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      Enterovirus 71‐induced autophagy detected in vitro and in vivo promotes viral replication

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          Enterovirus 71 (EV71) is an important pathogen causing death in children under 5 years old worldwide. However, the underlying pathogenesis remains unclear. This study reveals that EV71 infection in rhabdomyosarcoma (RD) and neuroblastoma (SK‐N‐SH) cells stimulated the autophagic process, which was demonstrated by an increase of punctate GFP‐microtubule‐associated protein 1 light chain 3 (GFP‐LC3), the level of autophagosome‐bound LC3‐II protein and double‐membrane autophagosome formation. EV71‐induced autophagy benefited EV71 replication, which was confirmed by the autophagic inducer rapamycin and the inhibitor 3‐methyladenine. Signaling pathway investigation revealed that the decreased expression of phosphorylated mTOR and phosphorylated p70S6K is involved in EV71‐induced autophagy in a cell‐specific manner. The expression of phosphorylated extracellular signal‐regulated kinase (Erk) was suppressed consistently in EV71‐infected cells. However it did not participate in the autophagic response of the cell. Other signaling pathway molecules, such as Erk, PI3K/Akt, Bcl‐2, BNIP3, and Beclin‐1 were not affected by infection with EV71. Electron microscopy showed co‐localization of autophagosome‐like vesicles with either EV71‐VP1 or LC3 protein in neurons of the cervical spinal cord in ICR mice infected with EV71. In conclusion, EV71 infection triggered autophagic flux and induced autophagosome formation both in vitro and in vivo. Autophagy induced by EV71 is beneficial for viral replication. Understanding the role of autophagy induced by EV71 in vitro and the formation of autophagosome‐like vesicle in vivo provide new insights into the pathogenesis of EV71 infection. J. Med. Virol. 81:1241–1252, 2009. © 2009 Wiley‐Liss, Inc.

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          Most cited references 34

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          Modification of intracellular membrane structures for virus replication

          Key Points Plus-stranded RNA viruses induce large membrane structures that might support the replication of their genomes. Similarly, cytoplasmic replication of poxviruses (large DNA viruses) occurs in associated membranes. These membranes originate from the endoplasmic reticulum (ER) or endosomes. Membrane vesicles that support viral replication are induced by a number of RNA viruses. Similarly, the poxvirus replication site is surrounded by a double-membraned cisterna that is derived from the ER. Analogies to autophagy have been proposed since the finding that autophagy cellular processes involve the formation of double-membrane vesicles. However, molecular evidence to support this hypothesis is lacking. Membrane association of the viral replication complex is mediated by the presence of one or more viral proteins that contain sequences which associate with, or integrate into, membranes. Replication-competent membranes might contain viral or cellular proteins that contain amphipathic helices, which could mediate the membrane bending that is required to form spherical vesicles. Whereas poxvirus DNA replication occurs inside the ER-enclosed site, for most RNA viruses the topology of replication is not clear. Preliminary results for some RNA viruses suggest that their replication could also occur inside double-membrane vesicles. We speculate that cytoplasmic replication might occur inside sites that are 'enwrapped' by an ER-derived cisterna, and that these cisternae are open to the cytoplasm. Thus, RNA and DNA viruses could use a common mechanism for replication that involves membrane wrapping by cellular cisternal membranes. We propose that three-dimensional analyses using high-resolution electron-microscopy techniques could be useful for addressing this issue. High-throughput small-interfering-RNA screens should also shed light on molecular requirements for virus-induced membrane modifications.
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            Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells.

            3-Methyladenine which stops macroautophagy at the sequestration step in mammalian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity raising the possibility that PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelárová, H., and Meijer, A. J. (1997) Eur. J. Biochem. 243, 240-246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I PI3K products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipids (dipalmitoyl phosphatidylinositol 3, 4-bisphosphate and dipalmitoyl phosphatidylinositol 3,4, 5-triphosphate) or by stimulating the enzymatic activity by interleukin-13 reduced macroautophagy. In contrast, an increase in the class III PI3K product (phosphatidylinositol 3-phosphate), either by feeding cells with a synthetic lipid or by overexpressing the p150 adaptor, stimulates macroautophagy. Transfection of a specific class III PI3K antisense oligonucleotide greatly inhibited the rate of macroautophagy. In accordance with a role of class III PI3K, wortmannin (an inhibitor of PI3Ks) inhibits macroautophagic sequestration and protein degradation in the low nanomolar range (IC(50) 5-15 nM). Further in vitro enzymatic assay showed that 3-methyladenine inhibits the class III PI3K activity. Dipalmitoyl phosphatidylinositol 3-phosphate supplementation or p150 overexpression rescued the macroautophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of the Galpha(i3) protein suggesting that both class III PI3K and trimeric G(i3) protein signaling are required in the control macroautophagy in HT-29 cells. In conclusion, our results demonstrate that distinct classes of PI3K control the macroautophagic pathway in opposite directions. The roles of PI3Ks in macroautophagy are discussed in the context of membrane recycling.
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              Autophagic machinery activated by dengue virus enhances virus replication

              Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that is favorable for viral replication.

                Author and article information

                J Med Virol
                J. Med. Virol
                Journal of Medical Virology
                Wiley Subscription Services, Inc., A Wiley Company (Hoboken )
                27 May 2009
                July 2009
                : 81
                : 7 ( doiID: 10.1002/jmv.v81:7 )
                : 1241-1252
                [ 1 ]Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
                Author notes
                [* ]Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.===
                Copyright © 2009 Wiley‐Liss, Inc.

                This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

                Page count
                Figures: 7, Tables: 0, References: 45, Pages: 12, Words: 1376
                Funded by: National Science Council, Taiwan
                Award ID: NSC 96‐2628‐B‐006‐003‐MY3
                Research Article
                Custom metadata
                July 2009
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.0 mode:remove_FC converted:15.04.2020

                Microbiology & Virology

                ev71, autophagy, autophagosome


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