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Dysregulation of SGs has been associated with cancer, neurodegeneration, and viral infection. GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is a central SG component that regulates granule assembly and dynamics through protein-RNA and protein\u2013protein interactions (PPIs). During viral infection, SGs can be hijacked to enhance viral replication and suppress host defense. In this study, we show that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N) binds G3BP1 and localizes to G3BP1-positive SGs in infected cells. We hypothesize that small-molecule ligands targeting G3BP1 can disrupt the G3BP1-N interaction and reduce viral replication. To test this, we developed a robust, miniaturized, 384-well time-resolved fluorescence resonance energy transfer (TR-FRET) assay for high-throughput screening (HTS). Screening a library of 2560 FDA-approved drugs yielded 17 hits. Subsequent counterscreening, validation, and biophysical binding studies identified 7 PPI inhibitors with IC50 values ranging from 0.8 to 31 \u03bcM. In cell-based assays, two compounds\u2500dexlansoprazole and citalopram\u2500disrupted the G3BP1-N interaction with minimal cytotoxicity. Both compounds showed antiviral activity in human lung epithelial cells infected with either the WA strain or an Omicron (BA.1) strain of SARS-CoV-2. This work establishes a strategy to modulate SGs by targeting a host\u2013virus PPI, highlighting G3BP1 as a potential therapeutic node for antiviral intervention.<\/jats:p>","DOI":"10.1021\/acsinfecdis.5c01026","type":"journal-article","created":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T10:19:31Z","timestamp":1775816371000},"page":"3322-3334","source":"Crossref","is-referenced-by-count":2,"title":["Targeting G3BP1-Mediated\nStress Granules to Suppress\nSARS-CoV-2 Replication"],"prefix":"10.1021","volume":"12","author":[{"given":"Rukayat","family":"Aromokeye","sequence":"first","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisha","family":"Kuang","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martha","family":"Ackerman-Berrier","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Savio","family":"Cardoza","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cameron","family":"Holberg","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megan","family":"Carver","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunlong","family":"Ma","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anil Mathew","family":"Tharappel","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongmin","family":"Li","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]},{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]},{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]},{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Wang","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gregory R. J.","family":"Thatcher","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]},{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]},{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5498-6694","authenticated-orcid":true,"given":"Haining","family":"Zhu","sequence":"additional","affiliation":[{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]},{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]},{"name":"University of Arizona , , , ,","place":["Tucson, Arizona, United States, 85721"]},{"name":"Research Service , , , ,","place":["Tucson, Arizona, United States, 85713"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,4,10]]},"reference":[{"key":"2026100909101207700_cit1","doi-asserted-by":"publisher","first-page":"18886","DOI":"10.1074\/jbc.M117.800706","article-title":"Histone\narginine demethylase JMJD6 is linked to stress granule assembly through\ndemethylation of the stress granule-nucleating protein G3BP1","volume":"292","author":"Tsai","year":"2017","journal-title":"J. Biol. Chem."},{"key":"2026100909101207700_cit2","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1083\/jcb.201408092","article-title":"G3BP1 promotes stress-induced RNA granule interactions to preserve\npolyadenylated mRNA","volume":"209","author":"Aulas","year":"2015","journal-title":"J. Cell Biol."},{"key":"2026100909101207700_cit3","doi-asserted-by":"publisher","first-page":"11056","DOI":"10.1021\/jacs.3c00165","article-title":"Global Discovery of Covalent Modulators of Ribonucleoprotein Granules","volume":"145","author":"Ciancone","year":"2023","journal-title":"J. Am. Chem. Soc."