1) Structure/function analysis of the Pneumoviruses polymerase complex Polymerization of N along the genomic and anti-genomic RNAs is concomitant to replication and requires the supply of neosynthesized N protein. This N protein is maintained monomeric and RNA-free through the interaction with the viral phosphoprotein P that plays the role of a chaperone, forming a soluble N0-P complex. The interaction between P and N in this complex differs from the interaction between N-RNA and P. The team has recognized expertise in the structural and functional characterization of N, P, and their interactions. Our data allowed to reveal the strong structural homology between RSV and MPV N0-P complex (Esneau et al., 2019, JBC), and to investigate the role of RNA in the specificity of RNA encapsidation by RSV N (Gonnin L., 2022, JBC). In parallel, in collaboration with I. Gutsche (IBS, CNRS), we managed to isolate RSV NC purified from insect cells and to obtain the first structure of high resolution of these NC (Gonnin, L., 2023, Nat.Comm). In line with these results, we are now focusing on the functional and structural characterization of RSV NC in cells and in vitro in the presence of P and L proteins.
Furthermore, we have characterized the interaction between HMPV N-RNA and P protein (Decool H, 2022, J.Virol), and obtained the first structure of this interaction by cryo-EM (collaboration with M. Renner (Sweden) (Whitehead, JD, 2023, Nat comm)).
Of most interest, we have also collaborated with the group of S. McLellan (Austin Universtiy, Texas) and contributed to the first cryo-EM structure resolution of the RSV L-P complex (Gilman MSA, 2019, Science). Although the structure of the C-terminal part of L still remains to be determined, these structural data open the way to the rational design of inhibitors of the L polymerase, but also allowed to reveal the complexity of interaction between P and L. The RSV P protein plays a central role within the polymerase complex by interacting with L, N, and M2-1, and cellular partners such as the phosphatase PPI. One main objective of our team is now to gain information on the dynamics of all these interactions, and their role for replication, transcription and assembly.
2) Structure and dynamics of Pneumoviruses viral factories Replication and transcription of most of the MNV take place within cytoplasmic viral factories (VF), which are viro-induced membrane-less organelles formed by liquid-liquid phase separation (LLPS). In 2020, we demonstrated the liquid nature of RSV VFs and managed to reconstitute pseudo-VF in vitro (Galloux M, 2020, mBio). Based on our results, we have determined the minimal elements required for LLPS i.e the tetrameric C-terminal part of P and the oligomeric form of N complexed to RNA (Gonnin L, 2022, JBC). We have recently upgraded the reconstitution of pseudo-VFs by adding the M2-1 protein, and showed that M2-1 impacts the dynamics of P (Diot C, 2023, IJMS). Of most interest, this study allowed to demonstrate that cyclopamine, a natural compound which was shown to harden RSV VFs and presents antiviral activity in vivo in a mouse model (Risso-Ballester, 2021, Nature), modifies M2-1/P interaction thus freezing the dynamics of RSV LLPS.
We are currently studying the role of the post-translational modifications of N protein in the functioning and dynamics of VFs. Based on our capacity to purify the different viral proteins, our main challenging goal now is to reconstitute in vitro functional pseudo-VFs, by adding the viral polymerase L.
3) Mechanism of virions assembly Pneumoviruses are suggested to share an overall common strategy with other MNV for its assembly and budding. The M proteins organize viral assembly by bridging between the RNPs localized in VFs and the viral glycoproteins expressed in the endoplasmic-Golgi and addressed to the plasma membrane. However, these steps remain poorly characterized. For many closely related Paramyxoviruses, specific M-N interaction is required for the RNP to be packed into viral pseudo-particles. However, for RSV, co-expression of the viral P, M, and F proteins is sufficient to induce virus-like particles (VLP) formation. One objective of our team is to characterize protein-protein interactions involving M, P and F, which has the potential to break new ground in the field of virus budding mechanisms and may provide new targets for antivirals. We have demonstrated and characterized a direct interaction between M and P proteins (Bajorek M, 2021, J. Virol). Furthermore, we have shown that the nature of lipids is critical for M-lipid interaction and specific lipid clustering at budding sites (Swain J, 2023, JBC). Based on these results, we are now focusing on how the M protein functions as the RSV assembly hub via interaction with lipids and recruiting other viral components. Specifically, we investigate the role of M phosphorylation, the effect of P on M-PS lipid binding and clustering in vitro on biomimetic membranes.
