Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China.
Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China.
Vet Res. 2023 Mar 14;54(1):23. doi: 10.1186/s13567-023-01152-2.
Tembusu virus (TMUV) is an emerging flavivirus that has broken out in different regions of China. TMUV infection has been reported to induce autophagy in duck embryo fibroblast cells. However, the molecular mechanisms underlying this autophagy induction remain unclear. Here, we explored the interactions between autophagy and TMUV and the effects of the structural and nonstructural proteins of TMUV on autophagy in vitro. Among our results, TMUV infection enhanced autophagy to facilitate viral replication in HEK293T cells. After pharmacologically inducing autophagy with rapamycin (Rapa), the replication of TMUV increased by a maximum of 14-fold compared with the control group. To determine which TMUV protein primarily induced autophagy, cells were transfected with two structural proteins and seven nonstructural proteins of TMUV. Western blotting showed that nonstructural proteins 2B (NS2B) and 4 A (NS4A) of TMUV significantly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3) from LC3-I to LC3-II in HEK293T cells. In addition, through immunofluorescence assays, we found that NS2B and NS4A significantly increased the punctate fluorescence of GFP-LC3-II. Furthermore, we found that both NS2B and NS4A interacted with polyubiquitin-binding protein sequestosome 1 (SQSTM1/p62) in a coimmunoprecipitation assay. Moreover, the autophagic degradation of p62 and LC3 mediated by NS2B or NS4A was inhibited by treatment with the autophagic flux inhibitor chloroquine (CQ). These results confirmed the vital effects of NS2B and NS4A in TMUV-induced complete autophagy and clarified the importance of complete autophagy for viral replication, providing novel insight into the relationship between TMUV and autophagy.
腾格里病毒（TMUV）是一种新兴的黄病毒，已在中国不同地区爆发。有报道称，TMUV 感染可诱导鸭胚成纤维细胞发生自噬。然而，这种自噬诱导的分子机制尚不清楚。在这里，我们研究了自噬与 TMUV 之间的相互作用，以及 TMUV 的结构蛋白和非结构蛋白对体外自噬的影响。我们的研究结果表明，TMUV 感染增强了自噬作用，从而促进了 HEK293T 细胞中的病毒复制。用雷帕霉素（Rapa）诱导自噬后，与对照组相比，TMUV 的复制增加了 14 倍。为了确定 TMUV 蛋白主要诱导自噬的蛋白，我们用 TMUV 的两个结构蛋白和七个非结构蛋白转染细胞。Western blot 显示，TMUV 的非结构蛋白 2B（NS2B）和 4A（NS4A）显著诱导 HEK293T 细胞中微管相关蛋白 1 轻链 3（LC3）从 LC3-I 向 LC3-II 的转化。此外，通过免疫荧光分析，我们发现 NS2B 和 NS4A 显著增加 GFP-LC3-II 的点状荧光。进一步研究发现，NS2B 和 NS4A 均与多聚泛素结合蛋白 SQSTM1/p62 相互作用。此外，在共免疫沉淀实验中，NS2B 或 NS4A 介导的 p62 和 LC3 的自噬降解被自噬流抑制剂氯喹（CQ）抑制。这些结果证实了 NS2B 和 NS4A 在 TMUV 诱导的完全自噬中的重要作用，阐明了完全自噬对病毒复制的重要性，为 TMUV 与自噬之间的关系提供了新的见解。