},{"key":"2026100909101207700_cit4","doi-asserted-by":"publisher","first-page":"361","DOI":"10.1083\/jcb.201302044","article-title":"Stress granules\nas crucibles of ALS pathogenesis","volume":"201","author":"Li","year":"2013","journal-title":"J. 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Cell.\nNeurosci."},{"key":"2026100909101207700_cit8","doi-asserted-by":"publisher","first-page":"649","DOI":"10.1038\/s41583-019-0222-5","article-title":"Stress granules and\nneurodegeneration","volume":"20","author":"Wolozin","year":"2019","journal-title":"Nat. Rev. Neurosci."},{"key":"2026100909101207700_cit9","doi-asserted-by":"publisher","first-page":"791074","DOI":"10.3389\/fphar.2022.791074","article-title":"The Association Between\nProton Pump Inhibitors and COVID-19 is Confounded\nby Hyperglycemia in a Population-Based Study","volume":"13","author":"Shafrir","year":"2022","journal-title":"Front. 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Adv."},{"key":"2026100909101207700_cit26","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1007842","article-title":"Separate domains of G3BP promote efficient clustering of alphavirus\nreplication complexes and recruitment of the translation initiation\nmachinery","volume":"15","author":"Gotte","year":"2019","journal-title":"PLoS Pathog."},{"key":"2026100909101207700_cit27","doi-asserted-by":"publisher","first-page":"10873","DOI":"10.1128\/JVI.01506-12","article-title":"Chikungunya virus nsP3 blocks stress granule assembly\nby recruitment\nof G3BP into cytoplasmic foci","volume":"86","author":"Fros","year":"2012","journal-title":"J. Virol."},{"key":"2026100909101207700_cit28","doi-asserted-by":"publisher","first-page":"160078","DOI":"10.1098\/rsob.160078","article-title":"Combined\nstructural, biochemical\nand cellular evidence demonstrates that both FGDF motifs in alphavirus\nnsP3 are required for efficient replication","volume":"6","author":"Schulte","year":"2016","journal-title":"Open Biol."},{"key":"2026100909101207700_cit29","doi-asserted-by":"publisher","first-page":"48","DOI":"10.1016\/j.arcmed.2020.10.002","article-title":"Role of Chikungunya\nnsP3 in Regulating G3BP1 Activity, Stress Granule Formation and Drug\nEfficacy","volume":"52","author":"Lu","year":"2021","journal-title":"Arch. Med. Res."},{"key":"2026100909101207700_cit30","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0005775","article-title":"Zika virus\ninhibits\neIF2alpha-dependent stress granule assembly","volume":"11","author":"Amorim","year":"2017","journal-title":"PLoS Negl Trop Dis"},{"key":"2026100909101207700_cit31","doi-asserted-by":"publisher","first-page":"2991","DOI":"10.1007\/s00705-015-2578-9","article-title":"Dengue\nvirus infection induces formation of G3BP1 granules in human\nlung epithelial cells","volume":"160","author":"Xia","year":"2015","journal-title":"Arch. Virol."},{"key":"2026100909101207700_cit32","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.00520-19","article-title":"Zika Virus Subverts\nStress Granules To Promote and\nRestrict Viral Gene Expression","volume":"93","author":"Bonenfant","year":"2019","journal-title":"J. Virol."},{"key":"2026100909101207700_cit33","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1004659","article-title":"Viral and cellular proteins containing FGDF\nmotifs bind G3BP to block\nstress granule formation","volume":"11","author":"Panas","year":"2015","journal-title":"PLoS Pathog."},{"key":"2026100909101207700_cit34","doi-asserted-by":"publisher","first-page":"3528","DOI":"10.1128\/JVI.06713-11","article-title":"Herpes simplex\nvirus 1 tegument protein US11 downmodulates the RLR signaling pathway\nvia direct interaction with RIG-I and MDA-5","volume":"86","author":"Xing","year":"2012","journal-title":"J. Virol."},{"key":"2026100909101207700_cit35","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.02246-18","article-title":"The US11\nGene of Herpes Simplex Virus 1 Promotes Neuroinvasion and Periocular\nReplication following Corneal Infection","volume":"93","author":"Charron","year":"2019","journal-title":"J.\nVirol."},{"key":"2026100909101207700_cit36","doi-asserted-by":"publisher","first-page":"649","DOI":"10.1177\/1040638713501675","article-title":"Emergence of Porcine epidemic diarrhea virus\nin the United States:\nclinical signs, lesions, and viral genomic sequences","volume":"25","author":"Stevenson","year":"2013","journal-title":"J. Vet. Diagn. Invest."