key: cord-0978656-xunjl6p3
authors: Laurent, Estelle M.N.; Sofianatos, Yorgos; Komarova, Anastassia; Gimeno, Jean-Pascal; Tehrani, Payman Samavarchi; Kim, Dae-Kyum; Abdouni, Hala; Duhamel, Marie; Cassonnet, Patricia; Knapp, Jennifer J.; Kuang, Da; Chawla, Aditya; Sheykhkarimli, Dayag; Rayhan, Ashyad; Li, Roujia; Pogoutse, Oxana; Hill, David E.; Calderwood, Michael A.; Falter-Braun, Pascal; Aloy, Patrick; Stelzl, Ulrich; Vidal, Marc; Gingras, Anne-Claude; Pavlopoulos, Georgios A.; Van Der Werf, Sylvie; Fournier, Isabelle; Roth, Frederick P.; Salzet, Michel; Demeret, Caroline; Jacob, Yves; Coyaud, Etienne
title: Global BioID-based SARS-CoV-2 proteins proximal interactome unveils novel ties between viral polypeptides and host factors involved in multiple COVID19-associated mechanisms
date: 2020-08-29
journal: bioRxiv
DOI: 10.1101/2020.08.28.272955
sha: aeb27fc4496176dbd8d126047c280b07e229bb08
doc_id: 978656
cord_uid: xunjl6p3

The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

Over a century after the Spanish flu pandemic, and despite undeniable medical advances in infectiology, modern human societies and economies are deeply shaken by the magnitude of the current worldwide SARS-CoV-2 outbreak. Unprecedented and coordinated research efforts are needed to overcome the current crisis. While we keep learning daily on COVID-19 clinical features, brute-force drug screening and therapeutic trials have not yet proven their efficacy. A fine understanding of SARS-CoV-2 pathogenesis at a molecular level is thus required to: (i) supervise and understand drug assays to combat viral mechanisms; and (ii), prepare potential future pandemics. To date, three major proteomic studies define most of our direct knowledge of SARS-CoV-2 protein interactions with host cell components 1,2,3 .

These works rely on classical immunoprecipitation (IP) methods followed by mass spectrometry (MS)based identification of physical interactors. The major limitation of IP-MS is the requirement of maintaining protein-protein interactions (PPIs) throughout the purification process. This implies using gentle lysis buffer to solubilize physically associated proteins without disrupting PPIs, hence precluding the detection of poorly soluble protein partners, or for which PPIs have low affinity. The proximity-dependent biotinylation technique (BioID) 4 circumvents most of these caveats. Briefly, it consists of fusing an abortive mutant of E. coli biotin ligase (BirA R118G; or BirA*) at the N-or C-terminal end of a protein of interest.

Upon addition of biotin in the culture media of living cells expressing the BirA*-tagged protein of interest, the BirA* moiety activates biotin to biotinoyl-AMP in an ATP-dependent manner. Due to the low affinity of mutated BirA* for this highly reactive species, it diffuses away and conjugates to nearby free amine groups within an estimated radius of ~10 nm 5 . Since proximal proteins are covalently biotinylated, it is no longer required to maintain stable interaction during the purification process. This enables stringent lysis conditions that can solubilize proteins from all compartments, including e.g. transmembrane proteins.

Biotinylated species, i.e. proteins that have been in proximity of the bait protein in living cells, can then be readily captured on streptavidin affinity columns and identified by MS following on-bead digestion, as described 6 . Allowing the identification of PPIs through covalent labeling of proximal partners in living cells, regardless of their solubility or affinity, proximity-dependent interactomics techniques are of outstanding interest to gather biochemical insights on how the SARS-CoV-2 proteins operate to hijack host cell.

