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

Area of Science:

• Virology
• Immunology
• Molecular Biology

Background:

• Interferon-induced proteins with tetratricopeptide repeats (IFITs) are crucial for antiviral defense against RNA viruses.
• The specific mechanisms by which IFIT3 recognizes viral RNA are not well understood.

Purpose of the Study:

• To elucidate how IFIT3 recognizes and binds RNA to restrict hepatitis C virus (HCV) infection.
• To investigate the role of RNA modifications, specifically N6-methyladenosine (m⁶A), in IFIT3-mediated antiviral activity.

Main Methods:

• Transcriptome-wide mapping of IFIT3 and m⁶A sites using HyperTRIBE-seq during HCV infection.
• Biochemical assays including RNA probe binding and co-purification experiments.
• Structure-function analyses to identify key RNA-binding regions of IFIT3.

Main Results:

• IFIT3 preferentially binds to m⁶A-modified HCV genomic RNA and host transcripts.
• Significant overlap was observed between IFIT3 binding sites and m⁶A sites.
• Specific structural regions of IFIT3, including TPR1-2 and a helical hairpin, were identified as essential for RNA binding and antiviral function.
• IFIT3's antiviral activity against HCV requires both RNA binding and interaction with IFIT2.

Conclusions:

• N6-methyladenosine (m⁶A) modification on RNA promotes recognition by IFIT3, highlighting a novel role for RNA modifications in antiviral immunity.
• IFIT3 restricts HCV infection through an m⁶A-dependent mechanism, independent of IFIT1 but requiring IFIT2 interaction for full antiviral function.
• These findings expand the understanding of RNA modifications in regulating antiviral effector protein function.