COVID-19 has emerged in December 2019 in Wuhan, China, caused by a novel RNA virus: SARS-CoV-2. As of March 22 2020, ~300,000 cases of COVID-19 and nearly 12,000 deaths (fatality rate ~4%) have been reported in the world. The high proportion of cases requiring respiratory assistance in intensive care units (~10%) catastrophically overwhelms health systems worldwide. The COVID-19 pandemic is a global health emergency and represents a never seen before challenge for the medical community but also for the scientific community which is appealed to provide prophylactics and treatment for this dreadful pathogen. Tremendous efforts are put together to counteract SARS-CoV-2 entry mechanisms and identify suitable targets for vaccine development. While generic treatments used for RNA virus infections (HIV, HCV, EBOV, ZIKV, HeV and NiV) and hydroxychloroquine are currently being investigated, no vaccine or effective treatment for SARS-CoV-2 is available or proven efficient as of today. We propose here an original and alternative contribution to the scientific effort aiming at combatting SARS-CoV-2/host-cell innate immune escape. Identifying the virus-host protein-protein interactions (PPIs) critical for infection will reveal mechanisms involved in pathogenesis and define promising drug targets. Through a unique combination of orthogonal state-of-the-art interactomic approaches conducted by highly qualified teams, N2H and BioID, we will timely profile the accurate interplay between SARS-CoV-2 proteins and human factors involved in anti-viral immune and consecutive inflammatory responses. As such, this project will quickly provide a wide network of potential host targets for innate immunity targeting treatment option. In a conceptually new manner, we will implement cutting-edge network modeling approaches aiming at delineating druggable disease modules. Of note, for potential transfer towards clinical applications, we will focus our testing strategies on repurposing approved drugs. Our approach could thus be exploited for developing rational and combined therapies targeting multiple elements within a disease module, at suboptimal doses, to counteract this deadly infectious process. Thus, our proposal is aligned with the topic “search for therapeutic targets and drug candidates”, especially turned here to restore the beneficial primary immune response and counter deleterious secondary inflammatory response to SARS-CoV-2 infection. Our works are of utmost importance: (i) due to the broad distribution of COVID-19 cases over the globe and its high degree of contagiousness, the virology community expects a seasonal recurrence of such infection. It is therefore important to tackle this major issue with all means and angles available; (ii) we hope a cure for SARS-CoV-2 infection will be discovered along the course of this project. However, in case of non-satisfactory response found within the next few months, or failure to treat all the patients because of the variety of the cases, our approach will bring essential rationale to the medical and scientific communities for immediate finding of efficient cure; and (iii), assembling such workforce enabling rapid characterization of emerging infectious agent, we are deeply convinced that our works will serve as a framework easily implementable to dramatically and timely expand basic knowledge on emerging infectious mechanisms. Hence, our proposal is of major importance to bring novel insights and rationales for future therapeutic approaches. Importantly, our project will bring an unprecedented knowledge on how the SARS-CoV-2 proteins hijack the innate immune response, and because of its semi-supervised nature, will greatly expand our knowledge of SARS-CoV-2 associated mechanisms beyond innate-immunity.

