IFIT3 Associates with m⁶A-Modified RNA to Restrict Hepatitis C Virus Infection

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 (m⁶A)-modified hepatitis C virus (HCV) genomic RNA and host transcripts to restrict HCV infection. IFIT3 cellular RNA binding sites and m⁶A sites, mapped transcriptome-wide by HyperTRIBE-seq during HCV infection, showed significant overlap. This m⁶A preference was further supported by findings that IFIT3 binding sites significantly overlapped those of established m⁶A-binding proteins; that inhibiting m⁶A installation reduced IFIT3 association with m⁶A-modified HCV RNA and cellular transcripts; that IFIT3 co-purified more efficiently with m⁶A-modified short RNA probes than with unmodified controls; and that mutating m⁶A consensus motifs in the HCV genome reduced IFIT3 association with viral RNA. Structure-function analyses identified two regions required for RNA probe binding: tetratricopeptide repeat domains 1-2 (TPR1-2) 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, TPR1-2 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 m⁶A-linked restriction of late stages of infection, extracellular HCV RNA showed reduced m⁶A and decreased IFIT3 association relative to intracellular RNA. Together, these findings define an m⁶A-linked mechanism by which IFIT3 engages viral RNA and reveal an unexpected role for m⁶A in antiviral effector function.

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 ⁶ 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 ⁶ A as a new regulator of IFIT protein function and broaden our understanding of how RNA modifications shape antiviral restriction.