site to another within the same dodecanucleotide, depending on the position of the TG dinucleotide and the nature of its close neighbors. Moreover, our data suggest that binding of NC even to close binding sites shows no strong cooperativity.