{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:45:53Z","timestamp":1785415553762,"version":"3.56.0"},"posted":{"date-parts":[[2026,3,26]]},"group-title":"Immunology","reference-count":69,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T00:00:00Z","timestamp":1774483200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"id":[{"id":"https:\/\/ror.org\/01d35cw23","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01AI125416"],"award-info":[{"award-number":["R01AI125416"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01CA258589"],"award-info":[{"award-number":["R01CA258589"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T32CA009111"],"award-info":[{"award-number":["T32CA009111"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/02e463172","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000928","name":"Welch Foundation","doi-asserted-by":"crossref","award":["I-2115--20220331"],"award-info":[{"award-number":["I-2115--20220331"]}],"id":[{"id":"10.13039\/100000928","id-type":"DOI","asserted-by":"crossref"}]},{"award":["RP210041"],"award-info":[{"award-number":["RP210041"]}],"id":[{"id":"https:\/\/ror.org\/0003xa228","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,26]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Interferon-induced proteins with tetratricopeptide repeats (IFITs) are RNA-binding effectors that restrict infection by diverse RNA viruses. Among the IFIT family, how IFIT3 recognizes RNA remains the least understood. Here, we identify IFIT3 as preferentially associating with N6-methyladenosine (m\u2076A)\u2013modified hepatitis C virus (HCV) genomic RNA and host transcripts to restrict HCV infection. IFIT3 cellular RNA binding sites and m\u2076A sites, mapped transcriptome-wide by HyperTRIBE-seq during HCV infection, showed significant overlap. This m\u2076A preference was further supported by findings that IFIT3 binding sites significantly overlapped those of established m\u2076A-binding proteins; that inhibiting m\u2076A installation reduced IFIT3 association with m\u2076A-modified HCV RNA and cellular transcripts; that IFIT3 co-purified more efficiently with m\u2076A-modified short RNA probes than with unmodified controls; and that mutating m\u2076A consensus motifs in the HCV genome reduced IFIT3 association with viral RNA. Structure\u2013function analyses identified two regions required for RNA probe binding: tetratricopeptide repeat domains 1\u20132 (TPR1\u20132) and a previously uncharacterized predicted helical hairpin between TPRs 6 and 7. In infected cells, the helical hairpin was required for IFIT3 association with HCV RNA but dispensable for interactions with other IFIT proteins. Conversely, TPR1\u20132 was dispensable for HCV RNA binding but essential for IFIT2 interaction, establishing that these functions are structurally separable. Loss of either region diminished antiviral activity, as indicated by increased levels of HCV RNA in clarified supernatants. Consistent with models of m\u2076A-linked restriction of late stages of infection, extracellular HCV RNA showed reduced m\u2076A and decreased IFIT3 association relative to intracellular RNA. Together, these findings define an m\u2076A-linked mechanism by which IFIT3 engages viral RNA and reveal an unexpected role for m\u2076A in antiviral effector function.<\/jats:p>\n                <jats:sec>\n                  <jats:title>SIGNIFICANCE<\/jats:title>\n                  <jats:p>\n                    RNA-binding proteins are critical effectors of antiviral defense, yet for many of these proteins the mechanisms of viral RNA recognition remain unclear. Here, we show that m\n                    <jats:sup>6<\/jats:sup>\n                    A modification on both host and viral RNA promotes recognition by the interferon stimulated gene, IFIT3. RNA recognition did not require interaction with IFIT1 or IFIT2, although IFIT3 antiviral function required both RNA binding and interaction with IFIT2. These findings identify m\n                    <jats:sup>6<\/jats:sup>\n                    A as a new regulator of IFIT protein function and broaden our understanding of how RNA modifications shape antiviral restriction.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.25.714224","type":"posted-content","created":{"date-parts":[[2026,3,27]],"date-time":"2026-03-27T05:25:39Z","timestamp":1774589139000},"source":"Crossref","is-referenced-by-count":0,"title":["IFIT3 Associates with m\u2076A-Modified RNA to Restrict Hepatitis C Virus Infection"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3813-9254","authenticated-orcid":false,"given":"Matthew","family":"Thompson","sequence":"first","affiliation":[{"name":"Department of Integrated Immunobiology, Duke University School of Medicine, Durham, NC 27710, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Moonhee","family":"Park","sequence":"additional","affiliation":[{"name":"Department of Integrated Immunobiology, Duke University School of Medicine, Durham, NC 27710, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-4098-2334","authenticated-orcid":false,"given":"Netanya S.","family":"Schlamowitz","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Department of Biophysics, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew R.","family":"Lanahan","sequence":"additional","affiliation":[{"name":"Department of Integrated Immunobiology, Duke University School of Medicine, Durham, NC 27710, 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