title: The p17 nonstructural protein of avian reovirus triggers autophagy enhancing virus replication via activation of phosphatase and tensin deleted on chromosome 10 (PTEN) and AMP-activated protein kinase (AMPK), as well as dsRNA-dependent protein kinase (PKR)/eIF2α signaling pathways.
abstract: Autophagy has been shown to facilitate replication or production of avian reovirus (ARV); nevertheless, how ARV induces autophagy remains largely unknown. Here, we demonstrate that the nonstructural protein p17 of ARV functions as an activator of autophagy. ARV-infected or p17-transfected cells present a fast and strong induction of autophagy, resulting in an increased level of autophagic proteins Beclin 1 and LC3-II. Although autophagy was suppressed by 3-methyladenine or shRNAs targeting autophagic proteins (Beclin 1, ATG7, and LC3) as well as by overexpression of Bcl-2, viral transcription, σC protein synthesis, and virus yield were all significantly reduced, suggesting a key role of autophagosomes in supporting ARV replication. Furthermore, we revealed for the first time that p17 positively regulates phosphatase and tensin deleted on chromosome 10 (PTEN), AMP-activated protein kinase (AMPK), and dsRNA dependent protein kinase RNA (PKR)/eIF2α signaling pathways, accompanied by down-regulation of Akt and mammalian target of rapamycin complex 1, thereby triggering autophagy. By using p53, PTEN, PKR, AMPK, and p17 short hairpin RNA (shRNA), activation of signaling pathways and LC3-II levels was significantly suppressed, suggesting that p17 triggers autophagy through activation of p53/PTEN, AMPK, and PKR signaling pathways. Furthermore, colocalization of LC3 with viral proteins (p17 and σC), p62 with LAMP2 and LC3 with Rab7 was observed under a fluorescence microscope. The expression level of p62 was increased at 18 h postinfection and then slightly decreased 24 h postinfection compared with mock infection and thapsigargin treatment. Furthermore, disruption of autophagosome-lysosome fusion by shRNAs targeting LAMP2 or Rab7a resulted in inhibition of viral protein synthesis and virus yield, suggesting that formation of autolysosome benefits virus replication. Taken together, our results suggest that ARV induces formation of autolysosome but does not induce complete autophagic flux.
authors.affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Pei I
authors.initials: PI
authors.lastname: Chi
authors.forename: Wei R
authors.initials: WR
authors.lastname: Huang
authors.forename: I H
authors.initials: IH
authors.lastname: Lai
authors.forename: Ching Y
authors.initials: CY
authors.lastname: Cheng
authors.forename: Hung J
authors.initials: HJ
authors.lastname: Liu
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008954:Models, Biological; D030101:Orthoreovirus, Avian / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D010588:Phagosomes / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*; D019892:eIF-2 Kinase / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D036022:Capsid Proteins; D015852:Eukaryotic Initiation Factor-2; D008869:Microtubule-Associated Proteins; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D017361:Viral Nonstructural Proteins; C031728:sigma 1 protein, reovirus; D019892:eIF-2 Kinase; D048051:p38 Mitogen-Activated Protein Kinases; D055372:AMP-Activated Protein Kinases; D051059:PTEN Phosphohydrolase