Morphogenesis, organization and functions of liquid viral factories formed by Mononegavirales – LiquidFact

To carry out the 6 objectives of the project, alongside more traditional approaches in molecular and cellular biology, we will use various cutting-edge techniques.
1) Super-resolution microscopy and focused ion beam scanning electron microscopy will be used to characterize the sub-micrometric organization of Negri bodies (CN). The structure of CNs will also be studied by methods using synchrotron radiation sources (e.g. scanning transmission X-ray microscopy or µ-SAXS coupled with correlation imaging).
2) The dynamics of CN will be characterized by live cell imaging approaches using spinning disc confocal microscopy as well as fluorescence recovery after photobleaching (FRAP) approaches.
3) Using systems reconstituted in vitro and a combination of fluorescence and NMR techniques, we will identify the physicochemical principles inducing SPL and the formation of the viral plant.
3) To characterize the CN proteome, we will use proximity biotinylation followed by protein identification by mass spectrometry. We will also identify the resident RNAs of CNs by cross-linking, immunoprecipitation, and sequencing (NGS) approaches.
5) To identify the ribosomes active in the cell, we will use ribopuromycylation approaches and antibodies specifically binding puromycin.

Significant results after 18 months of the project:
1) Reconstitution of a minimal in vitro system in which P forms liquid structures with properties similar to CNs.
2) Identification of RABV N domains involved in liquid phase separation.
3) Identification of a cellular kinase involved in the signaling pathway leading to the induction of interferon which is sequestered by the P of RABV in the CNs, resulting in the inhibition of the pathway.
4) Demonstration that this kinase and a number of other players in this pathway form biocondensates.

All these results will be the subject of two amanuscripts corrently in preparation (submission in September 2021). The minimal in vitro system is now ready to be finely characterized by NMR (just started) and fluorescence approaches or with methods using synchrotron radiation sources.
Finally, during the coming period, we wish to develop proximity labeling approaches in order to determine the proteome of CNs but also that of the protein condensates formed by the proteins acting in the signaling pathway leading to the production of interferon.

Nevers Q, Albertini AA, Lagaudrière-Gesbert C, Gaudin Y. Negri bodies and other virus membrane-less replication compartments. Biochim Biophys Acta Mol Cell Res. 2020 Dec;1867(12):118831. doi: 10.1016/j.bbamcr.2020.118831.

Replication of many viruses occurs in specialized compartments formed during infection and known as viral factories. The physicochemical nature of these factories and the molecular basis of their morphogenesis and organization are poorly understood.
The Mononegavirales (MNV) order includes several important human pathogens (Rabies virus -RABV-, Measles Virus -MeV-, Ebola virus…). All these viruses have a single strand RNA genome of negative polarity which is encapsidated by the nucleoprotein (N) to form the ribonucleoprotein that is associated with the RNA dependent RNA polymerase and its cofactor the phosphoprotein (P).
RABV factories are the Negri bodies (NBs) which are cytoplasmic inclusions housing the synthesis of viral RNA (mRNAs and genomic RNAs). We have demonstrated that NBs constitute a new category of membrane-less liquid organelles. Liquid organelles are formed by liquid-liquid phase separation (LLPS) and contribute to the cell compartmentalization. They are involved in a wide range of cellular processes. So far, the general principles leading to LLPS are poorly understood.
Published experimental data indicate that the liquid nature of viral factories can be generalized to other MNVs. This is a paradigm shift which opens new research horizons in the field of MNV replication and invites us to revisit the interplay between viral factories and the components of the cellular innate immunity.
This proposal aims to characterize the morphogenesis, the internal organization, the composition, the dynamics and the functions of RABV and MeV viral factories. It brings together 3 teams: a team of virologists and biochemists specialized in rhabdoviruses, another which develops new methods for cell biology on synchrotron radiation sources and a third one developing state-of-the-art solution state NMR and fluorescence approaches to investigate the dynamics and interactions of highly flexible proteins with a strong focus on negative strand RNA viruses structure.
The project has six major objectives:
1) Using super-resolution microscopy and focused ion beam scanning electron microscopy, we will characterize the submicrometer organization of NBs and how it evolves all along the viral cycle. NBs ultrastructure will also be investigated by various methods developed on synchrotron radiation sources (e.g. scanning transmission X-ray microscopy or µ-SAXS coupled to correlative imaging). We will also determine viral proteins structural elements which are required for NBs formation.
2) Using in vitro reconstituted systems and a combination of fluorescence techniques and NMR, we will identify the physicochemical principles underlying the LLPS leading to the formation of the viral factory.
3) As LLPS enriches NBs in specific factors, we will characterize NBs’ proteome using a proximity biotinylation assay and identification of proteins by mass spectrometry. We will also identify RNAs which are NBs’ residents.
4) We will characterize the interplay between NBs and cellular innate immunity. Indeed, the sequestration of viral RNAs in NBs raises the question of their accessibility to pathogen recognition receptors such as RIG-I and MDA5. Alternatively, liquid factories might constitute a signature of viral infection and cells might have evolved a mechanism allowing the sensing of such structures and/or their destabilization.
5) Experimental data suggest that RABV factories might also harbor viral proteins translation. We will investigate where viral mRNAs are translated in infected cells and if they use an unconventional mechanism of translation initiation to escape the translation inhibition induced by innate immunity.
5) At some stage, the RNPs must leave the viral factory to form new virions. We will investigate the molecular bases of the processes by which this happens.
Beyond its impact in virology and innate immunity, this project should have an impact in cellular biology by increasing our understanding of liquid organelles assembly.