Protéines NCp7 et Gag de VIH-1 : mécanismes et inhibition Y. Mély

The nucleocapsid protein NCp7 plays key roles in the condensation and protection of the genomic RNA (gRNA) and the viral DNA. NCp7 also chaperones the reverse transcription and integration steps during HIV-1 replication (for a review see 1,2). To further understand the properties and functions of NCp7, we have explored several research directions.

1.1. Role of NCp7 in viral particles

Using the recently developed 2-thienyl-3-hydroxychromone (3HCnt) and thienoguanosine (thG) nucleoside surrogates, we investigated in depth the NCp7-promoted (+)/(-)PBS annealing of the second strand transfer reaction in reverse transcription (3). These fluorescent nucleoside surrogates were introduced in the (-)PBS loop. This approach allowed us to recover the full set of kinetic rate constants governing the (+)/(-)PBS annealing both in the absence and the presence of wild-type and mutant NCp7 proteins. Through a specific binding, NCp7 exposes the PBS nucleotides to the solvent, allowing the formation of a loop–loop kissing complex. NCp7 also destabilizes the stem, which facilitates the conversion of the loop–loop intermediate into the final duplex.
In collaboration with F. Westerlund (Gothenburg), nanofluidic channels were used to investigate at the single molecule level the delicate balance between DNA condensation and chaperone activity of NCp7 (4). The first ten amino acids were found to be important for both activities. Moreover, we also investigated the initial step of compaction of single dsDNA molecules (Jiang et al, submitted). NCp7 was found to initiate compaction from the ends of long dsDNA, but compaction can also occur in the interior of the dsDNA, preferentially at its most flexible AT-rich regions.

1.2 Cytoplasmic Release of NCp7 from viral particles 

To visualize viruses in the host cell during infection, we developed a specific labeling protocol for correlative light-electron microscopy (CLEM). This aim was quite challenging because the labelling approaches developed for fluorescence microscopy are usually not suited for transmission electron microscopy (TEM). To overcome this bottleneck, we adapted a labeling approach with a tetracystein tag (TC) and a biarsenical resorufin-based label (ReAsH) for monitoring the HIV-1 particles during the early steps of HIV-1 infection by CLEM (5). In this approach, the ReAsH fluorophore triggers the photo-conversion of 3,3-diaminobenzidine tetrahydrochloride (DAB), generating a precipitate sensitive to osmium tetroxide staining that can be visualized by TEM. HeLa cells, infected by ReAsH-labeled pseudoviruses containing NCp7-TC proteins exhibit strong fluorescent cytoplasmic spots that overlap with dark precipitates in the TEM sections. Using this approach, single viral cores were observed all over the cytoplasm, and notably near microtubules and nuclear pores.
Using the same HIV-1 pseudoviruses containing NCp7-TC proteins labeled by FlAsH, we quantitatively monitored the NCp7 concentration in the viral cores during the early stages of infection 6. This approach was based on the strong quenching of FLAsH resulting from the high packaging of NCp7-TC in the viral cores. A strong fluorescence increase of individual FlAsH-labeled pseudoviruses containing NCp7-TC proteins was observed in infected cells at 8 and 16 h post-infection. This increase was abolished by inhibition of reverse transcription indicating that the release of NCp7 molecules from the viral complexes is likely connected to viral DNA synthesis. NCp7-TC release was more pronounced in the perinuclear space, where capsid disassembly can occur.  

1.3. Fate of NCp7 released in the cytoplasm

To investigate the fate of the NCp7 proteins released in the cytoplasm of infected cells, we used HeLa cells transfected with eGFP-labeled NCp7 as a model 7. To monitor its intracellular distribution and dynamics of NCp7-eGFP,  we used a series of quantitative fluorescence fluctuation based microscopy techniques (fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectroscopy, and raster imaging correlation spectroscopy). NCp7-eGFP was found to localize mainly in the cytoplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Thus, NCp7 molecules released in newly infected cells are thought to primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection.

During HIV-1 assembly, the Gag NC domain is required for the specific selection, encapsidation and packaging of the genomic HIV-1 RNA (gRNA) via the binding to the y domain that comprises four closely spaced stem loops (SL1-4) located within the 5’ untranslated region of the gRNA. This binding promotes Gag oligomerization and targeting to the plasma membrane. In this context, our main goal is by using advanced imaging technologies (FRET-FLIM, superresolution microscopy, FCS), to further characterize the roles of the Gag NC domain in the assembly process of HIV-1. 

