key: cord-0886905-9h5cwq4v authors: Martin-Sancho, Laura; Lewinski, Mary K.; Pache, Lars; Stoneham, Charlotte A.; Yin, Xin; Becker, Mark E.; Pratt, Dexter; Churas, Christopher; Rosenthal, Sara B.; Liu, Sophie; Weston, Stuart; De Jesus, Paul D.; O’Neill, Alan M.; Gounder, Anshu P.; Nguyen, Courtney; Pu, Yuan; Curry, Heather M.; Oom, Aaron L.; Miorin, Lisa; Rodriguez-Frandsen, Ariel; Zheng, Fan; Wu, Chunxiang; Xiong, Yong; Urbanowski, Matthew; Shaw, Megan L.; Chang, Max W.; Benner, Christopher; Hope, Thomas J.; Frieman, Matthew B.; García-Sastre, Adolfo; Ideker, Trey; Hultquist, Judd F.; Guatelli, John; Chanda, Sumit K. title: Functional Landscape of SARS-CoV-2 Cellular Restriction date: 2021-04-13 journal: Mol Cell DOI: 10.1016/j.molcel.2021.04.008 sha: 9959a38e0b68c5301686fc8108e60f1aed1046f4 doc_id: 886905 cord_uid: 9h5cwq4v A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of ER-Golgi-resident ISGs inhibiting viral assembly-egress. These included broad-acting antiviral ISGs, and eight ISGs that specifically inhibited SARS-CoV-2 and -1 replication. Amongst the broad-acting ISGs was BST2/tetherin, which impeded viral release, and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19. ). Next, we evaluated the ability of these 399 ISGs to inhibit SARS-CoV-2 replication using 144 ectopic expression screening. These studies were conducted using the human epithelial cell line 293T, 145 as these cells can be transfected with high efficiency, support productive replication of SARS-CoV-2 146 when expressing the viral entry factors ACE2 and TMPRSS2 (Hoffmann et al., 2020) , and respond to 147 IFN treatment, which reduced SARS-CoV-2 replication in a dose-response manner (Fig S1C) (Fig 1A) . cDNA encoding chloramphenicol acetyltransferase (CAT) was included 152 on each plate as negative control, and cDNA encoding the SARS-CoV-2 negative regulator LY6E 153 (Pfaender et al., 2020) was included as positive control (Fig 1A, B) . We first confirmed that cDNA treated cells (Fig S1D) . Screens were then conducted in duplicate and showed good reproducibility 157 with a Pearson correlation coefficient (r) = 0.81 (Fig 1C) . After applying cut-off criteria for infection 158 (log2FC at least four standard deviations lower than that of CAT negative control) and cell viability (at 159 least 70% number of cells of the negative control), we identified 65 ISGs that inhibited SARS-CoV-2 160 replication (Fig 1B) . This list of antiviral factors include upstream regulators as well as downstream 161 effectors of the IFN response. The former include the signaling adaptor MYD88, signal transducers 162 STAT1 and STAT2, helicase DDX60, and E3 ubiquitin ligase TRIM21, all of which were shown to 163 stimulate ISG54 production upon expression (Fig S1E) . Conversely, we found several downstream 164 effectors including BST2, IFITM2, and IFITM3, which likely harbor direct antiviral activities, and 165 accordingly did not result in ISG54 stimulation (Fig S1E) . Cross-comparison of these 65 factors with 166 published datasets of upregulated genes from COVID-19 patient samples and in vitro infected lung 167 cell models revealed a small but significant overlap (Fig S1F) induce an antiviral response, we measured if their expression stimulate ISG54 production. In addition 187 to ELF1, overexpression of C-type lectin CLEC4D, the E3 ubiquitin ligase TRIM21, and the 188 transcription factor REC8 resulted in a significant increase in ISG54 mRNA levels (Fig S1H) , suggesting that these factors likely exert indirect antiviral activities. (Table S3) . From 194 the 11 lines generated, 9 showed a significant reduction in SARS-CoV-2 replication compared to 195 parental cells (Fig 1F) . Subsequently, we