{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T22:48:22Z","timestamp":1768517302061,"version":"3.49.0"},"posted":{"date-parts":[[2024,12,13]]},"group-title":"Microbiology","reference-count":79,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2024,12,13]],"date-time":"2024-12-13T00:00:00Z","timestamp":1734048000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,12,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>SARS coronavirus 2 (SARS-CoV-2) non-structural protein 14 (Nsp14) possesses an N-terminal exonuclease (ExoN) domain that provides a proofreading function for the viral RNA-dependent RNA polymerase and a C-terminal N7-methyltransferase (N7-MTase) domain that methylates viral mRNA caps. Nsp14 also modulates host functions. This includes the activation of NF-\u03baB and downregulation of interferon alpha\/beta receptor 1 (IFNAR1). Here we demonstrate that Nsp14 exerts broader effects, activating not only NF-\u03baB responses but also ERK, p38 and JNK MAP kinase (MAPK) signaling, promoting cytokine production. Further, Nsp14 downregulates not only IFNAR1 but also IFN-\u03b3 receptor 1 (IFNGR1), impairing cellular responses to both IFN\u03b1 and IFN\u03b3. IFNAR1 and IFNGR1 downregulation is via a lysosomal pathway and also occurs in SARS-CoV-2 infected cells. Analysis of a panel of Nsp14 mutants reveals a consistent pattern. Mutants that disable ExoN function remain active, whereas N7-MTase mutations impair both pro-inflammatory pathway activation and IFN receptor downregulation. Innate immune modulating functions also require the presence of both the ExoN and N7-MTase domains likely reflecting the need for the ExoN domain for N7-MTase activity. We further identify multi-functional host protein Tollip as an Nsp14 interactor. Interaction requires the phosphoinositide-binding C2 domain of Tollip and sequences C-terminal to the C2 domain. Full length Tollip or regions encompassing the Nsp14 interaction domain are sufficient to counteract both Nsp14-mediated and Nsp14-independent activation of NF-\u03baB. Knockdown of Tollip partially reverses IFNAR1 and IFNGR1 downregulation in SARS-CoV-2 infected cells, suggesting relevance of Nsp14-Tollip interaction for Nsp14 innate immune evasion functions.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance<\/jats:title>\n                  <jats:p>SARS-CoV-2 protein Nsp14 both activates NF-\u03baB, which promotes virus replication and inflammation, and downregulates IFNAR1, which can render infected cells resistant to the antiviral effects of IFN-\u03b1\/\u03b2. Our study demonstrates that Nsp14 also activates MAPK signaling and downregulates IFNGR1, causing broader impacts than previously recognized. Data from a panel of Nsp14 mutants suggests a common underlying effect of Nsp14 may be responsible for its multiple innate immune activities. We further describe a novel interaction between Nsp14 and Tollip, a selective autophagy receptor. We show that Tollip expression downregulates Nsp14 activation of NF-\u03baB and that Tollip knockdown reverses IFNAR1 and IFNGR1 downregulation in SARS-CoV-2 infection, suggesting that Tollip functions as a regulator of Nsp14 innate immune modulation.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2024.12.12.628214","type":"posted-content","created":{"date-parts":[[2024,12,14]],"date-time":"2024-12-14T05:20:16Z","timestamp":1734153616000},"source":"Crossref","is-referenced-by-count":0,"title":["SARS-CoV-2 Nsp14 binds Tollip and activates pro-inflammatory pathways while downregulating interferon-\u03b1 and interferon-\u03b3 receptors"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-9537-8423","authenticated-orcid":false,"given":"Naveen","family":"Thakur","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Poushali","family":"Chakraborty","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2785-1572","authenticated-orcid":false,"given":"JoAnn M.","family":"Tufariello","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4195-425X","authenticated-orcid":false,"given":"Christopher F.","family":"Basler","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2024121601502928000_2024.12.12.628214v1.1","unstructured":"Anonymous (2024) WHO COVID-19 dashboard. (World Health Organization)."},{"key":"2024121601502928000_2024.12.12.628214v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.04.011"},{"key":"2024121601502928000_2024.12.12.628214v1.3","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2020.1719902"},{"key":"2024121601502928000_2024.12.12.628214v1.4","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1201130109"},{"key":"2024121601502928000_2024.12.12.628214v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/J.JMB.2020.11.024"},{"key":"2024121601502928000_2024.12.12.628214v1.6","doi-asserted-by":"publisher","DOI":"10.1111\/febs.15815"},{"key":"2024121601502928000_2024.12.12.628214v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2021.101518"},{"key":"2024121601502928000_2024.12.12.628214v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-18463-z"},{"key":"2024121601502928000_2024.12.12.628214v1.9","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1000896"},{"key":"2024121601502928000_2024.12.12.628214v1.10","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01296-07"},{"key":"2024121601502928000_2024.12.12.628214v1.11","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003565"},{"key":"2024121601502928000_2024.12.12.628214v1.12","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1000863"},{"key":"2024121601502928000_2024.12.12.628214v1.13","first-page":"793","article-title":"The