Oncolytic avian reovirus regulates the TLR3-IRF3-IFN-γ-JAK-STAT1 and TLR3-NF-κB-IFN-γ-JAK-STAT1 pathways inducing autophagy in murine melanoma cells

Chao-Yu Hsu; Zi-Yang Lo; Yi-Ying Wu; Wei-Ru Huang; Tz-Chuen Ju; Kuo-Pin Chuang; Lon-Fye Lye; Muhammad Munir; Hung-Jen Liu

doi:10.1016/j.vetmic.2025.110624

Oncolytic avian reovirus regulates the TLR3-IRF3-IFN-γ-JAK-STAT1 and TLR3-NF-κB-IFN-γ-JAK-STAT1 pathways inducing autophagy in murine melanoma cells

Vet Microbiol. 2025 Jun 26:307:110624. doi: 10.1016/j.vetmic.2025.110624. Online ahead of print.

Authors

Chao-Yu Hsu¹, Zi-Yang Lo², Yi-Ying Wu³, Wei-Ru Huang³, Tz-Chuen Ju⁴, Kuo-Pin Chuang⁵, Lon-Fye Lye⁶, Muhammad Munir⁷, Hung-Jen Liu⁸

Affiliations

¹ Division of Urology, Department of Surgery, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan.
² Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
³ Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan; The iEGG and Animal Biotechnology Center, National Chung Hsing University, Taichung, Taiwan.
⁴ Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan; Ph.D Program in Translational Medicine, National Chung Hsing University, Taichung, Taiwan.
⁵ Graduate Institute of Animal Vaccine Technology, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.
⁶ Department of Medical Research, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan.
⁷ Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancashire, UK.
⁸ Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan; The iEGG and Animal Biotechnology Center, National Chung Hsing University, Taichung, Taiwan; Ph.D Program in Translational Medicine, National Chung Hsing University, Taichung, Taiwan; Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, Taiwan; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan. Electronic address: hjliu5257@nchu.edu.tw.

PMID: 40580702
DOI: 10.1016/j.vetmic.2025.110624

Abstract

Oncolytic avian reovirus (ARV) has been identified as a virus capable of selectively infecting and inducing cell death in various cancer cell lines. This study investigates the role of ARV in activating innate immune responses in B16-F10 murine melanoma cells, focusing on the TLR3-IRF3-IFN-γ-JAK-STAT1 and TLR3-NF-κB-IFN-γ-JAK-STAT1 pathways. Our results revealed that the σC protein of ARV interacts with toll-like receptor 3 (TLR3) in the cytoplasm, leading to nuclear translocation of IRF3 and NF-κB as well as the upregulation of IFN-γ, as confirmed by quantitative real-time reverse transcription and polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), proximity ligation assay (PLA), and Western blot. Inhibition assays targeting TLR3, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and interferon regulatory factor 3 (IRF3) further validated the involvement of the TLR3-IRF3 and TLR3-NF-κB pathways in IFN-γ activation. Additionally, cells treated with signal transducer and activator of transcription 1 (STAT1) shRNA and Janus kinase (JAK) inhibitor revealed that ARV promotes autophagy via the IFN-γ-JAK-STAT1 pathway. Immunofluorescence staining and LC3-mCherry transfection further confirm ARV's role in triggering autophagy via TLR3-IRF3-IFN-γ-JAK-STAT1 and TLR3-NF-κB-IFN-γ-JAK-STAT1 pathways. Our results revealed that oncolytic ARV induces autophagy and apoptosis in middle to late stages of virus life cycle in murine melanoma cells. These findings highlight the potential of ARV as a novel oncolytic virotherapy through immune pathway activation in cancer cells.

Keywords: Autophagy; Avian reovirus; B16-F10 melanoma cells; IFN-γ; IRF3; NF-κB; Toll like receptor 3.