West Nile virus capsid protein inhibits autophagy by AMP-activated protein kinase degradation in neurological disease development

Autoři: Shintaro Kobayashi aff001;  Kentaro Yoshii aff001;  Wallaya Phongphaew aff002;  Memi Muto aff001;  Minato Hirano aff001;  Yasuko Orba aff002;  Hirofumi Sawa aff002;  Hiroaki Kariwa aff001
Působiště autorů: Laboratory of Public Health, Faculty of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan aff001;  Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan aff002;  Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo, Japan aff003;  Global Virus Network, Baltimore, Maryland, United States of America aff004
Vyšlo v časopise: West Nile virus capsid protein inhibits autophagy by AMP-activated protein kinase degradation in neurological disease development. PLoS Pathog 16(1): e32767. doi:10.1371/journal.ppat.1008238
Kategorie: Research Article
doi: 10.1371/journal.ppat.1008238


West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease.

Klíčová slova:

Autophagic cell death – Cell staining – DAPI staining – Immunoblotting – Neuronal death – Plasmid construction – Protein expression – West Nile virus


1. Fields BN, Knipe DM, Howley PM, Cohen JI. Fields virology. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2013. 2 v. (xx, 2456, 82 p.) p.

2. Kobayashi S, Orba Y, Yamaguchi H, Kimura T, Sawa H. Accumulation of ubiquitinated proteins is related to West Nile virus-induced neuronal apoptosis. Neuropathology. 2012;32(4):398–405. doi: 10.1111/j.1440-1789.2011.01275.x 22129084.

3. Schafernak KT, Bigio EH. West Nile virus encephalomyelitis with polio-like paralysis & nigral degeneration. Can J Neurol Sci. 2006;33(4):407–10. doi: 10.1017/s0317167100005370 17168167.

4. Taylor JP, Hardy J, Fischbeck KH. Toxic proteins in neurodegenerative disease. Science. 2002;296(5575):1991–5. doi: 10.1126/science.1067122 12065827.

5. Alirezaei M, Kiosses WB, Flynn CT, Brady NR, Fox HS. Disruption of neuronal autophagy by infected microglia results in neurodegeneration. PLoS One. 2008;3(8):e2906. doi: 10.1371/journal.pone.0002906 18682838; PubMed Central PMCID: PMC2483417.

6. Riley BE, Kaiser SE, Shaler TA, Ng AC, Hara T, Hipp MS, et al. Ubiquitin accumulation in autophagy-deficient mice is dependent on the Nrf2-mediated stress response pathway: a potential role for protein aggregation in autophagic substrate selection. J Cell Biol. 2010;191(3):537–52. doi: 10.1083/jcb.201005012 21041446; PubMed Central PMCID: PMC3003313.

7. Mizushima N, Hara T. Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration? Autophagy. 2006;2(4):302–4. doi: 10.4161/auto.2945 16874082.

8. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature. 2008;451(7182):1069–75. doi: 10.1038/nature06639 18305538; PubMed Central PMCID: PMC2670399.

9. Shoji-Kawata S, Levine B. Autophagy, antiviral immunity, and viral countermeasures. Biochim Biophys Acta. 2009;1793(9):1478–84. doi: 10.1016/j.bbamcr.2009.02.008 19264100; PubMed Central PMCID: PMC2739265.

10. Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature. 2006;441(7095):885–9. doi: 10.1038/nature04724 16625204.

11. Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, et al. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature. 2006;441(7095):880–4. doi: 10.1038/nature04723 16625205.

12. Kobayashi S, Orba Y, Yamaguchi H, Takahashi K, Sasaki M, Hasebe R, et al. Autophagy inhibits viral genome replication and gene expression stages in West Nile virus infection. Virus Res. 2014;191:83–91. doi: 10.1016/j.virusres.2014.07.016 25091564.

13. Orvedahl A, Alexander D, Talloczy Z, Sun Q, Wei Y, Zhang W, et al. HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe. 2007;1(1):23–35. doi: 10.1016/j.chom.2006.12.001 18005679.