Study overview. In our study we implemented BioID to capture the proximal interactors of the 28 SARS-CoV-2 polypeptides: (i) ORF1a/b, two polyproteins encoding 16 non-structural proteins NSP1-16. Notably, NSP11 was excluded from our analysis due to its small size (12 AAs); (ii) four structural proteins: Spike (S) This arbitrary filter reduced the number of interactors from 2,598 to 2,128, and the total number of interactions dropped from 10,198 to 5,415 (Supplemental Table 1 and online dashboard). Expectedly, the core was the most affected region of the network structure. This filtering allowed its relaxation (Fig.1) , revealing the connection of highly enriched groups of interactor/protein complexes to specific viral bait or subsets of viral bait proteins. For each viral protein, we summarized the main locations of their interactors ( Fig.2.A and B) . In addition to gene ontology and pathway enrichment analysis (Supplemental Table 1 ;

Supplemental Table 2 and Supplemental Table 3 ), we manually curated the data for each viral bait proteins and assigned them to main predicted functions based on their most notable interactomes features ( Fig.2 .C). Since our experimental design consisted in expressing a single BirA*-tagged viral protein at a time, we describe each proximal interactome profile separately. For each bait protein (Figs.3-31 ), a Venn diagram shows the hit distribution between the different experimental settings. Given the complexity of the observed network, we reasoned that the ability to interactively explore the data would be superior to static networks, and implemented an online interactive 3D map (http://www.sars-cov-2interactome.org/) which can be used to identify network regions, the distribution of enriched categories as well as isolate subgroups of interactions or interactors. Considering that BioID bait proteins label proximal partners within a sphere of ~10 nm radius in living cells, we argue that a data-driven network topology in three-dimensional space is an excellent representation for capturing viral protein and host factors organization. We further hypothesize that our analysis defines cellular volumes populated by identified proteins which are at the core of viral-host interplay. the human cellular machineries, revealing a complex nexus of interactions that could serve as targets to counteract the infectious cycle. 

Apart from a few amino-acid substitutions, SARS-CoV-1 and SARS-CoV-2 NSP1 are highly similar, and they are expected to ensure comparable functions. SARS-CoV-1 NSP1 has been reported to promote host mRNA degradation and to inhibit translation 10 . Upon SARS-CoV-1 infection, NSP1 thus appear as an important virulence factor, since it is very likely to provide the type I IFN response mRNA translation blockade 10 . In addition to previously reported interactions (e.g. PKP2, POLA1 and POLA2 1 ) that are not involved in translation, we identify 12 components of the eukaryotic translation initiation (eIF) 3 complex uniquely associated with SARS-CoV-2 NSP1. SARS-CoV-1 NSP1 has been reported to block translation through interacting with the 40S ribosomal subunit 11 , of which we identified RPS10 as a high confidence interactor. In the previous interactomic studies, very few SARS-CoV-2 NSP1 interactors were identified 1,2,3 . Our data strongly suggest that NSP1 binds to the 40S subunit through the eIF3 complex, which is well described to modulate translational activity 12 . Besides interacting with the 40S associated proteins, we detected the RNA helicase DDX3X which has been involved in innate immune response activation in HBV 13 , VACV 14 , HCV 15 and HIV-1 16 infections. Of note, we observed striking toxicity expressing N-ter BirA* tagged NSP1, but not with the C-ter tagged version. Data obtained with the N-ter tagged NSP1 samples are thus only produced through transient transfection, since we were unable to generate a stable cell line for this construct. In line with this observation, the C-ter tagged version did capture only 2/12 eIF3 complex components, suggesting that NSP1 is binding to this complex mainly through its C-ter end, and that an excessive binding of NSP1 to eIF3 complex is detrimental for the cell.

Under poly(I:C) treatment, NSP1 interactome was slightly modified. It gained a few interactors with no obvious involvement in viral life cycle. 

Little is known on coronaviruses NSP2 proteins functions. In our analysis, we retrieved 43 ( MIB1 is reported to interact with Notch receptor and regulates the internalization of Notch ligand. NSP4, through interacting with trafficking machineries, appears as a main host membrane remodeler and could also impair membrane embedded protein homeostasis upon SARS-CoV-2 infection.

The NSP5 protease (3CLPro) is the main factor processing the Orf1a/b-encoded polyproteins 34 . It is therefore an essential protein for virus protein maturation. However, our analysis only identified 14 high confidence NSP5 interactors, and we did not observe noticeable or shared features amongst 36 . TOLLIP (Toll-interacting protein) is involved in TLR and IL-1 signaling, and acts as an adapter between ATG8 and ubiquitinated substrates, playing a major role in clearing protein aggregates 37 .

