2. Academic Validation
  3. Nonstructural proteins 2B and 4A of Tembusu virus induce complete autophagy to promote viral multiplication in vitro

Nonstructural proteins 2B and 4A of Tembusu virus induce complete autophagy to promote viral multiplication in vitro

  • Vet Res. 2023 Mar 14;54(1):23. doi: 10.1186/s13567-023-01152-2.
Wangyang Tan # 1,  Senzhao Zhang # 1,  Yu He 1,  Zhen Wu 1,  Mingshu Wang 1 2 3,  Renyong Jia 1 2 3,  Dekang Zhu 2 3,  Mafeng Liu 1 2 3,  Xinxin Zhao 1 2 3,  Qiao Yang 1 2 3,  Ying Wu 1 2 3,  Shaqiu Zhang 1 2 3,  Juan Huang 1 2 3,  Sai Mao 1 2 3,  Xumin Ou 1 2 3,  Qun Gao 1 2 3,  Di Sun 1 2 3,  Bin Tian 2 3,  Shun Chen 4 5 6,  Anchun Cheng 7 8 9
Affiliations

Affiliations

  • 1 Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China.
  • 2 Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China.
  • 3 Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China.
  • 4 Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. [email protected].
  • 5 Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. [email protected].
  • 6 Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. [email protected].
  • 7 Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. [email protected].
  • 8 Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. [email protected].
  • 9 Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China. [email protected].
  • # Contributed equally.
PMID: 36918952 DOI: 10.1186/s13567-023-01152-2
Abstract

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.

Keywords

Tembusu virus; autophagy; nonstructural protein 2B; nonstructural protein 4A; p62.

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