used nine of these ISG-encoding lentiviruses to transduce 196 primary human epithelial tracheobronchial epithelial cells (HTBE). Each line was then differentiated 197 at an air-liquid interface to produce a mucociliary epithelium similar to that of the native human with SARS-CoV-2 and evaluated for their ability to support viral growth. All these four lines showed 202 a significant reduction in viral growth relative to control cultures transduced with GFP (Fig 1G) . Together, these results suggest that a subset of these factors exert antiviral activities in several cellular (Table S4) . Using these analyses, we identified densely 216 interconnected protein clusters that have significant enrichment in specific cellular biological 217 processes (Raudvere et al., 2019) . As expected, we found strong association with pathways that 218 stimulate IFN signaling, including cytosolic pattern recognition receptors (p value = 8.553 x 10 -5 ) and 219 regulators of STAT phosphorylation (p value = 1.542 x 10 -23 ), as well as pathways linked to the type I 220 IFN response (p value = 1.573 x 10 -23 ), the cellular response to viral infection (p value = 8.891 x 10 -19 ), 221 and cytokine signaling (p value = 1.588 x 10 -9 ) (Fig 2A, blue boxes) . We also observed an enrichment To understand how these antiviral effectors impact viral replication, the 37 validated ISGs 246 ( Fig 1D) were tested for their ability to inhibit specific stages of the SARS-CoV-2 infectious cycle. Firstly, we used a pseudotyped VSV expressing SARS-CoV-2 S protein (VSV-S), alongside a second 248 VSV expressing its natural glycoprotein (VSV-G) to measure viral entry (Fig 3A, diagram) . Second, we assessed viral translation and RNA replication by measuring viral RNA at 8 h post-infection (Fig 250 3B ). Lastly, we infected naïve cells with viral supernatants that were collected at 18 h post infection to 251 assess late stage activity, encompassing viral assembly and egress (Fig 3C) . These experimental data 252 were integrated with manually curated bioinformatic resources that provided information on 253 subcellular localization and membership to biological pathways to establish a visual model that 254 predicts the impact of these ISGs on the SARS-CoV-2 infectious cycle (Fig 4) . (Fig 3A, 4) . Of these, CLEC4D, ELF1, and REC8 promoted induction of ISG54 expression (Fig S1H) . Both ELF1 and CLEC4D have been shown to inhibit pathogen replication by controlling production of ISGs that also reduced entry of VSV-S were LY6E, UBD, and FAM46C (Fig 3A) . Overexpression of these factors did not significantly induce ISG54 production (Fig S1H) , suggesting 270 they might directly inhibit viral entry. FAM46C showed no effect on VSV-G (Fig S2A) , indicating it 271 could target a specific feature of S-mediated entry. Notably, LY6E and UBD showed a contrasting 272 ability to reduce entry of VSV-S but increase that of VSV-G (Fig S2A) . LY6E was previously shown 273 to restrict entry of live SARS-CoV-2 by inhibiting viral S protein fusion at the membrane (Pfaender et The IFIT-family includes five members (IFIT1, IFIT1B, IFIT2, IFIT3, and IFIT5), which 291 prevent active viral RNA replication by detection and sequestering of single-stranded 5′-ppp or 2′O-292 unmethylated RNA (Metz et al., 2013) . In this study, we identified three members of this family, 293 IFIT1, IFIT3, and IFIT5, to inhibit SARS-CoV-2 replication (Fig 3B) , suggesting this family plays an 294 important role in the restriction of SARS-CoV-2. Viral RNA levels were also reduced by the RNA 295 binding protein SPAT2SL (Fig 3B) . Following stress stimuli, SPAT2SL is recruited to cytoplasmic 296 stress granules, where viral RNA can be sequestered