ZC3HAV1 (or ZAP, Zinc finger CCCH-type antiviral protein 1) induces the recruitment of host RNA degradation machinery to the viral RNA 38 . It also positively regulates RIG-I downstream signaling, which activates IRF3 and triggers the expression of type I IFN stimulated genes (ISGs) 39 . TAX1BP1 is also involved in TNF, IL-1 and NFB signaling 40 . CYLD is involved in inflammation and innate immune response through its ability of inhibiting NFB nuclear translocation 41 . TMEM9B is a key regulator of proinflammatory cytokines production in response to TNF, IL-1 and TLR ligands 42 cell vesicular trafficking. Interestingly, whereas both N-and C-ter tagged NSP7 detected interactions with cytoskeletal components, only the C-ter tagged form labeled nucleoplasmic proteins (N-terminally tagged form showed in Fig. 9 ). This observation suggests that a non-canonical nuclear localization sequence (NLS) could be localized in the N-ter region of NSP7 (impaired by the N-ter tag), or that NSP7 can be cleaved, with a C-ter fragment translocated in the nucleus. This discrepancy is quite uncommon for a nonmembrane associated protein, and further experiments are required to elucidate the molecular etiology.

NSP7 nuclear interactors were essentially involved in DNA-templated transcription, DNA repair, mRNA processing and histone modification. These results suggest that NSP7 could impair host mRNA production, thus precluding proper antiviral transcriptional program and/or reducing the pool of endogenous mRNA (i.e. lighten host mRNA translational burden and enhance host cell capacity for viral RNAs translation). The C-ter tagged NSP7 also specifically captured important innate immune and inflammatory responses modulators under basal culture conditions, such as HERC5, PPM1B, TRIM56 and TBK1. Of interest, TBK1

(Serine/threonine-protein kinase TBK1) is activated by TLR following viral or bacterial sensing. TBK1 then associates with TANK and TRAF3 and phosphorylates the IFN regulatory factors 3 and 7 (IRF3 and IRF7).

These modifications lead to the activation of pro-inflammatory and antiviral genes, including type I IFN [46] [47] [48] . PPM1B is a phosphatase able to dephosphorylate TBK1, inhibiting its activation 49 . The pre-treatment with poly(I:C) did not induce major NSP7 interactome rewiring. (both reported 2 ). C-ter tagged NSP8 interacts with PSMC3IP and histones (H2A6, H1-0 and H1-2) which might indicate an association with DNA that is in line with the observed localization of NSP8 interactors in the nucleus (Fig.10 ). These interactions could suggest a preferential binding of NSP8 to HECT-domain No previously reported NSP10 interactors were identified in our analysis. According to our immunostaining, more than half of NSP10 BioID interactors are nuclear, including chromatin remodelers, transcription factors and RNA processing proteins. Surprisingly, N-ter BioID tagged NSP10 did not localize in the nucleus. We thus hypothesize it could interfere with nuclear factors either which shuttle to the nucleus or upon mitosis. Of note, NSP10 detected TLE1, an inhibitor of NFB regulated gene expression 53 and AZI2 (or NAP1), another regulator of NFB-dependent antiviral innate immune response 54 . Alike NSP7- NSP8, NSP10 expression in the absence of its viral partners NSP14 or NSP16 renders this interactome hard to interpret. Poly(I:C) treatment did not induce noticeable gains of NSP10 interactors.

NSP12 is an RdRp binding NSP7-NSP8 55 . Due to its crucial function in viral RNA replication 56 COVID-19. TAB1 mediates signaling between TGF receptor and MAP3K7, and TAB2 acts as an adapter between MAP3K7 and TRAF6 and promotes the activation of MAP3K7 downstream IL-1 activation 59, 60 .

Besides the gain of the IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), no obvious NSP12 interactome change was observed following poly(I:C) treatment. Together, these interactors could link NSP12 to a wider range of signaling processes than expected, including antiviral and inflammatory pathways.

The Previous interactomics data showed an association between NSP13 and centrosomal proteins. Although we did not identify the same partners, our proximal interactome was also enriched in centrosome components (e.g. CCP110, CEP76, POC1B). Besides these interactions, we did not detect specific features revealed by proximal partners enrichment analysis, regardless of the poly(I:C) treatment. Importantly, a recent functional study has suggested that SARS-CoV-2 NSP13 is an IFN signaling antagonist 63 . However, we did not detect any NSP13

interactor which could explain this phenotype. We hypothesize that this role can be dependent on the molecular context. Future experiments in SARS-CoV-2 infected cells will reveal additional and pathologically relevant interactions of this essential viral protein.

