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Home page > Biophotonics Team > Biomolecular interactions: (...)

Biomolecular interactions: Nucleocapsid protein of HIV-1

Last update 15 April 2013

Head : Yves Mély

The HIV-1 nucleocapsid protein, NCp7 is a small basic protein, characterized by two zinc fingers, that plays a key role in HIV-1 life cycle and constitutes thus an interesting target for an antiviral therapy. Since many years, a major research axis of our team is based on the characterization of the structure, the properties and the functions of this protein

Mechanism of the nucleic acid chaperone properties of NCp7

Through its chaperone properties, NCp7 rearranges the nucleic acids into their most stable conformation. We are characterizing at the single molecule level the mechanisms of the nucleic acid chaperone properties of NCp7, in particular during the two obligatory strand transfers of reverse transcription.

We have shown that NCp7 chaperones the first strand transfer by promoting the annealing of the complementary TAR RNA and cTAR DNA stem-loop sequences. This critically relies on NCp7 ability to preferentially recognizes unpaired guanines exhibiting a high degree of mobility (1) and to transiently melt their terminal base pairs (2–5). The destabilization activity of NCp7 is mediated by a hydrophobic plateau at the surface of the folded zinc fingers, which restricted the oligonucleotide flexibility and the local dynamics of the bases (6, 7). More specifically, NCp7 preferentially destabilizes the penultimate double-stranded segment in the cTAR stem, through a zinc-finger-dependent binding of NCp7 to two G residues (8). NCp7 also modifies the (+)/(-)PBS annealing mechanism in the second strand transfer by activating a loop-loop kissing pathway that is negligible without NCp7 (9, 10). This NCp7-directed switch of (+)/(-)PBS annealing is due to a drastic restriction of the local DNA dynamics, and is critical to ensure the specificity and fidelity of the second strand transfer (11). Moreover, we found that the opposite binding polarities of NCp7 to RNA and DNA sequences are mediated by the interaction of NCp7 with the sugar residues (12). Currently, we are completing our characterization at the molecular level of the fine mechanism of NCp7 and more specifically of its finger motifs, in both strand transfers in order to find new clues to inhibit these key steps in reverse transcription. In addition, we investigate in parallel the chaperone properties of Tat, to further understand its mechanism and role in the viral cycle (13, 14).

Interaction of NCp7 with viral and host proteins

Though a large part of the ubiquitous functions of NCp7 relies on its interactions with nucleic acid targets, it becomes more and more obvious that interactions of NCp7 with other HIV-1 proteins and host proteins play also a key role. Our objective is to characterize these interactions and identify their functions in the viral life cycle.

We are interested in how NCp7 cooperates with the reverse transcriptase (RT) in reverse transcription. We showed that NCp7 enhances the stability of RT-substrate complexes by reducing the observed dissociation rate constants (15). Thus, NCp7 not only indirectly assists the reverse transcription process by its nucleic acid chaperoning activity but also by positively affecting the RT-catalyzed nucleotide incorporation reaction, presumably via a physical interaction of the two viral proteins. We are currently investigating by single molecule techniques the molecular mechanisms of the role of NCp7 in assisting RT.

PNG - 34.8 kb
Gag assembly by FLIM (A) and interaction with a cell protein (B) by confocal microscopy

Moreover, we are looking for cellular partners that interact with NCp7 during the different steps of the viral cycle. Our objective is to identify these partners, demonstrate their interaction with NCp7 using biochemical and imaging techniques and describe the role of these interactions in HIV-1 replication. We also further characterize the role of the NCp7 domain within the Gag polyprotein in the assembly of the viral particles.

Bioactive molecules directed against NCp7

Due to its high conservation and its critical roles in various steps of the viral life cycle, NCp7 appears as an ideal target for an anti-HIV-1 therapy, with the promise to inhibit multiple steps in HIV-1 replication with one compound and raise low or no resistance (16, 17). On the basis of the NCp7 chaperone properties, we are developing different classes of molecules able to inhibit NCp7 and thus, the viral life cycle.