to reduce viral genome synthesis (Miller, 2011; 297 Zhu et al., 2008) . Finally, the ISG DNAJC6, a member of the heat shock protein 40 (HSP40) family, 298 was also determined to impact the SARS-CoV-2 replicative stage (Fig 3B) . HSP40 family members 299 are known to play critical roles in protein transport, folding, and structural disassembly, and have been Notably, BST2 expression reduced SARS-CoV-2 RNA replication (53% reduction compared 356 to control cells) followed by a more potent reduction of viral release (74% reduction) (Fig 3B, C) . To 357 further characterize the impact of BST2 on late stage replication, we evaluated viral egress in the 358 presence or absence of BST2 using a virus-like particle (VLP) system that bypasses viral entry and 359 viral RNA replication (Siu et al., 2008) . We confirmed that this system can recapitulate virus egress, (Fig 5E) , and that both Orf7a and BST2 were incorporated into the VLP particles (Fig 5E) 373 (Fitzpatrick et al., 2010) . Notably, Orf7a-mediated antagonism of BST2 activity did not involve 374 reduction of BST2 cell surface expression, in contrast with HIV-1 Vpu (Fig S3G) . We further 375 investigated the location of BST2 and Orf7a in the cell and observed that BST2 and Orf7a colocalized 376 in the perinuclear region (Fig 5G) . In silico docking of SARS-CoV-2 Orf7a and BST2 ectodomains 377 revealed a potential interface at the N-terminal region of BST2 ectodomain (Fig 5H, 5I) , suggesting 378 J o u r n a l P r e -p r o o f that these two proteins could physically interact. To evaluate this hypothesis, we assessed BST2 and 379 Orf7a interaction through immunoprecipitation studies. These were conducted using wild type (WT) or 380 a BST2 mutant defective for N-linked glycosylation (N2Q), as a previous report suggested that the 381 interaction between SARS-CoV-1 Orf7a and BST2 was glycosylation-dependent (Taylor et al., 2015) . In contrast to SARS-CoV-1, these data revealed an association between SARS-CoV-2 Orf7a and 383 BST2 independently of BST2 glycosylation status (Fig 5J) . To further investigate Orf7a antagonism of BST2, we infected parental or BST2 293T stable 386 cells with either WT SARS-CoV-2 or a recombinant SARS-CoV-2 that was engineered to replace 387 Orf7a with nanoluciferase (dOrf7a) (kindly provided by Ralph Baric) (Hou et al., 2020) . While WT 388 and dOrf7a viruses grew similarly in parental cells (Fig 5K) and showed comparable levels of SARS-389 CoV-2 N protein (Fig S3H) , the replication of dOrf7a virus was significantly attenuated in BST2-390 expressing cells at 48 h post-infection (Fig 5K) . Overall, these data establish BST2 as a potent (Fig 6) . In 399 addition, we experimentally evaluated if expression of these factors could inhibit the replication of 400 SARS-CoV-1 (Fig 6) . Ten anti SARS-CoV-2 ISGs were found to reduce replication of four or more 401 viruses (Fig 6) . These include well described IFN signaling transducers, signaling molecules, and 402 innate immune sensors, including STAT2 and MYD88, the inhibitors of viral entry IFITM2 and 403 IFITM3 (Brass et al., 2009) , and the viral nucleic acid binders ZBP1 and IFIT1. Conversely, a cluster 404 of 8 ISGs harbored selective activities for SARS-CoV-2 (Fig 6) , all of which also inhibited SARS- Data for SARS-CoV-2 were generated within this study (see Table S2 ). Data for the remaining 20 PIV3 HMPV NDV RSV MV EAV BUNV PV CBV VV IFITM3 ZBP1 MYD88 IFITM2 STAT2 NRN1 BST2 RETREG1 JADE2 IFIT1 MSR1 FNDC4 NT5C3A ISG20 TMEM268 IFIT3 ETV6 TAGAP TENT5C TENT5A SPATS2L MAX CCND3 RAB27A UPP2 ST3GAL4 CLEC4D GBP3 B4GALT5 FZD5 ARNTL TRIM21 NAPA CRP APOL2 ERLIN1 CASP7 DDX60 Systems biological assessment of 958 immunity to mild versus