NSP14 is a component of the replication-transcription complex. It is a dual functional enzyme, bearing both 3'-5' exonuclease (ExoN) and a guanine N7-methyltransferase (N7-MTase) activities 64 . ExoN ensures a proofreading function responsible of high-fidelity SARS-CoV-1 replication through its ssRNA and with machineries leading to mRNA degradation could explain why NSP14 was also identified as an IFN response antagonist 63 . We could hypothesize that NSP14 could either sequester host decapping and deadenylation machineries or stimulate host mRNA degradation. ORF3a is a key accessory protein showing a pro-apoptotic activity 76 complex which directs endosomes towards lysosome and is crucial in multivesicular body formation. These proteins are dedicated to downregulating membrane receptors abundance 95 . Interestingly, the ESCRT-0 complex has also been involved in IL-2 and GM-CSF/L signal transduction 96 . ORF3b also identified multiple components of the SNARE complex, but did not detect HOPS, CORVET or TRAPP complexes subunits, highlighting their differential involvement of ORF3a and ORF3b in SARS-CoV-2 pathogenesis. Amongst 

The (Erbb2-interacting protein) inhibits NOD2-dependent NFB signaling and proinflammatory cytokine secretion 114 . TBC1D23 (TBC1 domain family member 23, also known as HCV non-structural protein 4Atransactivated protein 1) may act as a general inhibitor of innate immunity signaling, strongly inhibiting multiple TLR and dectin/CLEC7A-signaling pathways 115 . B2M (Beta-2-microglobulin) is a component of the respectively, and are responsible of membrane-cytoskeleton binding and involved in spectrin-actin assembly 117 . EHD1 and EHD4 (EH domain-containing protein 1 and 4) control membrane tubulation in an ATP hydrolysis-dependent manner 118 . SNX1/2 (Sorting nexins 1 and 2) are involved in retromer transport and in sensing membrane curvature 119, 120 . FCHO2 (F-BAR domain only protein 2) can recruit membranes and modify their curvature as well 121 . Finally, SH3GL3 (Endophilin A3) has been reported to recruit other proteins to membranes with high curvature 122 . Together, these data show that M can impact diverse pathways, such as MAPK, NFB, innate immunity, antigen presentation and membrane curvature regulation. This later process is of interest since M is known to regulate virion membrane shaping 123 . 

Little 140, 141 . In addition to these proximal interactions of interest, we identified several factors involved in processes potentially related to anosmia and ageusia symptoms 142 .

ORF7a identified the REEP proteins which are involved in membrane receptors and/or olfactory receptors regulation mechanisms 143 . Along with ubiquitin-proteasome system interactions, an attractive hypothesis anosmia symptom, we detected GRID1, the glutamate receptor ionotropic, delta-1, which is essential for olfactory cells signal transmission 148 . In summary, ORF7b seems involved in similar mechanisms as ORF7a, but targets different factors. It seems more specialized in cell adhesion and could act on different components of olfactory signaling cascades. Following poly(I:C) treatment, ORF7b interactome is greatly impacted with 91 interactors increased/gained (including e.g. GRID1) and 67 decreased/lost, suggesting a regulation upon IFN pathway activation. More than half of these gained hits are assigned to the plasma membrane. Amongst enriched/gained interactors, we identified e.g.: ROBO2, which is involved in olfactory bulb neuron development 149 ; IFIT1, TOLLIP, BTN3A2/3A3 (immunity); and CD83 (MHC-II antigen presentation 150 ). ORF7b thus could be involved in multiple host pathway deregulations, such as inflammation, IFN pathway activation and olfaction. ubiquitin ligase targeting IRF3 for proteasomal degradation and bridging TBK1 and NEMO during infection 160 . In basal condition, N is thus able to interact with RNA binding and processing host-factors, including several stress granules biogenesis factors. We see these interactions as a viral strategy to either protect vRNA from host nucleases, or to control vRNA detection by pattern recognition receptors (PRRs), or to regulate vRNA transcription/replication/translation. The interactions with TRIM26 and PRKRA could reveal N-mediated inhibition of both IFN and NFB innate immune response pathways. In the poly(I:C)stimulated condition, N captured additional interactors of outstanding interest: EIF2AK2 (PKR) and its interactors STAU1/2; ANKRD17; TARBP2 and ZNF346; TRIM56; TRIM26; and IFIT1/2/3/5. It appears logical that N, being the first viral protein released in host cells, directly targets these fundamental innate immune response proteins. In SARS-CoV-1, N was found to inhibit IFN production interfering with TRIM25, an Ub E3 ligase mediating RIG-I degradation 161 . Our data suggest that N SARS-CoV-2 could use additional or different mechanisms, such as interfering with RIG-I stabilization by binding ANKRD17 162 , impairing viral RNA recognition through IFIT1/2/5 inhibition 163 , and/or precluding RIG-I downstream response targeting MAVS or IRF3 (IFIT3, TRIM26), and/or, upstream MAVS, blocking the TRIM56-mediated activation of STING and FAM173A. PKR is an IFN-induced dsRNA-dependent serine/threonine-protein kinase that detects viral dsRNA to promote multiple proinflammatory cytokines production 165 . PKR is also able to block translation 164 . Together, our data strongly support a central role of N in impairing host antiviral response.

