Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities

•Numerous SARS-CoV-2 proteins synergize to suppress immune sensing and signaling•Nsp14 targets IFNAR1 for lysosomal degradation•ORF3a ORF7a block autophagy by different mechanisms•Synergistic treatment with IFN-γ -λ1 is highly effective against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate responses but may still be vulnerable some. Here, we systematically analyze the impact of on interferon (IFN) autophagy. We show that counteract anti-viral responses. For example, Nsp14 type I IFN receptor degradation, ORF3a prevents fusion autophagosomes lysosomes, interferes autophagosome acidification. Most activities are evolutionarily conserved. However, Nsp15 antagonizes signaling less efficiently than orthologs closely related RaTG13-CoV SARS-CoV-1. Overall, more II or III signaling, infection experiments confirm potent inhibition -λ1. Our results define repertoire selected mechanisms antagonists also reveal vulnerability help develop safe approaches. The severe a zoonotic, novel emerged at end 2019 (Andersen et al., 2020Andersen K.G. Rambaut A. Lipkin W.I. Holmes E.C. Garry R.F. proximal origin SARS-CoV-2.Nat. Med. 2020; 26: 450-452Crossref PubMed Scopus (2692) Google Scholar; Wu 2020Wu F. Zhao S. Yu B. Chen Y.M. Wang W. Song Z.G. Hu Y. Tao Z.W. Tian J.H. Pei Y.Y. al.A new associated human disease in China.Nature. 579: 265-269Crossref (6145) Zhou 2020aZhou P. Yang X. X.G. Zhang L. Si H.R. Zhu Li Huang C.-L. pneumonia outbreak probable bat origin.Nature. 270-273Crossref (12018) Scholar). Infection causes (COVID-19) (Huang 2020Huang C. Ren J. Fan G. Xu Gu al.Clinical features patients infected Wuhan.Lancet. 395: 497-506Abstract Full Text PDF (27324) virus rapidly spread all over world owing its higher transmission rates (Ferretti 2020Ferretti Wymant Kendall M. Nurtay Abeler-Dörner Parker Bonsall D. Fraser Quantifying suggests epidemic control digital contact tracing.Science. 368: eabb6936Crossref (1384) Scholar), as well lower morbidity case fatality (CFRs; 3%–4%) (WHO, 2020WHOInfluenza COVID-19—similarities differences. World Health Organization, 2020Google Scholar) those previous coronaviruses such SARS-CoV-1 (CFR, 11%) middle east (MERS-CoV; CFR, 35%) (Chan-Yeung Xu, 2003Chan-Yeung R.-H. SARS: epidemiology.Respirology. 2003; 8: S9-S14Crossref (424) Petersen 2020Petersen E. Koopmans Go U. Hamer D.H. Petrosillo N. Castelli Storgaard Al Khalili Simonsen Comparing SARS-CoV influenza pandemics.Lancet Infect. Dis. 20: e238-e244Abstract (663) Zumla 2015Zumla Hui D.S. Perlman Middle East syndrome.Lancet. 2015; 386: 995-1007Abstract (761) pathogenicity much “common cold” CoVs HKU1 229E (Corman 2018Corman V.M. Muth Niemeyer Drosten Hosts Sources Endemic Human Coronaviruses.Adv. Virus Res. 2018; 100: 163-188Crossref (530) date, has caused 3 million deaths (https://coronavirus.jhu.edu/map.html). Upon target cell, recognized sensors like RIG-like receptors (RLRs) (Kell Gale, 2015Kell A.M. Gale Jr., RIG-I RNA recognition.Virology. 479–480: 110-121Crossref (265) Subsequently, these activate cell-intrinsic defenses (hereafter referred system) (Frieman 2008Frieman Heise Baric R. SARS immunity.Virus 2008; 133: 101-112Crossref (181) Totura Baric, 2012Totura A.L. R.S. pathogenesis: host viral antagonism interferon.Curr. Opin. Virol. 2012; 2: 264-275Crossref (296) (Takeuchi Akira, 2009Takeuchi O. Akira Innate immunity infection.Immunol. Rev. 2009; 227: 75-86Crossref (906) Activation RLRs induces cascades ultimately lead release IFNs other pro-inflammatory cytokines induction effectors (Koepke 2021Koepke Gack M.U. Sparrer K.M. antiviral TRIM proteins.Curr. Microbiol. 