14. Kyei GB, Dinkins C, Davis AS, Roberts E, Singh SB, Dong C, et al. Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages. J Cell Biol. 2009;186(2):255–68. doi: 10.1083/jcb.200903070 19635843; PubMed Central PMCID: PMC2717652.

15. Johnston JA, Ward CL, Kopito RR. Aggresomes: a cellular response to misfolded proteins. J Cell Biol. 1998;143(7):1883–98. doi: 10.1083/jcb.143.7.1883 9864362; PubMed Central PMCID: PMC2175217.

16. Shen D, Coleman J, Chan E, Nicholson TP, Dai L, Sheppard PW, et al. Novel cell- and tissue-based assays for detecting misfolded and aggregated protein accumulation within aggresomes and inclusion bodies. Cell Biochem Biophys. 2011;60(3):173–85. doi: 10.1007/s12013-010-9138-4 21132543; PubMed Central PMCID: PMC3112480.

17. Raju I, Kumarasamy A, Abraham EC. Multiple aggregates and aggresomes of C-terminal truncated human alphaA-crystallins in mammalian cells and protection by alphaB-crystallin. PLoS One. 2011;6(5):e19876. doi: 10.1371/journal.pone.0019876 21589881; PubMed Central PMCID: PMC3093407.

18. Kobayashi S, Yoshii K, Hirano M, Muto M, Kariwa H. A novel reverse genetics system for production of infectious West Nile virus using homologous recombination in mammalian cells. J Virol Methods. 2017;240:14–20. doi: 10.1016/j.jviromet.2016.11.006 27865748.

19. Wang S, Liu H, Zu X, Liu Y, Chen L, Zhu X, et al. The ubiquitin-proteasome system is essential for the productive entry of Japanese encephalitis virus. Virology. 2016;498:116–27. doi: 10.1016/j.virol.2016.08.013 27567260.

20. Byk LA, Iglesias NG, De Maio FA, Gebhard LG, Rossi M, Gamarnik AV. Dengue Virus Genome Uncoating Requires Ubiquitination. mBio. 2016;7(3). Epub 2016/06/30. doi: 10.1128/mBio.00804-16 27353759.

21. Pierson TC, Sanchez MD, Puffer BA, Ahmed AA, Geiss BJ, Valentine LE, et al. A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection. Virology. 2006;346(1):53–65. doi: 10.1016/j.virol.2005.10.030 16325883.

22. Momose I, Tatsuda D, Ohba S, Masuda T, Ikeda D, Nomoto A. In vivo imaging of proteasome inhibition using a proteasome-sensitive fluorescent reporter. Cancer Sci. 2012;103(9):1730–6. doi: 10.1111/j.1349-7006.2012.02352.x 22676179.

23. Hamasaki M, Furuta N, Matsuda A, Nezu A, Yamamoto A, Fujita N, et al. Autophagosomes form at ER-mitochondria contact sites. Nature. 2013;495(7441):389–93. doi: 10.1038/nature11910 23455425.

24. Mack HI, Zheng B, Asara JM, Thomas SM. AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization. Autophagy. 2012;8(8):1197–214. doi: 10.4161/auto.20586 22932492; PubMed Central PMCID: PMC3679237.

25. Shoji-Kawata S, Sumpter R, Leveno M, Campbell GR, Zou Z, Kinch L, et al. Identification of a candidate therapeutic autophagy-inducing peptide. Nature. 2013;494(7436):201–6. doi: 10.1038/nature11866 23364696; PubMed Central PMCID: PMC3788641.

26. Pineda CT, Ramanathan S, Fon Tacer K, Weon JL, Potts MB, Ou YH, et al. Degradation of AMPK by a cancer-specific ubiquitin ligase. Cell. 2015;160(4):715–28. doi: 10.1016/j.cell.2015.01.034 25679763; PubMed Central PMCID: PMC5629913.