SARS-CoV-1 ORF9b is encoded by an alternative open reading frame within N and has been linked to innate immune response impairment through its ability to target PCBP2 and AIP4 which leads to MAVS, TRAF3

and TRAF6 degradation 166 . The Kelhr laboratory also reports SARS-CoV-1 ORF9b property of triggering autophagy in an ATG5-dependent manner when highly expressed 166 . ORF9b is located at the outer mitochondrial membrane probably via its interaction with TOMM70A 167 . This recent study suggests that the ORF9b-TOMM70A interaction is essential for the type I IFN signaling inhibition role of ORF9b. In our BioID analysis, we identified TOMM70A amongst the top interactors of ORF9b, and MARK1/2/3 and DNM1L which were also previously reported 1,2,3 . Our data greatly expand our current knowledge, Whereas the C-ter tagged ORF14 interactome identified proximal interactors mostly localized at the ER and the mitochondrial membrane, the N-ter tagged bait labeled a majority of nucleolar components, which is in accordance with BirA*Flag-ORF14 exclusive localization in the nucleus (Fig.29 ). This discrepancy 170 .

Surprisingly, while the defect of ribosomes produced de novo efficiently inhibits the antiviral response, it did not seem to affect viral life cycle. Further investigation must be implemented to test the ribosome production rate in SARS-CoV-2 infected cells. In addition to these interactions, N-ter tagged ORF14 detected USP11 which is involved in NFB pathway regulation 171 

ORF10 is a predicted accessory protein of unknown function 169 This ongoing work will be subjected to several follow up studies to confirm the role of selected SARS-CoV-2 proteins in specific infectious mechanisms. Our interactome will be subjected to cross validation by orthogonal approaches and mined to identify putative drug targets candidates.

The operational costs of this project were supported by the Agence Nationale pour la Recherche BioID data acquisition. MS samples were prepared from three biological replicates of each bait protein fused either with a N-terminal or a C-terminal BirA*Flag epitope tag in basal condition or following poly(I:C) (Sigma-Aldrich; P1530) transfection at 2µg/mL for 5hrs using PolyJet reagent (Signagen) prior to tetracycline and biotin induction, and analyzed on a Thermo Q-Exactive mass spectrometer. Samples were separated by online reversed-phase chromatography using a Thermo Scientific Easy-nLC1000 system equipped with a Proxeon trap column (75 μm ID x 2 cm, 3 µm, Thermo Scientific,) and a C18 packed-tip column (Acclaim PepMap, 75 µm ID x 50 cm, 2 µm, Thermo Scientific). The digested peptides were separated using an increasing amount of acetonitrile in 0.1% formic acid from 2 to 30% for 2 hours at a flow rate of 300 nL/min. A voltage of 2. Table 1 .