4) Identification and characterization of RSV cellular partners Interactions between Pneumoviruses’ viral proteins and their cellular partners remain poorly characterized and play key roles in RNA polymerase functioning, RNPs traffic, or in the control of the host immune response. In the context of collaborations, the team has identified cellular partners of RSV N, P, M and NS1 proteins. Different projects of the team now focus on the role of these cellular proteins for RSV replication and host immune response. Most importantly, our data allowed to reveal two new interactions involved in the control of host immune response during RSV infection:
1. We have shown that N interacts with the protein TAX1BP1, and our results suggest that N-TAX1BP1 interaction could alter the production of type I interferon and of cytokines by alveolar macrophages during RSV infection (Descamps D, 2021, J.Virol). Our aim is to further characterise the role of TAX1BP1 and of its interaction with RSV N protein.
2. The severity of RSV infection depends on the host's immune response, particularly the production of type-I interferon. RSV elicits a weak innate immune response, partially mediated by the non-structural NS1 protein. NS1 is found in the cytosol and nucleus, where it appears to interfere with host gene transcription, but the molecular mechanisms remain unclear. We and others found that NS1 interacts with the MED25 subunit of the Mediator complex, a transcriptional coactivator of the RNA polymerase II machinery that regulates several host gene expressions, including antiviral genes (Dong J, 2022, JMB). This suggests that NS1 could modulate host transcription by interacting with the Mediator. NS1 consists of a globular core domain and a C-terminal helix α3. The latter modulates host gene expression. We showed that the NS1 α3 and α/β core domains act cooperatively to achieve a strong interaction with MED25-ACID in the nanomolar range. Our results suggest that this interaction is correlated with antiviral response antagonism, probably by blocking both TAD-binding faces of MED25-ACID.
3. In parallel, we are investigating the role co-chaperones of HSP70 in the viral cycle. During the respiratory syncytial virus (RSV) cycle, genome encapsidation and virions assembly are two critical steps which depend on fine regulation of the oligomerization of the viral nucleoprotein (N) and matrix (M) proteins. Both N and M protein are known to spontaneously oligomerize, suggesting a potential involvement of cellular chaperones in maintaining their solubility and proper oligomerization status. Hsp70 and Hsp90 are essential chaperones that play a key role in ensuring proper protein folding in the cell and have been shown to be involved in RSV replication. Interestingly, both Hsp70 and Hsp90 depend on upstream proteins for recognizing the misfolded or damaged client proteins and delivering them to these chaperones. These upstream proteins belong to the highly diverse J-domain protein (JDP/Hsp40) family, comprising 50 distinct chaperones with specialized client binding properties. Given the critical role of JDPs in determining the specificity of the Hsp70 and Hsp90, we hypothesize that there is a specific subset of JDPs that selectivity recognize the N and M proteins, facilitating their proper folding and assembly required for genome encapsidation and virion formation. Our recent study identified a part of the RSV-chaperones network. We showed that specific JDP proteins interact with RSV N and M proteins, affecting their oligomerization and proper function. Targeting these specific chaperones used by RSV could represents a new antiviral approach.
5) Identification of new targets and development of tools for the development of antiviral strategies For 15 years, the team has developed tools and assays dedicated to both the fundamental study of Pneumoviruses’ replication and to the screening of the antiviral activity of compounds. Most importantly, we have generated recombinant fluorescent and luminescent viruses which were used in the context of various collaborations with private and academic partners to screen/validate antivirals activity against human RSV in cells and in vivo in a mouse model respectively. During the last 5 years, the team has also generated bovine RSV (Fix J, 2023, Vet Research) and HMPV recombinant viruses.
Based on the structures of viral proteins and of their domains of interaction, the team has developed collaborations in order to either identify or relationally design antivirals targeting different viral proteins interactions: screening of protein-protein interaction libraries, use of peptido-mimetics, optimized stapled peptides (Galloux M, 2020, AAC), and in silico design of compounds.
Our systems also allowed to participate to studies which consist in the characterization of the mechanism of action of new antiviral molecules such as the cyclopamine (Risso-Ballester J, 2021, Nature; DI-RV-21-0070; Diot C., 2023, IJMS), or the molecule Retro-2.2 (Le Rouzic A, 2024, IJMS).
In parallel, the team has also participated to the development of new and original vaccine approaches including the use of AI. First, in collaboration with Sabine Riffault (V2I team), we have recently used nanorings formed by N-RNA purified from E.coli (patents WO/2006/117456 ; WO/2007/119011) as carrier for in silico designed epitopes for human RSV F protein (Sesterhenn F, 2019, PLoS Biol.; Sesterhenn F, 2020, Science). A similar approach is now led with in silico designed synthetic epitope for the bovine F protein in the context of the Neovacc project. Second, in collaboration with M. Rosa-Calatrava (CIRI, Lyon), we have tested the efficiency of recombinant HMPV virus expressing both the RSV and HMPV F proteins (Chupin C, 2021, Vaccines, and DI-RV-22-0035).
