Biology subjects

Richard, C.-A.

Publications and source records attributed to Richard, C.-A..

5 recordsLinked to original sources

bioRxiv2025

In vitro liquid-liquid phase separation induced by respiratory syncytial virus proteins and RNA

Respiratory syncytial virus (RSV) causes severe respiratory infections, with viral replication occurring in cytoplasmic membraneless viral factories formed by liquid-liquid phase separation (LLPS). The interactions between the RSV nucleoprotein N, phosphoprotein P, transcription factor M2-1, and RNA drive these condensates. Here we employed a microfluidic Phase Scan platform, together with biochemical and cellular assays to systematically characterise LLPS involving RSV proteins and RNA. We identified optimal stoichiometric ratios of N oligomers and P tetramers for condensate formation, demonstrated that monomeric N inhibits LLPS, and revealed that M2-1 enhances condensate formation by increasing multivalency. Notably, we discovered that M2-1 preferentially binds 5 capped RNA, distinguishing it from N, which binds uncapped RNA. These findings elucidate molecular determinants of RSV viral factory assembly and subcompartmentalisation, providing insights into viral replication mechanisms and informing potential antiviral strategies targeting LLPS processes.

microbiology↗

bioRxiv2025

Can Plitidepsin Be Used as an Antiviral Against RSV?

Human respiratory syncytial virus (HRSV) is a main cause of acute lower respiratory tract infections in infants, the elderly and the immunocompromised patients. Although vaccines have recently been approved for the elderly and for pregnant women, there is no curative treatment for HRSV. HRSV replicates in the cytoplasm of infected cells, and transcription and replication of the viral genome depend on the viral RNA polymerase complex, which recruits cellular factors for RNA synthesis. Among them, the eucaryotic translation elongation factor 1A (eEF1A) was previously shown to be critical for HRSV replication. eEF1A activity can be inhibited by plitidepsin (Aplidin), a cyclopeptide extracted from ascidian Aplidium albicans, which was shown highly potent against SARS-CoV-2, with a 50% inhibitory concentration (IC90) of 0.70 to 1.62 nM depending on the cell line. Here, we investigated whether plitidepsin could also inhibit HRSV replication. We found that plitidepsin inhibited HRSV replication with an IC50 of {approx}3 nM in cell cultures. However, further investigation revealed that plitidepsin has pleiotropic effects, affecting the translation of both cellular and viral proteins in a similar manner. Overall, our results show that plitidepsin blocks cellular translation and indicate that plitidepsin induces a proteasome-mediated degradation of eEF1A, also showing the dependance of HRSV replication to cellular factors such as eEF1A. These results thus highlight an original mechanism of action of plitidepsin on eEF1A, which render the use of this compound for antiviral therapy very risky.

microbiology↗

bioRxiv2023

Specific targeting and clustering of Phosphatidylserine lipids by RSV M protein is critical for virus particle production

Human Respiratory Syncytial virus (RSV) is the leading cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. The elderly and the immunosuppressed are also affected. It is a major unmet target for vaccines and anti-viral drugs. RSV assembles and buds from the host cell plasma membrane by forming infections viral particles which are mostly filamentous. A key interaction during RSV assembly is the interaction of plasma membrane lipids with the Matrix (M) protein layer forming at assembly sites. Although the structure of RSV M protein dimer is known, it is unclear how the viral M proteins interact with certain plasma membrane lipids to promote viral assembly. Here, we demonstrate that M proteins cluster at the plasma membrane by selectively binding with phosphatidylserine (PS). Our in vitro studies suggest that M binds PS lipid as dimers and M mutant with a phosphomimetic substitution inhibits interaction with PS lipids. The presence of other negatively charged lipids like PI(4, 5)P2 does not affect RSV M ability to bind, while cholesterol negatively affects M interaction with lipids. Moreover, we show that the initial binding of the RSV M protein with lipids is independent of the cytoplasmic tail of fusion (F) glycoprotein (FCT). It is the first in vitro study of M interaction with plasma membrane lipids. M binding to plasma membrane may represent a viable therapeutic strategy for small molecules that will block viral spread.

biochemistry↗

bioRxiv2021

Characterization of the interaction domains between the phosphoprotein and the nucleocapsid of human Metapneumovirus

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein, forming an RNA-N complex (NNuc), which serves as template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the nucleocapsid template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between P and NNuc. However, the HMPV P-NNuc interaction still remains to characterize. Here, we finely characterized the binding domains involved in HMPV P and NNuc interaction by studying binding between recombinant proteins, combined with the use of a functional assay of the polymerase complex activity and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc, that P binds the N-terminal domain of N (NNTD), and identified conserved N residues critical for the interaction. Our results allowed to propose a structural model of the HMPV P-NNuc interaction. IMPORTANCELike respiratory syncytial virus (RSV), human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Nowadays, no vaccine or efficient antiviral treatments are available for these two pneumoviruses. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction, in particular a pocket on the surface of the N protein that could be used as a template for the design of compounds interfering with N-P complexes and viral replication.

microbiology↗

bioRxiv2020

Tetramerization of Phosphoprotein is essential for Respiratory Syncytial virus budding while its N terminal region mediates direct interactions with the Matrix protein.

It was shown previously that the Matrix (M), Phosphoprotein (P), and the Fusion (F) proteins of Respiratory syncytial virus (RSV) are sufficient to produce virus-like particles (VLPs) that resemble the RSV infection-induced virions. However, the exact mechanism and interactions among the three proteins are not known. This work examines the interaction between P and M during RSV assembly and budding. We show that M interacts with P in the absence of other viral proteins in cells using a Split Nano Luciferase assay. By using recombinant proteins, we demonstrate a direct interaction between M and P. By using Nuclear Magnetic Resonance (NMR) we identify three novel M interaction sites on P, namely site I in the N2 region, site II in the 115-125 region, and the oligomerization domain (OD). We show that the OD, and likely the tetrameric structural organization of P, is required for virus-like filament formation and VLP release. Although sites I and II are not required for VLP formation, they appear to modulate P levels in RSV VLPs. ImportanceHuman RSV is the commonest cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. The lack of knowledge of RSV budding mechanism presents a continuing challenge for VLP production for vaccine purpose. We show that direct interaction between P and M modulates RSV VLP budding. This further emphasizes P as a central regulator of RSV life cycle, as an essential actor for transcription and replication early during infection and as a mediator for assembly and budding in the later stages for virus production.

microbiology↗