BioID data analysis. The matrix (Supplemental Table 1 ) shows the average log2 fold change against control and the corresponding p-and q-values for each bait and condition. The poly(I:C) data were also compared against control (green font when significant) and against the corresponding condition (N-terminal tag for all viral bait proteins, except for NSP1). The poly(I:C) column indicates the status of each interaction when compared to the basal condition. 'Gained' and 'Lost' are self-explanatory, and 'Decreased' and 'Increased'

were defined when the log2 fc against the basal condition was <(-1) or >(+1), respectively. 'Unchanged'

corresponds to log2fc values between -1 and +1, and empty cells depict interactors not detected in the basal and poly(I:C) condition. The InDegree column depicts the number of bait proteins detecting a given interactor, regardless of the condition (N-ter, C-ter or poly(I:C)). This criterion was chosen to filter out the most connected interactors (8+), likely to be organelle-specific background not filtered using the BirA* alone control samples.

interactors' table (Supplemental Table 1 ). In the 'High conf. Table 2 , which was generated using the Metascape annotation tool 5 (https://metascape.org/). For the Figures 3-31 , the Metascape Express Analysis report was used to automatically select the functional categories enriched for each given bait protein. The detail output of the functional enrichment analysis, including the gene list assigned to each category for each bait protein, are provided in Supplemental Table 3 . Finally, each viral this information is not available (such as the reported target-target interactions) we set this factor equal to 1. The node 3D coordinates obtained by minimizing the energy of the physical model are fed into code that creates a 3D visualization of the network by assigning a point to each node and a line connecting a pair of nodes when there is an interaction between the corresponding proteins. Our code is written in Python 3.8 7 and makes use of several modules, primarily: NetworkX 8 for graph operations, NumPy 9 for array manipulation and numerical computations, pandas 10 for data handling and Plotly 11 for visualization.

Online interactive web application. In order to make our results available to the scientific community in an usable and informative manner, we have implemented a web app accessible at http://www.sars-cov-2-interactome.org/, containing the interactive network visualization as well as multiple options for selecting and filtering the dataset to view subnetworks, providing the ability to focus on particular areas of interest and explore various levels of detail. In addition, we integrate and overlay information about the reported status of interactors and interactions with respect to SARS-CoV-1/2 or other coronaviruses, as well as the previously reported interactions between the targets (Supplemental Table 1 better representing their physical size and actual location in space. We plan to continually improve the dashboard by adding useful features in future updates. Integrating with networks from other related studies in a comparative manner could provide an even more comprehensive resource that could yield

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Lyn-dependent signaling regulates the innate immune response by controlling dendritic cell activation of NK cells

Lyn and Fyn function as molecular switches that control immunoreceptors to direct homeostasis or inflammation

A Disintegrin and Metalloproteinase 9 Domain (ADAM9) Is a Major Susceptibility Factor in the Early Stages of Encephalomyocarditis Virus Infection

IL-6: Relevance for immunopathology of SARS-CoV-2

An Autocrine Wnt5a Loop Promotes NF-κB Pathway Activation and Cytokine/Chemokine Secretion in Melanoma

Tyrosine Phosphatase PTPRJ/DEP-1 Is an Essential Promoter of Vascular Permeability, Angiogenesis, and Tumor Progression

Maintenance of murine platelet homeostasis by the kinase Csk and phosphatase CD148

SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant

Ubiquitin recognition in endocytic trafficking -with or without ESCRT-0

ESCRT-dependent cargo sorting at multivesicular endosomes

RalA but Not RalB Enhances Polarized Delivery of Membrane Proteins to the Basolateral Surface of Epithelial Cells

Novel role for ALCAM in lymphatic network formation and function

Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells

USP11 negatively regulates TNFalpha-induced NF-kappaB activation by targeting on IkappaBalpha

Coronavirus envelope (E) protein remains at the site of assembly

Factor XII and kininogen asymmetric assembly with gC1qR/C1QBP/P32 is governed by allostery

Mitochondrial p32/C1qbp Is a Critical Regulator of Dendritic Cell Metabolism and Maturation

The mitochondrial protein C1qbp promotes cell proliferation, migration and resistance to cell death

TMEM59 defines a novel ATG16L1-binding motif that promotes local activation of LC3

BTN1A1, the mammary gland butyrophilin, and BTN2A2 are both inhibitors of T cell activation

Heteromeric interactions regulate butyrophilin (BTN) and BTN-like molecules governing γδ T cell biology