severe COVID-19 infection in humans Imbalanced Host Response to SARS-CoV-961 Drives Development of COVID-19 The IFITM Proteins Mediate 964 Cellular Resistance to Influenza A H1N1 Virus, West Nile Virus, and Dengue Virus CHARMM: The biomolecular simulation 968 program Network propagation in 970 the cytoscape cyberinfrastructure A Single-Cell Atlas of the 973 Human Healthy Airways A gene ontology inferred from molecular networks Bulk and single-cell gene expression 979 profiling of SARS-CoV-2 infected human cell lines identifies molecular targets for 980 therapeutic intervention Coronaviruses: an overview of their replication and 982 pathogenesis The Interferon Inducible Gene: Viperin Direct Restriction of Virus Release and Incorporation of the Interferon-Induced Protein BST-987 2 into HIV-1 Particles Ten Strategies of Interferon Evasion by Viruses A SARS-CoV-2-Human Protein-992 Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing (Systems 993 Biology) Flavivirus internalization is regulated by a size-995 dependent endocytic pathway Impaired type I interferon activity and 998 inflammatory responses in severe COVID-19 patients The antiviral protein, viperin, localizes to lipid 1000 droplets via its N-terminal amphipathic -helix SARS-CoV-2 Cell Entry 1003 Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor SARS-CoV-2 Reverse Genetics 1007 Reveals a Variable Infection Gradient in the Respiratory Tract Clinical features of patients infected with 2019 novel coronavirus in Wuhan, 1010 China A protein-interaction network of 1013 interferon-stimulated genes extends the innate immune system landscape CRISPR-Cas9 genome engineering of primary CD4+ T 1017 cells for the interrogation of HIV-host factor interactions Triple combination of interferon beta-1b, lopinavir-1020 ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an 1021 open-label, randomised, phase 2 trial -SNAP stimulates disassembly of 1024 endosomal SNARE complexes and regulates endocytic trafficking pathways PIPER: An FFT-based protein 1027 docking program with pairwise potentials The ClusPro web server for protein-protein docking Inferring gene 1032 ontologies from pairwise similarity data CoV-2 productively infects human gut enterocytes Type I Interferon Susceptibility 1038 Distinguishes SARS-CoV-2 from SARS-CoV 1042 (2020). Presence of Genetic Variants Among Young Men With Severe COVID-19 LY6E mediates an evolutionarily conserved enhancement of 1045 virus infection by targeting a late entry step Deep immune profiling of 1048 COVID-19 patients reveals distinct immunotypes with therapeutic implications Interferon-stimulated genes 1051 and their role in controlling hepatitis C virus Stress Granules and Virus Replication Critical roles of FAM134B in ER-phagy and diseases The 1056 Cluster Has Emerged Recently in Evolution and Is Expressed in 1057 Human Vascular Tissue HSP40, and HSP60 bind to the 3′ untranslated region of the Murine hepatitis virus genome Tetherin inhibits retrovirus release and is 1062 antagonized by HIV-1 Vpu Ubiquitin-like 1064 modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I 1065 from its signaling platform Rab27a and Rab27b control different 1068 steps of the exosome secretion pathway Large-scale Multi-omic 1071 Analysis of COVID-19 Severity (Infectious Diseases The Human 1073 Cluster: Identification, Classification, and Sites of Distribution An 1076 Endoplasmic Reticulum (ER) Membrane Complex Composed of SPFH1 and SPFH2 1077 Mediates the ER-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors LY6E impairs coronavirus fusion and confers immune 1081 control of viral disease 1083 g:Profiler: a web server for functional enrichment analysis and conversions of