In recent years, MALDI Mass Spectrometry Imaging (MSI) has become a tool of choice to collect molecular information on tissues, in a spatially-resolved manner. MALDI-MSI can lead to the detection and localization of hundreds of biomolecules from a histological tissue section, in a label free manner and in a single experiment, avoiding tissue homogenization. However, despite all improvements, these approaches still show limitations for lower abundance proteins and the identification of Post Translational Modifications (PTMs). Moreover, Protein-Protein Interactions (PPIs), which are keys in biological signaling cannot be characterized. Regular bottom-up proteomics, guided by MALDI-MSI, have been used on tissues to bridge the spatial dimension of imaging and the strength in identification of large-scale proteomic approaches. However, even if these approaches have led to interesting results, they do not encompass the large-scale analysis of PTMs and PPIs. In the STRUCTURAL project, we therefore propose to develop specific structural proteomics approaches that will enable this important information to be obtained from tissues, where proteins are in their native microenvironment. PPIs will be addressed by developing appropriate cross-linking MS (XL-MS) strategy. For the characterization of protein PTMs, proteoforms will be characterized using dedicated Top-Down Proteomics (TDP) pipeline. These approaches will be optimized on rat brain, and further extended to the analysis of spinal cord injury (SCI). SCI is a devastating traumatic injury leading to severe sensory and motor deficits. The project relies on the complementary of two partners: PRISM Inserm U1192 lab which has lead expertise in MSI and Spatially-Resolved proteomics applied to clinical problematics including SCI and MSBio (Institut Pasteur Paris) which has strong expertise in structural proteomics and has already developed optimized pipelines in XL-MS and TDP. To achieve this goal, STRUCTRUAL is organized into 4 different tasks. The first one aims at translating the conventional XL-MS workflow to the tissue context for limited areas of the sections. The main challenge here will be to increase the sensitivity of the XL-MS approach to a level compatible with the quantity of protein present in tissue section of biological interest (less than 1 mm², 1-5 µg of protein) while increasing spatial resolution. The eXL-MS workflow already developed by the MSBio Unit, which is based on an original trifunctional cross-linkers for efficient purification of cross-linked peptides by click-chemistry, will serve as basis for further optimization. In the second task, we will develop an optimized TDP approach allowing the identification of proteoforms from very localized area of tissue sections (again, less than 1 mm2). This optimization will cover both the sample preparation and the LC-MS/MS analysis of the intact proteins. The data obtained from the XL-MS and TDP (using high resolution Orbitrap mass spectrometers) will be merged to build maps of protein networks at the proteoform level in rat brains. Finally, in Task 3, the workflows developed within task 1 & 2 will be applied to identify PPIs and detect differentially expressed proteoforms in the SCI to demonstrate the validity of the method. Spatiotemporal studies will be performed directly from thin rat SCI tissue sections sampled at the lesion site, rostral and caudal regions of the spine and different time points (1h, 12h, 1 day, 7 days and 10 days post-lesion) and the generated data will be used to improve the knowledge on the regeneration mechanisms. Task 4 will be dedicated to management. In a nutshell, the methodology developed will make it possible to go beyond the current limitations by bringing a completely novel dimension in Spatially-Resolved proteomics. This will provide better insights into physio or physio-pathological processes for a wide range of biological and clinical applications.

Borna disease virus (BoDV), a single-stranded RNA virus of negative polarity, has puzzled researchers for decades, because of its neurotropism and non-cytolytic multiplication strategy. Its recent recognition as a zoonotic agent causing severe encephalitis and brain dysfunction has provided further impetus for increasing our knowledge on this enigmatic pathogen. To date, very little is known regarding the BoDV replication modalities. It is the only animal Mononegavirales to replicate in the nucleus, where it assembles viral factories that, strikingly, are physically bound to the neuronal chromatin. The only available structural information is the X-ray structure of the RNA-free nucleoprotein and nothing is known regarding its recognition of the genomic RNA or the organization of its nucleocapsid. Intriguingly, sequence analysis predicts that the BoDV polymerase is radically different from that of other Mononegavirales, being 20 % shorter and not containing the canonical RNA-capping motifs. In particular, the methyltransferase domain seems absent, strongly suggesting the recruitment of specialized host factors at viral factories -such as the capping machinery- to complement this activity which is essential for the maturation of viral mRNAs. Finally, the functional consequences of the binding of viral factories onto the chromatin, notably its impact of neuronal epigenetics and neuronal communication, are also completely unexplored. In this context, the Bavarian project aims at providing an integrative and multiscale vision of the BoDV replication