2.1.   Role of NC domains on the interaction of Gag with genomic RNA

In collaboration with S. Bernacchi and R. Marquet (IBMC, Strasbourg), our aim was to further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the plasma membrane (PM) 8. To this end, we compare in live and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs). Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol.

2.2.    Role of the NC domain of Gag in the interaction with host proteins.

Gag proteins have been reported to interact with numerous host proteins. We have recently reviewed these interactions, highlighting those that are dependent on the NC domain of Gag 9. This review clearly shows that the NC domain plays a key role in the late steps of HIV-1 replication, not only by selecting and binding the gRNA, but also through its interaction with multiple cell proteins.
Among the possible interactants of Gag, we have been interested in RPL7, a major ribosomal protein, 10. We found that Gag/RPL7 interaction is mediated by the NC zinc fingers of Gag and the N- and C-termini of RPL7, respectively, but seems independent of RNA binding, Gag oligomerization and Gag interaction with the plasma membrane. RPL7 was also demonstrated to exhibit a stronger DNA/RNA chaperone activity than Gag. Finally, Gag and RPL7 were shown to function in concert to drive rapid nucleic acid hybridization.
To further understand how Gag and RPL7 act together, we investigated the mechanism of their nucleic acid chaperone activity individually and in concert 11. We used as a model system, the annealing between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence. Gag alone or complexed with RPL7 was found to act as a NA chaperone that destabilizes cTAR stem-loop and promotes its annealing with dTAR through the stem ends via a two-step pathway. In contrast, RPL7 alone acts as a NA annealer that promotes cTAR/dTAR annealing via two parallel pathways. In contrast to the isolated proteins, their complex promoted efficiently the annealing of cTAR with highly stable dTAR mutants. This was confirmed by the RPL7-promoted boost of the physiologically relevant Gag-chaperoned annealing of (+)PBS RNA to the highly stable tRNALys3 primer, favoring the notion that Gag recruits RPL7 to overcome major roadblocks in viral assembly.
The NA chaperone activity of Gag as well as its NA condensation activity in comparison with NCp7 were further examined using nanofluidic channels 4. In collaboration with F. Westerlund, we found that the condensation activity of Gag was more efficient than for NCp7 alone, while in contrast, the NA chaperone activity of Gag was far lower than the NCp7 one. Interestingly, the p6 region of Gag was found to partially inhibit the chaperone activity of Gag but not its condensation activity.
Finally, we investigated the role of the NC domain in the interaction of Gag with the ESCRT TSG101 protein involved in HIV-1 budding 12. Our data clearly highlighted that not only the p6 domain, but also the NC domain of Gag is crucial for the recruitment of TSG101 by Gag at the plasma membrane.

Due to its conservation in HIV-1 viral strains and its key roles in HIV-1 viral cycle, NCp7 appears as a particularly interesting target for developing new antiviral therapies which would be expected to induce limited drug resistance. This was the main objective of the European project Thinpad [2013-16, in collaboration with the teams of M. Botta (Sienna), J.M. Gattell (Barcelona) and two companies (IRBM and Virostatics)] and the COST action CM 1407 « Natchemdrugs » (2015-19). In these projects, we have explored a large range of chemical families and have found a number of NC inhibitors with potential antiviral activity. More than 20 papers have been published on this topic. Of particular interest are the (thia)calixarenephosphonic acids that were found to inhibit the NA chaperone activity of NCp7 with a new binding mode and to exhibit multitarget antiviral activity 13. 5,6-Dihydroxypyrimidine scaffolds 14, aminopyrrolic scaffolds 15and bifunctional aminothiazoles 16 were also identified as active NC inhibitors targeting the hydrophobic plateau at the top of the folded fingers. We also designed a library of peptides (10-17 amino acids) including most of the NCp7 structural determinants responsible for its specific NA binding and destabilizing activities 17. The most active peptide pE was found to inhibit the NCp7 destabilizing activity, with an IC50 in the nanomolar range, by competing with NCp7 for binding to its NA substrates. Formulated with a cell-penetrating peptide, pE was found to accumulate into HeLa cells, with low cytotoxicity. However, pE did not show any antiviral activity, probably due to its sequestration by cellular RNAs. Noteworthy, we also designed a one-step and one-pot FRET-based assay to screen inhibitors of HIV-1 reverse transcriptase and NCp7 18. Finally, we identified in collaboration with R. Hartmann (U. Saarbrucken), 2-ureidothiophene-3-carboxylic acids as dual bacterial RNA polymerase and viral reverse transcriptase inhibitors 19.