27. Soto-Acosta R, Bautista-Carbajal P, Cervantes-Salazar M, Angel-Ambrocio AH, Del Angel RM. DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target. PLoS Pathog. 2017;13(4):e1006257. doi: 10.1371/journal.ppat.1006257 28384260; PubMed Central PMCID: PMC5383345.

28. Hardie DG. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat Rev Mol Cell Biol. 2007;8(10):774–85. doi: 10.1038/nrm2249 17712357.

29. Zungu M, Schisler JC, Essop MF, McCudden C, Patterson C, Willis MS. Regulation of AMPK by the ubiquitin proteasome system. Am J Pathol. 2011;178(1):4–11. doi: 10.1016/j.ajpath.2010.11.030 21224036; PubMed Central PMCID: PMC3069915.

30. Sidhu JS, Rajawat YS, Rami TG, Gollob MH, Wang Z, Yuan R, et al. Transgenic mouse model of ventricular preexcitation and atrioventricular reentrant tachycardia induced by an AMP-activated protein kinase loss-of-function mutation responsible for Wolff-Parkinson-White syndrome. Circulation. 2005;111(1):21–9. doi: 10.1161/01.CIR.0000151291.32974.D5 15611370; PubMed Central PMCID: PMC2908313.

31. Khanal P, Kim G, Yun HJ, Cho HG, Choi HS. The prolyl isomerase Pin1 interacts with and downregulates the activity of AMPK leading to induction of tumorigenicity of hepatocarcinoma cells. Mol Carcinog. 2013;52(10):813–23. doi: 10.1002/mc.21920 22549912.

32. Dong X, Levine B. Autophagy and viruses: adversaries or allies? J Innate Immun. 2013;5(5):480–93. doi: 10.1159/000346388 23391695; PubMed Central PMCID: PMC3790331.

33. Orvedahl A, Levine B. Autophagy and viral neurovirulence. Cell Microbiol. 2008;10(9):1747–56. doi: 10.1111/j.1462-5822.2008.01175.x 18503639; PubMed Central PMCID: PMC2737270.

34. Munz C. Beclin-1 targeting for viral immune escape. Viruses. 2011;3(7):1166–78. Epub 2011/10/14. doi: 10.3390/v3071166 21994775; PubMed Central PMCID: PMC3185790.

35. Mouna L, Hernandez E, Bonte D, Brost R, Amazit L, Delgui LR, et al. Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins. Autophagy. 2016;12(2):327–42. Epub 2015/12/15. doi: 10.1080/15548627.2015.1125071 26654401; PubMed Central PMCID: PMC4836022.

36. Samuel MA, Diamond MS. Pathogenesis of West Nile Virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion. J Virol. 2006;80(19):9349–60. doi: 10.1128/JVI.01122-06 16973541; PubMed Central PMCID: PMC1617273.

37. Xu Q, Zhu N, Chen S, Zhao P, Ren H, Zhu S, et al. E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells. Scientific reports. 2017;7:45375. doi: 10.1038/srep45375 28349961; PubMed Central PMCID: PMC5368976.

38. Jimenez de Oya N, Blazquez AB, Casas J, Saiz JC, Martin-Acebes MA. Direct Activation of Adenosine Monophosphate-Activated Protein Kinase (AMPK) by PF-06409577 Inhibits Flavivirus Infection through Modification of Host Cell Lipid Metabolism. Antimicrob Agents Chemother. 2018;62(7). doi: 10.1128/AAC.00360-18 29712653; PubMed Central PMCID: PMC6021616.

39. Yang MR, Lee SR, Oh W, Lee EW, Yeh JY, Nah JJ, et al. West Nile virus capsid protein induces p53-mediated apoptosis via the sequestration of HDM2 to the nucleolus. Cell Microbiol. 2008;10(1):165–76. doi: 10.1111/j.1462-5822.2007.01027.x 17697133.

40. Bhuvanakantham R, Ng ML. West Nile virus and dengue virus capsid protein negates the antiviral activity of human Sec3 protein through the proteasome pathway. Cell Microbiol. 2013;15(10):1688–706. doi: 10.1111/cmi.12143 23522008.