mechanism of RNA capping by SARS-CoV-2","volume":"609","year":"2022","journal-title":"Nature"},{"key":"2024121601502928000_2024.12.12.628214v1.14","doi-asserted-by":"crossref","unstructured":"N. S. Ogando et al., Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication. Proc Natl Acad Sci U S A 118 (2021).","DOI":"10.1073\/pnas.2108709118"},{"key":"2024121601502928000_2024.12.12.628214v1.15","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.03662-21"},{"key":"2024121601502928000_2024.12.12.628214v1.16","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00542-16"},{"key":"2024121601502928000_2024.12.12.628214v1.17","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01257-21"},{"key":"2024121601502928000_2024.12.12.628214v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/J.CELL.2020.06.034"},{"key":"2024121601502928000_2024.12.12.628214v1.19","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.01007-23"},{"key":"2024121601502928000_2024.12.12.628214v1.20","doi-asserted-by":"publisher","DOI":"10.1016\/j.cyto.2023.156287"},{"key":"2024121601502928000_2024.12.12.628214v1.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.cca.2020.06.017"},{"key":"2024121601502928000_2024.12.12.628214v1.22","doi-asserted-by":"publisher","DOI":"10.1186\/s12985-022-01814-1"},{"key":"2024121601502928000_2024.12.12.628214v1.23","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-022-00839-1"},{"key":"2024121601502928000_2024.12.12.628214v1.24","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.1680"},{"key":"2024121601502928000_2024.12.12.628214v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/s41594-020-0511-8"},{"key":"2024121601502928000_2024.12.12.628214v1.26","doi-asserted-by":"publisher","DOI":"10.1126\/science.abc8665"},{"key":"2024121601502928000_2024.12.12.628214v1.27","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.80.2.785-793.2006"},{"key":"2024121601502928000_2024.12.12.628214v1.28","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00133-08"},{"key":"2024121601502928000_2024.12.12.628214v1.29","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.00266-21"},{"key":"2024121601502928000_2024.12.12.628214v1.30","doi-asserted-by":"crossref","unstructured":"K. Zhang et al., Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression. Sci Adv 7 (2021).","DOI":"10.1126\/sciadv.abe7386"},{"key":"2024121601502928000_2024.12.12.628214v1.31","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03610-3"},{"key":"2024121601502928000_2024.12.12.628214v1.32","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.10.004"},{"key":"2024121601502928000_2024.12.12.628214v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2020.10.034"},{"key":"2024121601502928000_2024.12.12.628214v1.34","doi-asserted-by":"crossref","unstructured":"C. P. Lapointe , R. Grosely , A. G. Johnson , J. Wang , I. S. Fernandez , J. D. Puglisi , Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation. Proc Natl Acad Sci U S A 118 (2021).","DOI":"10.1073\/pnas.2017715118"},{"key":"2024121601502928000_2024.12.12.628214v1.35","first-page":"253","article-title":"The viral protein NSP1 acts as a ribosome gatekeeper for shutting down host translation and fostering SARS-CoV-2 translation","volume":"27","year":"2020","journal-title":"RNA"},{"key":"2024121601502928000_2024.12.12.628214v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2020.108234"},{"key":"2024121601502928000_2024.12.12.628214v1.37","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2286-9"},{"key":"2024121601502928000_2024.12.12.628214v1.38","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2020.11.115"},{"key":"2024121601502928000_2024.12.12.628214v1.39","doi-asserted-by":"crossref","unstructured":"A. Addetia et al., SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98. mBio 12 (2021).","DOI":"10.1128\/mBio.00065-21"},{"key":"2024121601502928000_2024.12.12.628214v1.40","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2016650117"},{"key":"2024121601502928000_2024.12.12.628214v1.41","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2021.108916"},{"key":"2024121601502928000_2024.12.12.628214v1.42","doi-asserted-by":"crossref","unstructured":"G. Gori Savellini , G. Anichini , C. Gandolfo , M. G. Cusi , Nucleopore Traffic Is Hindered by SARS-CoV-2 ORF6 Protein to Efficiently Suppress IFN-beta and IL-6 Secretion. Viruses 14 (2022).","DOI":"10.3390\/v14061273"},{"key":"2024121601502928000_2024.12.12.628214v1.43","doi-asserted-by":"crossref","unstructured":"J. C. Hsu , M. Laurent-Rolle , J. B. Pawlak , C. B. Wilen , P. Cresswell , Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein. Proc Natl Acad Sci U S A 118 (2021).","DOI":"10.1073\/pnas.2101161118"},{"key":"2024121601502928000_2024.12.12.628214v1.44","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad483"},{"key":"2024121601502928000_2024.12.12.628214v1.45","doi-asserted-by":"crossref","unstructured":"M. Zaffagni , J. M. Harris , I. L. Patop , N. R. Pamudurti , S. Nguyen , S. Kadener , SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome. Elife 11 (2022).","DOI":"10.7554\/eLife.71945"},{"key":"2024121601502928000_2024.12.12.628214v1.46","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2022.1007089"},{"key":"2024121601502928000_2024.12.12.628214v1.47","doi-asserted-by":"publisher","DOI":"10.1038\/s41556-024-01388-w"},{"key":"2024121601502928000_2024.12.12.628214v1.48","doi-asserted-by":"crossref","unstructured":"M. J. Tofaute et al., SARS-CoV-2 NSP14 MTase activity is critical for inducing canonical NF-kappaB activation. Biosci Rep 44 (2024).","DOI":"10.1042\/BSR20231418"},{"key":"2024121601502928000_2024.12.12.628214v1.49","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01475-z"},{"key":"2024121601502928000_2024.12.12.628214v1.50","doi-asserted-by":"publisher","DOI":"10.1186\/s12964-024-01949-4"},{"key":"2024121601502928000_2024.12.12.628214v1.51","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-021-00575-7"},{"key":"2024121601502928000_2024.12.12.628214v1.52","doi-asserted-by":"publisher","DOI":"10.1016\/j.molimm.2024.09.001"},{"key":"2024121601502928000_2024.12.12.628214v1.53","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2020.1780953"},{"key":"2024121601502928000_2024.12.12.628214v1.54","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-17665-9"},{"key":"2024121601502928000_2024.12.12.628214v1.55","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2021.109126"},{"key":"2024121601502928000_2024.12.12.628214v1.56","doi-asserted-by":"publisher","DOI":"10.1007\/s00109-020-01999-4"},{"key":"2024121601502928000_2024.12.12.628214v1.57","doi-asserted-by":"publisher","DOI":"10.1126\/science.abi9310"},{"key":"2024121601502928000_2024.12.12.628214v1.58","doi-asserted-by":"crossref","unstructured":"N. S. Ogando , J. C. Zevenhoven-Dobbe , Y. van der Meer , P. J. Bredenbeek , C. C. Posthuma , E. J. Snijder , The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2. J Virol 94 (2020).","DOI":"10.1128\/JVI.01246-20"},{"key":"2024121601502928000_2024.12.12.628214v1.59","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.388009"},{"key":"2024121601502928000_2024.12.12.628214v1.60","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2006.09.062"},{"key":"2024121601502928000_2024.12.12.628214v1.61","doi-asserted-by":"publisher","DOI":"10.15252\/embj.2023114272"},{"key":"2024121601502928000_2024.12.12.628214v1.62","doi-asserted-by":"publisher","DOI":"10.1016\/j.micinf.2011.08.018"},{"key":"2024121601502928000_2024.12.12.628214v1.63","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M113.484170"},{"key":"2024121601502928000_2024.12.12.628214v1.64","doi-asserted-by":"publisher","DOI":"10.1038\/35014038"},{"key":"2024121601502928000_2024.12.12.628214v1.65","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M109537200"},{"key":"2024121601502928000_2024.12.12.628214v1.66","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.167.2.987"},{"key":"2024121601502928000_2024.12.12.628214v1.67","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2014.05.048"},{"key":"2024121601502928000_2024.12.12.628214v1.68","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0130818"},{"key":"2024121601502928000_2024.12.12.628214v1.69","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00061-13"},{"key":"2024121601502928000_2024.12.12.628214v1.70","doi-asserted-by":"crossref","unstructured":"D. K. Morrison , MAP kinase pathways. Cold Spring Harb Perspect Biol 4 (2012).","DOI":"10.1101\/cshperspect.a011254"},{"key":"2024121601502928000_2024.12.12.628214v1.71","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2023.104960"},{"key":"2024121601502928000_2024.12.12.628214v1.72","doi-asserted-by":"publisher","DOI":"10.1038\/s41577-020-0346-x"},{"key":"2024121601502928000_2024.12.12.628214v1.73","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-022-00713-0"},{"key":"2024121601502928000_2024.12.12.628214v1.74","doi-asserted-by":"publisher","DOI":"10.1111\/imr.13113"},{"key":"2024121601502928000_2024.12.12.628214v1.75","doi-asserted-by":"publisher","DOI":"10.1247\/csf.23.33"},{"key":"2024121601502928000_2024.12.12.628214v1.76","doi-asserted-by":"publisher","DOI":"10.1101\/2024.07.10.602835"},{"key":"2024121601502928000_2024.12.12.628214v1.77","doi-asserted-by":"publisher","DOI":"10.1189\/jlb.0309189"},{"key":"2024121601502928000_2024.12.12.628214v1.78","doi-asserted-by":"publisher","DOI":"10.1016\/j.molimm.2004.03.009"},{"key":"2024121601502928000_2024.12.12.628214v1.79","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-32957-y"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2024.12.12.628214","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T07:40:04Z","timestamp":1768462804000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2024.12.12.628214"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,12,13]]},"references-count":79,"URL":"https:\/\/doi.org\/10.1101\/2024.12.12.628214","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1128\/mbio.01071-25","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2024,12,13]]},"subtype":"preprint"}}