title: Muscovy duck reovirus σNS protein triggers autophagy enhancing virus replication.
abstract: Muscovy duck reovirus (MDRV) causes high morbidity and mortality in Muscovy ducklings at 10 days old and can persist in an infected flock until the ducklings of 6 weeks old. It shares common physicochemical properties with avian reovirus (ARV) and differs in coding assignment and pathogenicity. The ARV p17 protein has been shown to trigger autophagy via activation multiple signaling pathways, which benefits virus replication. Since MDRV lacks the p17 protein, whether and how MDRV induces autophagy remains unknown. The aim of this study was to explore whether MDRV induces autophagy and which viral proteins are involved in MDRV-induced autophagy.
  
  
  
  The autophagosome-like structures in MDRV-infected cells was observed under transmission electron microscopy. MDRV-induced autophagy was examined by analyzing the LC3-II level and phosphorylated form of mammalian target of rapamycin (mTOR) by Western blot assays. The effects of 3-methyladenine, rapamycin, chloroquine on viral yields were measured with quantitative(q) real-time reverse transcription (RT)-polymerase chain reaction (PCR) and 50% tissue culture infective dose (TCID50) assays, respectively. Additionally, to determine which viral protein is responsible for MDRV-induced autophagy, both p10.8- and σNS-encoding genes of MDRV were cloned into the pCI-neo-flag vector and transfected into DF-1 cells for detection of LC3-II.
  
  
  
  The typical double-membrane vesicles containing cytoplasmic inclusions were visible in MDRV-infected immortalized chicken embryo fibroblast (DF-1) cells under transmission electron microscopy. Both primary Muscovy duck embryo fibroblasts (MDEF) and DF-1 cells infected with MDRV exhibited a significant increased levels of LC3-II accompanied with downregulation of phosphorylated form of mTOR, further confirming that MDRV is capable of inducing autophagy. Autophagy could be suppressed by 3-methylademine and induced by rapamycin and chloroquine. Furthermore, we found that σNS induces an increased levels of LC3-II, suggesting that the MDRV σNS protein is one of viral proteins involved in induction of autophagy. Both qRT-PCR and TCID50assays showed that virus yield was increased in rapamycin treated DF-1 cells following MDRV infection. Conversely, when infected cells were pretreated with chloroquine, virus yield was decreased.
  
  
  
  The MDRV σNS nonstructural protein is responsible for MDRV-induced autophagy and benefits virus replication.
authors.affiliation: College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People's Republic of China.|Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou, 350002, People's Republic of China.
authors.forename: Yijian
authors.initials: Y
authors.lastname: Wu
authors.affiliation: College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People's Republic of China.
authors.forename: Longping
authors.initials: L
authors.lastname: Cui
authors.affiliation: College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People's Republic of China.|Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou, 350002, People's Republic of China.
authors.forename: Erpeng
authors.initials: E
authors.lastname: Zhu
authors.affiliation: College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People's Republic of China.|Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou, 350002, People's Republic of China.
authors.forename: Wuduo
authors.initials: W
authors.lastname: Zhou
authors.affiliation: College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People's Republic of China.|Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou, 350002, People's Republic of China.
authors.forename: Quanxi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People's Republic of China.|Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou, 350002, People's Republic of China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Wu
authors.affiliation: College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People's Republic of China.|Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou, 350002, People's Republic of China.
authors.forename: Baocheng
authors.initials: B
authors.lastname: Wu
authors.affiliation: College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People's Republic of China.|Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou, 350002, People's Republic of China.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung, 402, Taiwan. hjliu5257@nchu.edu.tw.|Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, 402, Taiwan. hjliu5257@nchu.edu.tw.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung, 402, Taiwan. hjliu5257@nchu.edu.tw.
authors.forename: Hung-Jen
authors.initials: HJ
authors.lastname: Liu
mesh_terms: D000818:Animals; D000071182:Autophagosomes / Q000648:ultrastructure; D001343:Autophagy; D015153:Blotting, Western; D002460:Cell Line; D002645:Chickens; D046529:Microscopy, Electron, Transmission; D008869:Microtubule-Associated Proteins / Q000032:analysis; D030101:Orthoreovirus, Avian / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction; D058570:TOR Serine-Threonine Kinases / Q000032:analysis; D019562:Viral Load; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D008869:Microtubule-Associated Proteins; D017361:Viral Nonstructural Proteins; D058570:TOR Serine-Threonine Kinases
keywords: Autophagy; LC3-II; Muscovy duck reovirus; Viral replication; σNS nonstructural protein