The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner

KATP Channels in the Cardiovascular System

ZAP-mediated mRNA degradation

The membrane protein of SARS-CoV suppresses NF-kappaB activation

The SARS-Coronavirus Membrane protein induces apoptosis through modulating the Akt survival pathway

Protein kinase D2 mediates lysophosphatidic acidinduced interleukin 8 production in nontransformed human colonic epithelial cells through NF-kappaB

A role for Erbin in the regulation of Nod2-dependent NF-kappaB signaling

Spatiotemporal inhibition of innate immunity signaling by the Tbc1d23 RAB-GAP

The MHC I loading complex: a multitasking machinery in adaptive immunity

Modulation of spectrin-actin assembly by erythrocyte adducin

ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling

Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors

Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of highcurvature membranes and 3-phosphoinositides

Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature

Mechanism of endophilin N-BAR domain-mediated membrane curvature

A structural analysis of M protein in coronavirus assembly and morphology

MHC I chaperone complexes shaping immunity

Characterization of the Antigen Processing Machinery and Endogenous Peptide Presentation of a Bat MHC Class I Molecule

Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists

Severe acute respiratory syndrome coronavirus ORF6 antagonizes STAT1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/Golgi membrane

The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway

The COMMD Family Regulates Plasma LDL Levels and Attenuates Atherosclerosis Through Stabilizing the CCC Complex in Endosomal LDLR Trafficking

Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling

COMMD proteins and the control of the NF kappa B pathway

Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3

Ubiquitin-induced oligomerization of the RNA sensors RIG-I and MDA5 activates antiviral innate immune response

Severe Acute Respiratory Syndrome Coronavirus ORF7a Inhibits Bone Marrow Stromal Antigen 2 Virion Tethering through a Novel Mechanism of Glycosylation Interference

SARS coronavirus 7a protein blocks cell cycle progression at G0/G1 phase via the cyclin D3/pRb pathway

The Chaperone UNC93B1 Regulates Toll-like Receptor Stability Independently of Endosomal TLR Transport

CD97 in leukocyte trafficking

Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b

A gamma-secretase-like intramembrane cleavage of TNFalpha by the GxGD aspartyl protease SPPL2b

A missense mutation in gamma-glutamyl carboxylase gene causes combined deficiency of all vitamin Kdependent blood coagulation factors

Venous and arterial thromboembolic complications in COVID-19 patients admitted to an academic hospital in

Anosmia and Ageusia: Common Findings in COVID-19 Patients

Members of RTP and REEP gene families influence functional bitter taste receptor expression

TRIM4 modulates type I interferon induction and cellular antiviral response by targeting RIG-I for K63-linked ubiquitination

The ORF7b protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is expressed in virus-infected cells and incorporated into SARS-CoV particles

Dimerization of the cytokine receptors gp130 and LIFR analysed in single cells

The role of furin cleavage site in SARS-CoV-2 spike protein-mediated membrane fusion in the presence or absence of trypsin

Functional architecture of olfactory ionotropic glutamate receptors

Robo1 and robo2 control the development of the lateral olfactory tract

Thymic CD4 T cell selection requires attenuation of March8-mediated MHCII turnover in cortical epithelial cells through CD83

The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I

Structural basis of Notch O-glucosylation and O-xylosylation by mammalian protein-O-glucosyltransferase 1 (POGLUT1)

Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells

TRIM2, a novel member of the antiviral family, limits New World arenavirus entry

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle

The Nucleocapsid Protein of SARS-CoV-2: a Target for Vaccine Development

Biochemical characterization of SARS-CoV-2 nucleocapsid protein

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

Autoubiquitination of TRIM26 links TBK1 to NEMO in RLR-mediated innate antiviral immune response. J. Interfering with TRIM25-Mediated RIG-I Ubiquitination

Ankrd17 positively regulates RIG-I-like receptor (RLR)-mediated immune signaling

Structural basis for viral 5'-PPP-RNA recognition by human IFIT proteins

The dsRNA protein kinase PKR: virus and cell control

SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome

SARS-CoV-2 ORF9b suppresses type I interferon responses by targeting TOM70

The Interferon-Induced Exonuclease ISG20 Exerts Antiviral Activity through Upregulation of Type I Interferon Response Proteins

