Swine TRIM25 restricts vesicular stomatitis virus replication by activating type I interferon signaling and binding viral RNA

Importance: Vesicular stomatitis virus is a zoonotic rhabdovirus that infects livestock and can cause economically important disease.Tripartite motif-containing 25 (TRIM25) is an E3 ubiquitin ligase involved in innate antiviral signaling, but its role in pigs during vesicular stomatitis virus infection is unclear.Objective: To define the mechanism by which swine TRIM25 restricts vesicular stomatitis virus replication.Methods: Porcine 3D4/21 cells with TRIM25 overexpression or knockdown were infected with vesicular stomatitis virus.Viral replication was quantified by immunoblotting, quantitative reverse transcription polymerase chain reaction, and 50% tissue culture infectious dose assays.Type I interferon signaling was assessed by transcript quantification, interferon-beta and interferon-stimulated response element reporter assays, and co-immunoprecipitation.Viral RNA binding was tested by RNA immunoprecipitation.Results: TRIM25 overexpression reduced viral RNA and infectious titers, whereas TRIM25 knockdown increased replication.TRIM25 increased interferon-beta and interferonstimulated gene expression and enhanced interferon-beta and interferon-stimulated response element promoter activity.Mechanistically, TRIM25 promoted Lys63-linked ubiquitination of RIG-I and increased phosphorylation of TANK-binding kinase 1 and interferon regulatory factor 3. TRIM25 also bound vesicular stomatitis virus genomic RNA, and binding required the C-terminal region.Conclusions and Relevance: Porcine TRIM25 restricts vesicular stomatitis virus replication by amplifying type I interferon signaling and directly binding viral RNA.