gene lists Optimization of 1086 Normal Human Bronchial Epithelial (NHBE) Cell 3D Cultures for in vitro Lung Model Viral determinants in H5N1 influenza A virus enable productive infection of HeLa 1091 cells The Hsp70 chaperone 1093 network INTERFEROME v2.0: an updated database of annotated interferon-1096 regulated genes A diverse range of gene products are effectors of the type I interferon antiviral 1099 response Pan-viral specificity of IFN-1102 induced genes reveals new roles for cGAS in innate immunity The ETS transcription factor ELF1 1105 regulates a broadly antiviral program distinct from the type I interferon response Viperin Regulates Cellular Lipid Metabolism during 1108 Human Cytomegalovirus Infection Cytoscape: A Software Environment for Integrated Models of 1110 Biomolecular Interaction Networks Multiscale community detection in Cytoscape The M, E, and N Structural Proteins of the Severe 1115 Acute Respiratory Syndrome Coronavirus Are Required for Efficient Assembly, Trafficking, 1116 and Release of Virus-Like Particles The ubiquitin-like modifier FAT10 decorates autophagy-targeted Salmonella and contributes 1119 to Salmonella resistance in mice Comparative transcriptome analysis reveals the intensive early-stage 1122 responses of host cells to SARS-CoV-2 infection Severe Acute Respiratory Syndrome Coronavirus 1125 ORF7a Inhibits Bone Marrow Stromal Antigen 2 Virion Tethering through a Novel 1126 Mechanism of Glycosylation Interference Stimulation of NF-B Activity by the HIV Restriction Factor BST2 Turning the World Upside-Down in Cellulose for Improved 1130 Culturing and Imaging of Respiratory Challenges within a Human 3D Model The interferon-induced protein BST-2 restricts HIV-1 1133 release and is downregulated from the cell surface by the viral Vpu protein Severe acute respiratory syndrome coronavirus 1136 spike protein counteracts BST2-mediated restriction of virus-like particle release BST2/CD317 counteracts human 1139 coronavirus 229E productive infection by tethering virions at the cell surface Generation of VSV pseudotypes using recombinant ∆G-VSV for studies 1142 on virus entry, identification of entry inhibitors, and immune responses to vaccines ER-phagy: shaping up and destressing the endoplasmic reticulum The C-type 1148 lectin receptor CLECSF8/CLEC4D is a key component of anti-mycobacterial immunity Inhibition of HIV-1 Particle Assembly by 2′,3′-1152 Cyclic-Nucleotide 3′-Phosphodiesterase MDA5 Governs the Innate Immune Response to 1155 SARS-CoV-2 in Lung Epithelial Cells Reverse genetics with a full-length infectious 1158 cDNA of severe acute respiratory syndrome coronavirus Translation of Genotype to Phenotype by a 1162 Hierarchy of Cell Subsystems Cholesterol 25-hydroxylase suppresses SARS-CoV-2 1165 replication by blocking membrane fusion Influenza Virus Z-RNAs Induce 1168 ZBP1-Mediated Necroptosis HiDeF: 1170 identifying persistent structures in multiscale 'omics data Granule/Nucleolar Protein potentially involved in 5.8s rRNA processing/transport Highlights • IFN-mediated restriction of SARS-CoV-2 relies on a subset of 65 ISGs • ER-and Golgi-resident proteins are enriched amongst the inhibitory ISGs • BST2 inhibits SARS-CoV-2 release and is antagonized by virally-encoded Orf7a • Eight of the ISGs inhibit SARS-CoV-1 and -2 but no other related viruses eTOC blurb Deficient interferon responses to SARS-CoV-2 infection have been associated with severe COVID-19 utilized a gain-of-function screen to identify interferon-stimulated effectors that govern innate immune responses to SARS-CoV-2. These factors could underlie genetic predisposition to severe COVID-19 and can serve as candidates for development of antiviral therapies