Hsu Chao-Yu, Lo Zi-Yang, Wu Yi-Ying, Huang Wei-Ru, Ju Tz-Chuen, Chuang Kuo-Pin, Lye Lon-Fye, Munir Muhammad, Liu Hung-Jen
Division of Urology, Department of Surgery, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan.
Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
Vet Microbiol. 2025 Aug;307:110624. doi: 10.1016/j.vetmic.2025.110624. Epub 2025 Jun 26.
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.
溶瘤禽呼肠孤病毒（ARV）已被鉴定为一种能够选择性感染并诱导多种癌细胞系发生细胞死亡的病毒。本研究调查了ARV在激活B16-F10小鼠黑色素瘤细胞先天性免疫反应中的作用，重点关注TLR3-IRF3-IFN-γ-JAK-STAT1和TLR3-NF-κB-IFN-γ-JAK-STAT1信号通路。我们的结果显示，ARV的σC蛋白在细胞质中与Toll样受体3（TLR3）相互作用，导致IRF3和NF-κB发生核转位以及IFN-γ上调，这通过定量实时逆转录聚合酶链反应（qRT-PCR）、酶联免疫吸附测定（ELISA）、邻近连接测定（PLA）和蛋白质免疫印迹法得以证实。针对TLR3、活化B细胞核因子κB轻链增强子（NF-κB）和干扰素调节因子3（IRF3）的抑制试验进一步验证了TLR3-IRF3和TLR3-NF-κB信号通路参与IFN-γ的激活。此外，用转录信号转导子与激活子1（STAT1）短发夹RNA和Janus激酶（JAK）抑制剂处理的细胞显示，ARV通过IFN-γ-JAK-STAT1信号通路促进自噬。免疫荧光染色和LC3-mCherry转染进一步证实了ARV通过TLR3-IRF3-IFN-γ-JAK-STAT1和TLR3-NF-κB-IFN-γ-JAK-STAT1信号通路触发自噬的作用。我们的结果显示，溶瘤性ARV在小鼠黑色素瘤细胞病毒生命周期的中晚期诱导自噬和凋亡。这些发现突出了ARV通过激活癌细胞免疫途径作为一种新型溶瘤病毒疗法的潜力。