Dark Proteome of Newly Emerged SARS-CoV-2 in Comparison with Human and Bat Coronaviruses

Ribosome biogenesis restricts innate immune responses to virus infection and DNA

The deubiquitinating enzyme USP11 controls an IkappaB kinase alpha (IKKalpha)-p53 signaling pathway in response to tumor necrosis factor alpha (TNFalpha)

SUMO: a multifaceted modifier of chromatin structure and function

TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes

A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

The protein expression profile of ACE2 in human tissues

Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles

The current landscape of coronavirus-host protein-protein interactions

SARS-CoV-2 viral proteins coding sequences were cloned into pcDNA5 FRT/TO N-ter and C-ter

BirA*Flag vectors by Gateway cloning (as in 1 ) using our collection of Gateway-compatible entry clones as templates 2 (see Supplemental table 4 for sequences)

BioID samples were prepared as in 3 . Briefly, Flp-In™ T-REx™ HEK293 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS

:1,000 Turbonuclease (BPS Bioscience) and incubated on an end-over-end rotator at 4°C for 1 hour, briefly sonicated to disrupt any visible aggregates, then centrifuged at 45,000 x g for 30 min at 4°C. Supernatant was transferred to a fresh 15 mL conical tube. 25 μl of packed, pre-equilibrated by incubating the beads with 1 µg MS-grade TPCK trypsin

Following centrifugation at 2,000 x g for 2 min, the supernatant was collected and transferred to a fresh Eppendorf tube. Two additional washes were performed with 150 µL of 50 mM ammonium bicarbonate and pooled with the first eluate. The sample was lyophilized and

Confocal microscopy. Each N-ter tagged viral protein expression was inducted by 1µg/mL tetracycline and 50µM biotin for 24 hrs (in basal or poly(I:C) conditions) in stably transfected Flp-In™ T-REx™ HEK293 cells

Briefly, cells were grown on poly-lysine coated coverslips, rinsed with PBS before fixation with 4%

Acquisition was performed on a Zeiss LSM700 confocal microscope connected to a Zeiss Axiovert 200 M equipped with an EC Plan-Neofluar 40×/1.30 numerical aperture and an oil immersion objective (Carl Zeiss AG, Oberkochen, Germany). and 2598 targets (modulo isoforms), with 10198 interactions. Based on this data we construct a graph with interactors and interactions represented by the vertices (nodes) and edges (links) respectively of a directed graph

To visualize this graph, our objective is to determine an optimum placement of the nodes in threedimensional space that would minimize edge crossings and reveal the interactome's structure to the best possible extent. We employ a custom, multi-stage, force-directed graph layout algorithm

poly(I:C), when present for the interaction. For interactions where Supplemental Material and Method -References

High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies

A Flexible Genome-Scale Resource of SARS-CoV-2 Coding Sequence Clones. G3: Genes, Genomes, Genetics Early online August 6

Global interactomics uncovers extensive organellar targeting by Zika Virus

ToppCluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems

Metascape provides a biologist-oriented resource for the analysis of systems-level datasets

Efficient and high quality force-directed graph drawing

Python 3 Reference Manual

Exploring network structure, dynamics, and function using NetworkX

The NumPy Array: A Structure for Efficient Numerical Computation

Data Structures for Statistical Computing in Python, Proceedings of the 9th Python in Science Conference

Over 1 is considered as either enrichment or gain (if absent from the basal condition), between 1 and -1 corresponds to unchanged, and below -1 shows decreased or lost hits. When no value is indicated, it corresponds to interactions not detected in the poly(I:C) and the corresponding basal condition. The reported interactions columns display the bait-prey pairs previously identified in the literature (see tab D for details and references and main of all coronaviruses across the literature, with corresponding techniques and references. E. Comparative ToppCluster analysis (see Supplemental Method) output of all bait interactomes obtained in basal condition (union of N-and C-terminally tagged viral bait proteins BioID results). F. Input list for the analysis presented in E. G. Same as E, but with hits increased or gained after poly(I:C) treatment. H. Input enriched or gained interactors after poly(I:C) for the ToppCluster analysis presented in G. I. Raw output of the Perseus analysis of MaxQuant LFQ results. Columns G-JA: log2 transformed LFQ intensities for all biological replicates and conditions