2021; 59: 50-57Crossref (30) Released subsequently neighboring cells induce an transcriptional response eventually limit (van Gent 2018van K.M.J. their roles defenses.Annu. 5: 385-405Crossref (125) Gack, 2015Sparrer Intracellular detection nucleic acids.Curr. 1-9Crossref (82) Besides cytokine-mediated cascades, able directly eliminate invading pathogens activated, Autophagy capable targeting components even whole viruses degradation (Choi 2018Choi Bowman J.W. Jung J.U. during - double-edged sword.Nat. 16: 341-354Crossref (353) 2017Sparrer Gableske Zurenski M.A. Z.M. Baumgart G.J. Kato Pacheco-Rodriguez Liang Pornillos al.TRIM23 mediates virus-induced via activation TBK1.Nat. 2017; 1543-1557Crossref (118) Scholar, 2018Sparrer proteins: New players autophagy.PLoS Pathog. 14: e1006787Crossref (31) Eventually, recruits stimulates adaptive system, facilitating elimination (Iwasaki Medzhitov, 2010Iwasaki Medzhitov Regulation system.Science. 2010; 327: 291-295Crossref (1502) Iwasaki 2015Iwasaki Control system.Nat. Immunol. 343-353Crossref (1120) Inborn defects auto-antibodies high frequencies COVID-19, suggesting defense play major role (Bastard 2020Bastard Rosen L.B. Q. Michailidis Hoffmann H.-H. Dorgham K. Philippot Rosain Béziat V. al.HGID LabNIAID-USUHS Immune Response COVID GroupCOVID CliniciansCOVID-STORM CliniciansImagine GroupFrench Cohort Study GroupMilieu Intérieur ConsortiumCoV-Contact CohortAmsterdam UMC Covid-19 BiobankCOVID Genetic EffortAutoantibodies life-threatening COVID-19.Science. 370: 80Crossref (1277) 2020Zhang Bastard Liu Z. Le Pen Moncada-Velez Ogishi Sabli I.K.D. Hodeib Korol al.Inborn errors eabd4570Crossref (1127) Notably, infections numbers subclinical (up 80%; WHO, Indeed, recent evidence sensitive toward vitro (Lokugamage 2020Lokugamage Hage de Vries Valero-Jimenez Schindewolf Dittmann Rajsbaum Menachery Type susceptibility distinguishes from SARS-CoV.bioRxiv. https://doi.org/10.1101/2020.03.07.982264Crossref (0) underlying reasons differences between poorly understood. Recent reports suggest manipulates (Blanco-Melo 2020Blanco-Melo Nilsson-Payant B.E. W.C. Uhl Hoagland Møller Jordan T.X. Oishi Panis Sachs al.Imbalanced Host Drives Development COVID-19.Cell. 181: 1036-1045.e9Abstract (2262) Stukalov 2020Stukalov Girault Grass Bergant Karayel Urban Haas D.A. Oubraham al.Multi-level proteomics reveals host-perturbation strategies https://doi.org/10.1101/2020.06.17.156455Crossref ORF3b yet unknown mechanism (Konno 2020Konno Kimura I. Uriu Fukushi Irie T. Koyanagi Sauter Gifford R.J. Nakagawa Sato antagonist whose activity further increased naturally occurring elongation variant.bioRxiv. https://doi.org/10.1101/2020.05.11.088179Crossref In addition, non-structural protein 1 (Nsp1) shuts down cellular translation including (Schubert 2020Schubert Karousis E.D. Jomaa Scaiola Echeverria Gurzeler L.A. Leibundgut Thiel Mühlemann Ban Nsp1 binds ribosomal mRNA channel inhibit translation.Nat. Struct. Mol. Biol. 27: 959-966Crossref (242) Thoms 2020Thoms Buschauer Ameismeier Koepke Denk Hirschenberger Kratzat H. Hayn Mackens-Kiani Cheng al.Structural basis translational shutdown evasion SARS-CoV-2.Science. 369: 1249-1255Crossref Analysis interplay IFN-β revealed least 8 about 30 interfere (Lei 2020Lei Dong Ma Xiao al.Activation Commun. 11: 3810Crossref (497) Xia 2020Xia Cao Xie J.Y.-C. V.D. Shi P.