},{"key":"2026100909101207700_cit37","doi-asserted-by":"publisher","first-page":"108392","DOI":"10.1016\/j.vetmic.2019.108392","article-title":"GTPase-activating protein-binding\nprotein 1 (G3BP1) plays an antiviral\nrole against porcine epidemic diarrhea virus","volume":"236","author":"Pandey","year":"2019","journal-title":"Vet. Microbiol."},{"key":"2026100909101207700_cit38","doi-asserted-by":"publisher","first-page":"449","DOI":"10.3390\/v15020449","article-title":"Pro-Viral\nand Anti-Viral Roles of\nthe RNA-Binding Protein G3BP1","volume":"15","author":"Jayabalan","year":"2023","journal-title":"Viruses"},{"key":"2026100909101207700_cit39","doi-asserted-by":"publisher","DOI":"10.1101\/2024.07.29.605725","article-title":"Formation of stress granules and\nnon-canonical survival responses in arsenite-exposed cells","author":"Hirano","year":"2024","journal-title":"bioRxiv"},{"key":"2026100909101207700_cit40","doi-asserted-by":"publisher","first-page":"523","DOI":"10.3390\/v1030523","article-title":"Activation of the Antiviral Kinase PKR and Viral Countermeasures","volume":"1","author":"Dauber","year":"2009","journal-title":"Viruses"},{"key":"2026100909101207700_cit41","doi-asserted-by":"publisher","first-page":"112","DOI":"10.1186\/s12985-020-01362-6","article-title":"The role of host eIF2alpha\nin viral infection","volume":"17","author":"Liu","year":"2020","journal-title":"Virol. J."},{"key":"2026100909101207700_cit42","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1038\/s41421-021-00275-0","article-title":"SARS-CoV-2\nnucleocapsid protein impairs stress granule formation to promote viral\nreplication","volume":"7","author":"Zheng","year":"2021","journal-title":"Cell Discov"},{"key":"2026100909101207700_cit43","article-title":"HTS Assay Validation","volume-title":"Assay Guidance Manual","author":"Iversen","year":"2012"},{"key":"2026100909101207700_cit44","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.202308083","article-title":"Identification of small molecule inhibitors of G3BP-driven\nstress granule formation","volume":"223","author":"Freibaum","year":"2024","journal-title":"J. Cell Biol."},{"key":"2026100909101207700_cit45","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1009100","article-title":"Structural basis of RNA recognition by the\nSARS-CoV-2 nucleocapsid\nphosphoprotein","volume":"16","author":"Dinesh","year":"2020","journal-title":"PLoS Pathog."},{"key":"2026100909101207700_cit46","doi-asserted-by":"publisher","first-page":"823","DOI":"10.1021\/acs.biochem.4c00536","article-title":"Disruption of molecular interactions between G3BP1\nstress granule\nhost protein and nucleocapsid (NTD-N) protein impedes SARS-CoV-2 virus\nreplication","volume":"64","author":"Dhaka","year":"2024","journal-title":"Biochemistry"},{"key":"2026100909101207700_cit47","doi-asserted-by":"publisher","DOI":"10.1101\/2020.04.03.024257","article-title":"SARS-CoV-2 and SARS-CoV differ in their cell tropism\nand drug sensitivity\nprofiles","author":"Bojkova","year":"2020","journal-title":"bioRxiv"},{"key":"2026100909101207700_cit48","doi-asserted-by":"publisher","first-page":"2452","DOI":"10.1007\/s10620-020-06430-z","article-title":"Repurposed GI Drugs in the Treatment\nof COVID-19","volume":"65","author":"Aguila","year":"2020","journal-title":"Dig. Dis. Sci."},{"key":"2026100909101207700_cit49","doi-asserted-by":"publisher","first-page":"755600","DOI":"10.3389\/fphar.2021.755600","article-title":"Antidepressant and Antipsychotic Drugs Reduce Viral\nInfection by SARS-CoV-2 and Fluoxetine Shows Antiviral Activity Against\nthe Novel Variants in vitro","volume":"12","author":"Fred","year":"2022","journal-title":"Front. Pharmacol."},{"key":"2026100909101207700_cit50","doi-asserted-by":"publisher","first-page":"e42","DOI":"10.1016\/S2214-109X(21)00448-4","article-title":"Effect of early treatment with fluvoxamine on risk\nof emergency care\nand hospitalisation among patients with COVID-19: the TOGETHER randomised,\nplatform clinical trial","volume":"10","author":"Reis","year":"2022","journal-title":"Lancet Glob Health"},{"key":"2026100909101207700_cit51","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1177\/108705719900400206","article-title":"A Simple\nStatistical Parameter for\nUse in Evaluation and Validation of High Throughput Screening Assays","volume":"4","author":"Zhang","year":"1999","journal-title":"J. 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