Small molecules. Using a virtual screening protocol, two molecules able to bind to the NCp7 zinc fingers, without ejecting zinc, and showing antiretroviral activity at micromolar concentrations were identified (18). Moreover, screening of a chemical library on an assay based on NCp7 chaperone properties allowed us to identify a few hits inhibiting NCp7, without ejecting zinc (19). Our aim is to screen further compounds in order to find out molecules targeting NCp7 with improved efficiency. Through another and more general screening assay, the N,N’-bis(1,2,3-thiadiazol-5-yl)benzene-1,2-diamine (NV038) compound that efficiently blocks the replication of a wide spectrum of HIV and SIV strains was identified (20). NV038 interferes with an early reverse transcription step, by efficiently depleting zinc from NCp7 through a different mechanism than the previously reported zinc ejectors. We also identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) that ejects zinc from both zinc fingers, even when NCp7 is bound to oligonucleotides (21). WDO-217 inhibits infections of a wide spectrum of wild-type and drug-resistant HIV-1.

Oligonucleotides directed against NCp7. Small methylated oligoribonucleotides (mODN) that extensively inhibit the NCp7 chaperoning activity and impede HIV-1 replication in primary human cells at nanomolar concentrations were developed (22). These mODNs severely impair viral cDNA synthesis. The mODNs likely inhibit NCp7 by a competitive mechanism whereby they tightly bind to the hydrophobic platform of the NCp7 peptide (23). Therefore, the mONs may impair the process of the RT-directed viral DNA synthesis by sequestering NCp7 molecules, thus preventing the chaperoning of viral DNA synthesis by NCp7.

Peptides. Trp-rich hexapeptides have been identified by phage display. We showed that these peptides bind to the TAR and PBS sequences with affinities comparable to that of NCp7 and inhibit efficiently the destabilizing activity of NCp7 (24). These peptides show antiviral activities in the micromolar range (25). More efficient inhibitions of NCp7 chaperone activities were obtained with a second generation of peptides, which are currently investigated.

References

1. Bazzi,A., Zargarian,L., Chaminade,F., De Rocquigny,H., René,B., Mély,Y., Fossé,P. and Mauffret,O. (2012) Intrinsic nucleic acid dynamics modulates HIV-1 nucleocapsid protein binding to its targets. Plos One, 7, e38905.

2. Bernacchi,S., Stoylov,S., Piémont,E., Ficheux,D., Roques,B.P., Darlix,J.L. and Mély,Y. (2002) HIV-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of TAR and its complementary sequence. J. Mol. Biol., 317, 385–399.

3. Azoulay,J., Clamme,J.P., Darlix,J.L., Roques,B.P. and Mély,Y. (2003) Destabilization of the HIV-1 complementary sequence of TAR by the nucleocapsid protein through activation of conformational fluctuations. J. Mol. Biol., 326, 691–700.

4. Beltz,H., Clauss,C., Piémont,E., Ficheux,D., Gorelick,R.J., Roques,B., Gabus,C., Darlix,J.-L., de Rocquigny,H. and Mély,Y. (2005) Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis. J. Mol. Biol., 348, 1113–1126.

5. Godet,J., de Rocquigny,H., Raja,C., Glasser,N., Ficheux,D., Darlix,J.-L. and Mély,Y. (2006) During the early phase of HIV-1 DNA synthesis, nucleocapsid protein directs hybridization of the TAR complementary sequences via the ends of their double-stranded stem. J. Mol. Biol., 356, 1180–1192.

6. Avilov,S.V., Piemont,E., Shvadchak,V., de Rocquigny,H. and Mély,Y. (2008) Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine. Nucleic Acids Res., 36, 885–896.

7. Avilov,S.V., Godet,J., Piémont,E. and Mély,Y. (2009) Site-specific characterization of HIV-1 nucleocapsid protein binding to oligonucleotides with two binding sites. Biochemistry (Mosc.), 48, 2422–2430.

8. Godet,J., Kenfack,C., Przybilla,F., Richert,L., Duportail,G. and Mély,Y. (2013) Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer. Nucleic Acids Res., 10.1093/nar/gkt164.

9. Ramalanjaona,N., de Rocquigny,H., Millet,A., Ficheux,D., Darlix,J.-L. and Mély,Y. (2007) Investigating the mechanism of the nucleocapsid protein chaperoning of the second strand transfer during HIV-1 DNA synthesis. J. Mol. Biol., 374, 1041–1053.

10. Bourbigot,S., Ramalanjaona,N., Boudier,C., Salgado,G.F.J., Roques,B.P., Mély,Y., Bouaziz,S. and Morellet,N. (2008) How the HIV-1 nucleocapsid protein binds and destabilises the (-)primer binding site during reverse transcription. J. Mol. Biol., 383, 1112–1128.