-Y. Evasion Interferon SARS-CoV-2.Cell Rep. 33: 108234Abstract (406) so far only was analyzed some detail, our knowledge how being complete. Currently, explored clinical trials (Fragkou 2020Fragkou P.C. Belhadi Peiffer-Smadja Moschopoulos C.D. Lescure F.X. Janocha Karofylakis Yazdanpanah Mentré Skevaki al.Review currently testing prevention COVID-19.Clin. 988-998Abstract (68) Sallard 2020Sallard Mentre interferons potential COVID-19.Antiviral 178: 104791Crossref (340) Studies assessing effects IFNs, often combination treatment, promising Hung 2020Hung I.F.N. Lung K.C. Tso E.Y.K. Chung T.W.H. Chu M.Y. Ng Lo Chan Tam A.R. al.Triple beta-1b, lopinavir-ritonavir, ribavirin admitted hospital COVID-19: open-label, randomised, phase trial.Lancet. 1695-1704Abstract (988) Shalhoub, 2020Shalhoub beta-1b COVID-19.Lancet. 1670-1671Abstract (47) Thorlund 2020Thorlund Dron Park Hsu Forrest J.I. Mills E.J. A real-time dashboard COVID-19.Lancet Digit. Health. e286-e287Abstract (148) 2020Wang Zhan Hou Qiu Zou Wan al.Retrospective Multicenter Shows Early Therapy Is Associated Favorable Clinical Responses COVID-19 Patients.Cell Microbe. 28: 455-464.e2Abstract (173) 2020bZhou Shannon C.P. Wei X.S. Xiang Z.H. Tebbutt S.J. Kollmann T.R. Fish E.N. Interferon-alpha 2b Treatment COVID-19.Front. 40: 578-588Google receiving therapy suffer side psychological symptoms depression (Fried, 2002Fried M.W. Side hepatitis C management.Hepatology. 2002; 36: S237-S244Crossref Neri 2010Neri Bertino Petralia Giancarlo Rizzotto Calvagno G.S. Mauceri Abate Boemi Di Pino Multidisciplinary Therapeutic Approach Reducing Risk Psychiatric Effects Patients With Chronic Hepatitis Treated Pegylated α Ribavirin.J. Clin. Gastroenterol. 44 (17): e210Crossref (34) van Zonneveld 2005van Flink H.J. Verhey Senturk Zeuzem Akarca U.S. Cakaloglu Simon So T.M.K. Gerken al.HBV 99-01 GroupThe safety pegylated alpha-2b chronic B: predictive factors dose reduction discontinuation.Aliment. Pharmacol. Ther. 2005; 21: 1163-1171Crossref (63) Novel system manner while minimizing overall inflammation detrimental needed (Hung Thus, understanding give valuable clues vulnerabilities might exploited therapeutic control. 29 total SARS-CoV-2-encoded (Gordon 2020Gordon D.E. Jang G.M. Bouhaddou Obernier White O’Meara M.J. Rezelj V.V. Guo J.Z. Swaney D.L. interaction map drug repurposing.Nature. 583: 459-468Crossref (2261) Hachim 2020Hachim Kavian Cohen C.A. Chin A.W.H. D.K.W. Mok C.K.P. Tsang O.T.Y. Yeung Y.C. Perera R.A.P.M. Poon L.L.M. al.ORF8 antibodies accurate serological markers early late infection.Nat. 1293-1301Crossref (127) following branches system: induction, IFN/pro-inflammatory cytokine identified Nsp1, Nsp3, Nsp5, Nsp10, Nsp13, Nsp14, ORF3a, ORF6, ORF7a, ORF7b encoded SARS-CoV-2. Interference achieved diverse, synergistic ranging downregulation (IFNAR1) expression blockage interference autophagosome-lysosome acidification autophagosomes, respectively. function conserved SARS-CoV-1, SARS-CoV-2, RaTG13-CoV, one notable exception: significantly suppressing Altogether, analyses IFN-λ1 pathways antagonized least. Consequently, two were combined very low doses potentiated individual effect could improved anti-inflammatory activation. To examine immunity, used Strep II-tagged constructs coding 28 known (Nsp1, Nsp2, Nsp4, Nsp6, Nsp7, Nsp8, Nsp9, Nsp11, Nsp12, Nsp15, Nsp16, S, ORF3c, E, M, ORF7b, ORF8, ORF9b, N, ORF9c, ORF10) (Figure 1A). examined untagged Nsp3. Expression confirmed western blotting immunofluorescence (Figures S1A S1B). i.e., 1B S1C), 1C S1D), 1D S1E), quantitative reporter assays. All assays normalized cell viability