complex. Our multi-pronged proposal will describe the structural organization and explore the BoDV replication complex modus operandi within infected cells. To tackle these questions, our three teams will combine their expertise, implementing cutting-edge structural biology, biochemistry, virology and neuronal functional assays. Our complementary approaches will allow a fine characterization of: (i) the architecture of the BoDV replicative machinery, (ii) its viral-host polypeptides interplays and (iii) the impact of altering the viral replication on viral fitness, neuronal epigenetics and function. Our project will provide an unmatched deciphering of BoDV pathogenic mechanisms and, in a broader view, will represent a breakthrough in the field of RNA virus family evolutionary mechanisms. Indeed, Bavarian will focus on the only non-retroviral RNA virus known so far that needs to interact with the host DNA genome to persist and proliferate. To achieve its goals, our project groups three teams with complementary expertise. The team of the coordinator (IBS, Grenoble) is composed of biochemists and structural virologists, with recognized experience in viral replication. Partner-2 (Infinity, Toulouse) has a long-standing expertise in the analysis of the mechanisms and consequences of viral persistence, in particular BoDV, in the central nervous system. Partner-3 (Prism, Lille) has a strong background in proximal interactomics (notably BioID) and systems biology, illustrated by his recent description of whole proteome proximal interactomes of emerging viruses such as Zika and SARS-CoV-2. These recent months, the three teams have established a very efficient collaboration, which has led to a very convincing set of preliminary data that strongly ensures the chances of success of the proposed program. The recent SARS-CoV-2 pandemic is a vivid reminder that no pathogen should be under-estimated. Indeed, deciphering the interaction of viruses with target cells is essential to gather clues for therapeutic intervention. Notably, increasing our knowledge on the organization of viral replication complexes is instrumental, because it often allows to identify strategies to block not only one, but often several viruses. Obtaining original data on the unexplored BoDV system could therefore open the road for finding new antivirals and/or therapeutic strategies, which may be applicable to many pathogens.

Esophageal Atresia (EA) is a rare developmental defect of the foregut that presents with or without a Tracheo-Esophageal Fistula (TEF). The prevalence of EA/TEF over time and around the world has been relatively stable (> 165 new EA/year on average in France). EA/TEF is manifested in a broad spectrum of anomalies: in some patients it manifests as an isolated atresia, but in more than 60% of the cases it affects several organ systems. While the associated malformations are often those of the VACTERL spectrum (Vertebral, Anorectal, Cardiac, Tracheo-Esophageal, Renal and Limb), many patients are affected by other malformations, such as anomalies of the genitourinary, respiratory and gastrointestinal systems. Though EA/TEF is a genetically heterogeneous condition, recurrent genes and loci are sometimes affected. Trachea-Esophageal (TE) defects are in fact a variable feature in several known single gene disorders and in patients with specific recurrent Copy Number Variations and structural chromosomal aberrations. At present, a causal genetic aberration can be identified in less than 10% of patients. In most, EA/TEF is a sporadic finding; the familial recurrence rate is low (1%). As this suggests that epigenetic and environmental factors also contribute to the disease, non-syndromic EA/TEF is generally believed to be a multifactorial condition. Several population-based studies and case reports describe a wide range of associated risks, including age, diabetes, drug use, herbicides, smoking and fetal alcohol exposure. The phenotypical and genetic heterogeneity seen in EA/TEF patients indicates not one underlying cause, but several. In this project we will combine the French register of EA and multiomic studies in order to elucidate new causes or mechanisms in the etiology within specific sub-populations. Improved knowledge of predictive factors and molecular mechanisms may improve prediction and parental counseling and prevent co-morbidity. In this context, state of the art multi-omics will be performed from esophageal biopsies. Systemic/integrative biology will be then undertaken to establish predictive networks. Validation of EA pathways will be investigated using cross link coupled to mass spectrometry (XL-MS) and with BioID in order to evaluate the protein-protein partner involved in networks to better understand physiopathological mechanisms occurring in EA etiology. Integration of all the data using robust bioinformatics and biostatistics will give hypotheses for EA etiology. Based on these fundamental knowledges acquired on the EA pathology, a translational research step will be launched. Multi-omics analyses will be then performed on extracellular vesicles (EVs) issued from amniotic liquid. EVs have raised interest as a potential source of biomarker discovery because of the resemblance of their molecular content to that of the releasing cells. EVs in amniotic liquid will be the mirror of the EA pathology occurring in course of fetus development. Thus, multi-omic analyses of these EVs will be the first steps to improve the prenatal EA/TEF diagnostic.