1.  Darlix, J. L.; de Rocquigny, H.; Mely, Y., The multiple roles of the nucleocapsid in retroviral RNA conversion into proviral DNA by reverse transcriptase. Biochemical Society transactions 2016, 44 (5), 1427-1440.

2.  Nadeem. MF; Mely. Y, Nucleic acid chaperone properties of the HIV-1 NC protein: Dependence on the protein structure and mechanisctic aspects. In The role of the mini Zinc finger protein in the replication of the AIDS virus, Cambridge Scholars Publishing: 2021, in press.

3.  Sholokh, M.; Sharma, R.; Grytsyk, N.; Zaghzi, L.; Postupalenko, V. Y.; Dziuba, D.; Barthes, N. P. F.; Michel, B. Y.; Boudier, C.; Zaporozhets, O. A.; Tor, Y.; Burger, A.; Mely, Y., Environmentally Sensitive Fluorescent Nucleoside Analogues for Surveying Dynamic Interconversions of Nucleic Acid Structures. Chemistry 2018, 24(52), 13850-13861.

4.  Jiang, K.; Humbert, N.; Kk, S.; Lequeu, T.; Lin, Y. L.; Mely, Y.; Westerlund, F., Annealing of ssDNA and compaction of dsDNA by the HIV-1 nucleocapsid and Gag proteins visualized using nanofluidic channels. Quarterly reviews of biophysics 2019, 52, e2.

5.  Lysova, I.; Spiegelhalter, C.; Real, E.; Zgheib, S.; Anton, H.; Mely, Y., ReAsH/tetracystein-based correlative light-electron microscopy for HIV-1 imaging during the early stages of infection. Methods and applications in fluorescence 2018, 6 (4), 045001.

6.  Zgheib, S.; Lysova, I.; Real, E.; Dukhno, O.; Vauchelles, R.; Pires, M.; Anton, H.; Mely, Y., Quantitative monitoring of the cytoplasmic release of NCp7 proteins from individual HIV-1 viral cores during the early steps of infection. Scientific reports 2019, 9 (1), 945.

7.  Anton, H.; Taha, N.; Boutant, E.; Richert, L.; Khatter, H.; Klaholz, B.; Ronde, P.; Real, E.; de Rocquigny, H.; Mely, Y., Investigating the cellular distribution and interactions of HIV-1 nucleocapsid protein by quantitative fluorescence microscopy. PloS one 2015, 10 (2), e0116921.

8.  Boutant, E.; Bonzi, J.; Anton, H.; Nasim, M. B.; Cathagne, R.; Real, E.; Dujardin, D.; Carl, P.; Didier, P.; Paillart, J. C.; Marquet, R.; Mely, Y.; de Rocquigny, H.; Bernacchi, S., Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane. Biophysical journal 2020, 119 (2), 419-433.

9.  Klingler, J.; Anton, H.; Real, E.; Zeiger, M.; Moog, C.; Mely, Y.; Boutant, E., How HIV-1 Gag Manipulates Its Host Cell Proteins: A Focus on Interactors of the Nucleocapsid Domain. Viruses 2020, 12 (8).

10.       Mekdad, H. E.; Boutant, E.; Karnib, H.; Biedma, M. E.; Sharma, K. K.; Malytska, I.; Laumond, G.; Roy, M.; Real, E.; Paillart, J. C.; Moog, C.; Darlix, J. L.; Mely, Y.; de Rocquigny, H., Characterization of the interaction between the HIV-1 Gag structural polyprotein and the cellular ribosomal protein L7 and its implication in viral nucleic acid remodeling. Retrovirology 2016, 13 (1), 54.