S1F). Induction (IFN-α IFN-β) monitored using firefly luciferase controlled full IFN-α4 promotors (IFNA4 IFNB1) isolated binding sites transcription IRF3 nuclear factor κB (NF-κB) 1B). HEK293T Sendai virus, mimicking RLR Activity promotors, sites, NF-κB strongly impaired Stimulation IFN-α2, IFN-β, IFN-λ1, culminates genes containing element (ISRE) (Sparrer gamma activated sequence (GAS)-containing promotors. Pro-inflammatory (tumor necrosis alpha [TNF-α] IL-1α) promotor. Signaling (IFN-α2 IFN-β), (IFN-γ), (IFN-λ1), (TNF-α quantified reporters respective S1D). IFN-α2 ISRE promotor repressed similar set interfered average remaining pathway, calculated aggregated mean left presence proteins, showed efficiency which 1C). (average pathway 82% 89%, respectively) 22% 47% IFN-β). levels SARS-CoV-2-protein-expressing membrane association stably expressed GFP-LC3B S1E). During autophagy, cytoplasmic MAP1LC3B (LC3B) proteolytically processed lipidated (LC3B-II) decorate autophagosomal membranes (Klionsky 2016Klionsky D.J. Abdelmohsen Abe Abedin Abeliovich Arozena A.A. Adachi Adams C.M. P.D. Adeli al.Guidelines use interpretation monitoring (3rd edition).Autophagy. 2016; 12: 1-222Crossref (3972) 2020Koepke Winter Grenzner Regensburger Engelhart der Merwe J.A. Krebs Blum Kirchhoff An method high-throughput quantification mammalian cells.Sci. 10: 12241Crossref (10) Autophagosome under basal conditions either novo turnover 1D). differentiate processes, treated saturating amounts Bafilomycin A1, inhibits autophagic turnover. increase reduced compared non-blocking 1D), indicating turnover, rather it. Blockage co-expression induced death, responsible number autophagosomes. Unexpectedly, consistently reduced, Taken together, analysis antagonize ORF7b), (Nsp15, ORF7a). counteracted aimed identifying steps targeted pathways. focused top five inhibitors according Figures 1D. removed analysis, it general (Thoms overexpressing ORF7b. namely, STAT1 STAT2 2A), phosphorylation status. Basal not affected any tested 2B; S2A S2B). accumulates 2B 2D). tendency observed phospho-STAT2 S2C). Neither ORF6 nor 2B–2D This finding agrees (Kimura 2021Kimura Konno Sarbecovirus Proteins Hamper Blocking Nuclear Export.Cell 34: 108916Abstract (28) Lei Yuen 2020Yuen C.K. Lam J.Y. Wong W.M. Mak L.F. Cai J.P. Jin D.Y. K.K.W. J.F.W. al.SARS-CoV-2 nsp13, nsp14, nsp15 orf6 antagonists.Emerg. Microbes 9: 1418-1428Crossref (268) trafficking factors. (to lesser extent) endogenous 2C). decreased upon overexpression specifically level 2E 2F), remained unchanged 2G). seems modified post-translational modifications, high-molecular-weight isoforms detectable (HMW; Figure 2E). Influenza hemagglutinin (HA) positive (Xia 2016Xia Vijayan Pritzl C.J. Fuchs S.Y. McDermott A.B. Hahm Hemagglutinin Antagonizes Inducing Degradation Receptor 1.J. 90: 2403-2417Crossref (59) Inhibition A1 absence did affect S2D). rescued addition required Nsp14-mediated 2H). data demonstrate cascade levels. Mechanistically, essential IFNAR1, thereby preventing STAT LC3B-II decorated p62 degraded (autophagic turnover; 3A). 5 autophagy-modulating Levels beclin-1 ULK1, parts core machinery initiation, constant 3B, S2E, S2F). Overexpression led slight consistent decrease LCB3-II accumulation p62, blocks 3C 3D). line this finding, GFP-LC3B-puncta (= autophagosomes) per HeLa almost 0 3E 3F). LC3B 4- 7-fold 3C), whereas approximately 1.5-fold result indicates three 10-fold Curiously, although M altered 3B–3D). resulted perinuclear space, normally distributed As asked phenotype dominant du