41. Yang JS, Ramanathan MP, Muthumani K, Choo AY, Jin SH, Yu QC, et al. Induction of inflammation by West Nile virus capsid through the caspase-9 apoptotic pathway. Emerging infectious diseases. 2002;8(12):1379–84. Epub 2002/12/25. doi: 10.3201/eid0812.020224 12498651; PubMed Central PMCID: PMC2738518.

42. Dokland T, Walsh M, Mackenzie JM, Khromykh AA, Ee KH, Wang S. West Nile virus core protein; tetramer structure and ribbon formation. Structure. 2004;12(7):1157–63. doi: 10.1016/j.str.2004.04.024 15242592.

43. Nagata N, Iwata-Yoshikawa N, Hayasaka D, Sato Y, Kojima A, Kariwa H, et al. The pathogenesis of 3 neurotropic flaviviruses in a mouse model depends on the route of neuroinvasion after viremia. J Neuropathol Exp Neurol. 2015;74(3):250–60. doi: 10.1097/NEN.0000000000000166 25668565.

44. Tajima S, Yagasaki K, Kotaki A, Tomikawa T, Nakayama E, Moi ML, et al. In vitro growth, pathogenicity and serological characteristics of the Japanese encephalitis virus genotype V Muar strain. The Journal of general virology. 2015;96(9):2661–9. Epub 2015/06/07. doi: 10.1099/vir.0.000213 26048886.

45. de Wispelaere M, Frenkiel MP, Despres P. A Japanese encephalitis virus genotype 5 molecular clone is highly neuropathogenic in a mouse model: impact of the structural protein region on virulence. J Virol. 2015;89(11):5862–75. Epub 2015/03/20. doi: 10.1128/JVI.00358-15 25787283; PubMed Central PMCID: PMC4442416.

46. Shirato K, Miyoshi H, Goto A, Ako Y, Ueki T, Kariwa H, et al. Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus. The Journal of general virology. 2004;85(Pt 12):3637–45. Epub 2004/11/24. doi: 10.1099/vir.0.80247-0 15557236.

47. Kobayashi S, Suzuki T, Igarashi M, Orba Y, Ohtake N, Nagakawa K, et al. Cysteine residues in the major capsid protein, Vp1, of the JC virus are important for protein stability and oligomer formation. PLoS One. 2013;8(10):e76668. doi: 10.1371/journal.pone.0076668 24130786; PubMed Central PMCID: PMC3793911.

48. Kobayashi S, Suzuki T, Kawaguchi A, Phongphaew W, Yoshii K, Iwano T, et al. Rab8b Regulates Transport of West Nile Virus Particles from Recycling Endosomes. J Biol Chem. 2016;291(12):6559–68. doi: 10.1074/jbc.M115.712760 26817838; PubMed Central PMCID: PMC4813582.

49. Phongphaew W, Kobayashi S, Sasaki M, Carr M, Hall WW, Orba Y, et al. Valosin-containing protein (VCP/p97) plays a role in the replication of West Nile virus. Virus Res. 2017;228:114–23. doi: 10.1016/j.virusres.2016.11.029 27914931.

50. Pierson TC, Diamond MS, Ahmed AA, Valentine LE, Davis CW, Samuel MA, et al. An infectious West Nile virus that expresses a GFP reporter gene. Virology. 2005;334(1):28–40. Epub 2005/03/08. doi: 10.1016/j.virol.2005.01.021 15749120.

51. Kimura T, Kimura-Kuroda J, Nagashima K, Yasui K. Analysis of virus-cell binding characteristics on the determination of Japanese encephalitis virus susceptibility. Archives of virology. 1994;139(3–4):239–51. Epub 1994/01/01. doi: 10.1007/bf01310788 7832632.

52. Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014;343(6166):80–4. doi: 10.1126/science.1246981 24336569; PubMed Central PMCID: PMC3972032.

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