11.       Karnib, H.; Nadeem, M. F.; Humbert, N.; Sharma, K. K.; Grytsyk, N.; Tisne, C.; Boutant, E.; Lequeu, T.; Real, E.; Boudier, C.; de Rocquigny, H.; Mely, Y., The nucleic acid chaperone activity of the HIV-1 Gag polyprotein is boosted by its cellular partner RPL7: a kinetic study. Nucleic acids research 2020, 48 (16), 9218-9234.

12.       El Meshri, S. E.; Boutant, E.; Mouhand, A.; Thomas, A.; Larue, V.; Richert, L.; Vivet-Boudou, V.; Mely, Y.; Tisne, C.; Muriaux, D.; de Rocquigny, H., The NC domain of HIV-1 Gag contributes to the interaction of Gag with TSG101. Biochimica et biophysica acta. General subjects 2018, 1862 (6), 1421-1431.

13.       Humbert, N.; Kovalenko, L.; Saladini, F.; Giannini, A.; Pires, M.; Botzanowski, T.; Cherenok, S.; Boudier, C.; Sharma, K. K.; Real, E.; Zaporozhets, O. A.; Cianferani, S.; Seguin-Devaux, C.; Poggialini, F.; Botta, M.; Zazzi, M.; Kalchenko, V. I.; Mori, M.; Mely, Y., (Thia)calixarenephosphonic Acids as Potent Inhibitors of the Nucleic Acid Chaperone Activity of the HIV-1 Nucleocapsid Protein with a New Binding Mode and Multitarget Antiviral Activity. ACS infectious diseases 2020, 6 (4), 687-702.

14.       Malancona, S.; Mori, M.; Fezzardi, P.; Santoriello, M.; Basta, A.; Nibbio, M.; Kovalenko, L.; Speziale, R.; Battista, M. R.; Cellucci, A.; Gennari, N.; Monteagudo, E.; Di Marco, A.; Giannini, A.; Sharma, R.; Pires, M.; Real, E.; Zazzi, M.; Dasso Lang, M. C.; De Forni, D.; Saladini, F.; Mely, Y.; Summa, V.; Harper, S.; Botta, M., 5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid Protein. ACS medicinal chemistry letters 2020,11 (5), 766-772.

15.       Ciaco, S.; Humbert, N.; Real, E.; Boudier, C.; Francesconi, O.; Roelens, S.; Nativi, C.; Seguin-Devaux, C.; Mori, M.; Mely, Y., A Class of Potent Inhibitors of the HIV-1 Nucleocapsid Protein Based on Aminopyrrolic Scaffolds. ACS medicinal chemistry letters 2020, 11 (5), 698-705.

16.       Mori, M.; Dasso Lang, M. C.; Saladini, F.; Palombi, N.; Kovalenko, L.; De Forni, D.; Poddesu, B.; Friggeri, L.; Giannini, A.; Malancona, S.; Summa, V.; Zazzi, M.; Mely, Y.; Botta, M., Synthesis and Evaluation of Bifunctional Aminothiazoles as Antiretrovirals Targeting the HIV-1 Nucleocapsid Protein. ACS medicinal chemistry letters 2019, 10 (4), 463-468.

17.       Shvadchak, V.; Zgheib, S.; Basta, B.; Humbert, N.; Langedijk, J.; Morris, M. C.; Ciaco, S.; Maskri, O.; Darlix, J. L.; Mauffret, O.; Fosse, P.; Real, E.; Mely, Y., Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein. Biochemistry 2018, 57 (30), 4562-4573.

18.       Sharma, B. M.; Melymuk, L.; Bharat, G. K.; Pribylova, P.; Sanka, O.; Klanova, J.; Nizzetto, L., Spatial gradients of polycyclic aromatic hydrocarbons (PAHs) in air, atmospheric deposition, and surface water of the Ganges River basin. The Science of the total environment 2018, 627, 1495-1504.

19.       Elgaher, W. A.; Sharma, K. K.; Haupenthal, J.; Saladini, F.; Pires, M.; Real, E.; Mely, Y.; Hartmann, R. W., Discovery and Structure-Based Optimization of 2-Ureidothiophene-3-carboxylic Acids as Dual Bacterial RNA Polymerase and Viral Reverse Transcriptase Inhibitors. Journal of medicinal chemistry 2016, 59 (15), 7212-22.