11. Godet,J., Ramalanjaona,N., Sharma,K.K., Richert,L., de Rocquigny,H., Darlix,J.-L., Duportail,G. and Mély,Y. (2011) 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. Nucleic Acids Res., 39, 6633–6645.

12. Bazzi,A., Zargarian,L., Chaminade,F., Boudier,C., De Rocquigny,H., René,B., Mély,Y., Fossé,P. and Mauffret,O. (2011) Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC. Nucleic Acids Res., 39, 3903–3916.

13. Boudier,C., Storchak,R., Sharma,K.K., Didier,P., Follenius-Wund,A., Muller,S., Darlix,J.-L. and Mély,Y. (2010) The mechanism of HIV-1 Tat-directed nucleic acid annealing supports its role in reverse transcription. J. Mol. Biol., 400, 487–501.

14. Godet,J., Boudier,C., Humbert,N., Ivanyi-Nagy,R., Darlix,J.-L. and Mély,Y. (2012) Comparative nucleic acid chaperone properties of the nucleocapsid protein NCp7 and Tat protein of HIV-1. Virus Res., 169, 349–360.

15. Grohmann,D., Godet,J., Mély,Y., Darlix,J.-L. and Restle,T. (2008) HIV-1 nucleocapsid traps reverse transcriptase on nucleic acid substrates. Biochemistry (Mosc.), 47, 12230–12240.

16. De Rocquigny,H., Shvadchak,V., Avilov,S., Dong,C.Z., Dietrich,U., Darlix,J.-L. and Mély,Y. (2008) Targeting the viral nucleocapsid protein in anti-HIV-1 therapy. Mini Rev. Med. Chem., 8, 24–35.

17. Goldschmidt,V., Miller Jenkins,L.M., de Rocquigny,H., Darlix,J.-L. and Mély,Y. (2010) The nucleocapsid protein of HIV-1 as a promising therapeutic target for antiviral drugs. Hiv Ther., 4, 179–198.

18. Mori,M., Manetti,F. and Botta,M. (2010) Predicting the Binding Mode of Known NCp7 Inhibitors To Facilitate the Design of Novel Modulators. J. Chem. Inf. Model., 10.1021/ci100393m.

19. Shvadchak,V., Sanglier,S., Rocle,S., Villa,P., Haiech,J., Hibert,M., Van Dorsselaer,A., Mély,Y. and de Rocquigny,H. (2009) Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein. Biochimie, 91, 916–923.

20. Pannecouque,C., Szafarowicz,B., Volkova,N., Bakulev,V., Dehaen,W., Mély,Y. and Daelemans,D. (2010) Inhibition of HIV-1 replication by a bis-thiadiazolbenzene-1,2-diamine that chelates zinc ions from retroviral nucleocapsid zinc fingers. Antimicrob. Agents Chemother., 54, 1461–1468.

21. Vercruysse,T., Basta,B., Dehaen,W., Humbert,N., Balzarini,J., Debaene,F., Sanglier-Cianférani,S., Pannecouque,C., Mély,Y. and Daelemans,D. (2012) A phenyl-thiadiazolylidene-amine derivative ejects zinc from retroviral nucleocapsid zinc fingers and inactivates HIV virions. Retrovirology, 9, 95.

22. Grigorov,B., Bocquin,A., Gabus,C., Avilov,S., Mély,Y., Agopian,A., Divita,G., Gottikh,M., Witvrouw,M. and Darlix,J.-L. (2011) Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex. Nucleic Acids Res., 39, 5586–5596.

23. Avilov,S.V., Boudier,C., Gottikh,M., Darlix,J.-L. and Mély,Y. (2012) Characterization of the inhibition mechanism of HIV-1 nucleocapsid protein chaperone activities by methylated oligoribonucleotides. Antimicrob. Agents Chemother., 56, 1010–1018.

24. Raja,C., Ferner,J., Dietrich,U., Avilov,S., Ficheux,D., Darlix,J.-L., de Rocquigny,H., Schwalbe,H. and Mély,Y. (2006) A tryptophan-rich hexapeptide inhibits nucleic acid destabilization chaperoned by the HIV-1 nucleocapsid protein. Biochemistry (Mosc.), 45, 9254–9265.

25. Dietz,J., Koch,J., Kaur,A., Raja,C., Stein,S., Grez,M., Pustowka,A., Mensch,S., Ferner,J., Möller,L., et al. (2008) Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi. ChemMedChem, 3, 749–755.