1887

Abstract

Microtubule-associated protein 1 light chain 3 (MAP1LC3) is a protein with a well-defined function in autophagy, but still incompletely understood roles in several other autophagy-independent processess. Studies have shown MAP1LC3 is a host-dependency factor for the replication of several viruses. Japanese encephalitis virus (JEV), a neurotropic flavivirus, replicates on ER-derived membranes that are marked by autophagosome-negative non-lipidated MAP1LC3 (LC3-I). Depletion of LC3 exerts a profound inhibition on virus replication and egress. Here, we further characterize the role of LC3 in JEV replication, and through immunofluorescence and immunoprecipitation show that LC3-I interacts with the virus capsid protein in infected cells. This association was observed on capsid localized to both the replication complex and lipid droplets (LDs). JEV infection decreased the number of LDs per cell indicating a link between lipid metabolism and virus replication. This capsid-LC3 interaction was independent of the autophagy adaptor protein p62/Sequestosome 1 (SQSTM1). Further, no association of capsid was seen with the Gamma-aminobutyric acid receptor-associated protein family, suggesting that this interaction was specific for LC3. High-resolution protein-protein docking studies identified a putative LC3-interacting region in capsid, FTAL and other key residues that could mediate a direct interaction between the two proteins.

Funding
This study was supported by the:
  • Department of Biotechnology, Ministry of Science and Technology, http://dx.doi.org/10.13039/501100001407
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2020-10-23
2024-04-16
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References

  1. Uchil PD, Satchidanandam V. Architecture of the flaviviral replication complex. protease, nuclease, and detergents reveal encasement within double-layered membrane compartments. J Biol Chem 2003; 278:24388–24398 [View Article] [PubMed]
    [Google Scholar]
  2. Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK et al. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell host Microbe 2009; 5:365–375 [View Article] [PubMed]
    [Google Scholar]
  3. Neufeldt CJ, Cortese M, Acosta EG, Bartenschlager R. Rewiring cellular networks by members of the Flaviviridae family. Nat Rev Microbiol 2018; 16:125–142 [View Article] [PubMed]
    [Google Scholar]
  4. Hoenen A, Gillespie L, Morgan G, van der Heide P, Khromykh A et al. The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein. Virology 2014; 448:104–116 [View Article] [PubMed]
    [Google Scholar]
  5. Overby AK, Popov VL, Niedrig M, Weber F. Tick-Borne encephalitis virus delays interferon induction and hides its double-stranded RNA in intracellular membrane vesicles. J Virol 2010; 84:8470–8483 [View Article] [PubMed]
    [Google Scholar]
  6. Aktepe TE, Liebscher S, Prier JE, Simmons CP, Mackenzie JM. The host protein reticulon 3.1A is utilized by flaviviruses to facilitate membrane remodelling. Cell Rep 2017; 21:1639–1654 [View Article] [PubMed]
    [Google Scholar]
  7. Miller S, Krijnse-Locker J. Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol 2008; 6:363–374 [View Article] [PubMed]
    [Google Scholar]
  8. Mackenzie JM, Khromykh AA, Jones MK, Westaway EG. Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A. Virology 1998; 245:203–215 [View Article] [PubMed]
    [Google Scholar]
  9. Mackenzie JM, Jones MK, Young PR. Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 1996; 220:232–240 [View Article] [PubMed]
    [Google Scholar]
  10. Miller S, Kastner S, Krijnse-Locker J, Bühler S, Bartenschlager R. The non-structural protein 4A of dengue virus is an integral membrane protein inducing membrane alterations in a 2K-regulated manner. J Biol Chem 2007; 282:8873–8882 [View Article] [PubMed]
    [Google Scholar]
  11. Xie X, Zou J, Zhang X, Zhou Y, Routh AL et al. Dengue NS2A protein orchestrates virus assembly. Cell Host Microbe 2019; 26:e608606–622 [View Article] [PubMed]
    [Google Scholar]
  12. Bhullar D, Jalodia R, Kalia M, Vrati S. Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication. PLoS One 2014; 9:e114931 [View Article] [PubMed]
    [Google Scholar]
  13. Nain M, Mukherjee S, Karmakar SP, Paton AW, Paton JC et al. Grp78 is an important host factor for Japanese encephalitis virus entry and replication in mammalian cells. J Virol 2017; 91: [View Article] [PubMed]
    [Google Scholar]
  14. Teo CS, Chu JJ. Cellular vimentin regulates construction of dengue virus replication complexes through interaction with NS4A protein. J Virol 2014; 88:1897–1913 [View Article] [PubMed]
    [Google Scholar]
  15. Ferraris P, Blanchard E, Roingeard P. Ultrastructural and biochemical analyses of hepatitis C virus-associated host cell membranes. J Gen Virol 2010; 91:2230–2237 [View Article] [PubMed]
    [Google Scholar]
  16. Lee JY, Cortese M, Haselmann U, Tabata K, Romero-Brey I et al. Spatiotemporal coupling of the hepatitis C virus replication cycle by creating a lipid Droplet- proximal membranous replication compartment. Cell Rep 2019; 27:e36053602–3617 [View Article] [PubMed]
    [Google Scholar]
  17. Zhang J, Lan Y, Sanyal S. Modulation of lipid droplet metabolism-a potential target for therapeutic intervention in Flaviviridae infections. Front Microbiol 2017; 8:2286 [View Article] [PubMed]
    [Google Scholar]
  18. Boulant S, Targett-Adams P, McLauchlan J. Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus. J Gen Virol 2007; 88:2204–2213 [View Article] [PubMed]
    [Google Scholar]
  19. Appel N, Zayas M, Miller S, Krijnse-Locker J, Schaller T et al. Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 2008; 4:e1000035 [View Article] [PubMed]
    [Google Scholar]
  20. Ishida K, Goto S, Ishimura M, Amanuma M, Hara Y et al. Functional correlation between subcellular localizations of Japanese encephalitis virus capsid protein and virus production. J Virol 2019; 93: [View Article] [PubMed]
    [Google Scholar]
  21. Samsa MM, Mondotte JA, Iglesias NG, Assunção-Miranda I, Barbosa-Lima G et al. Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathog 2009; 5:e1000632 [View Article] [PubMed]
    [Google Scholar]
  22. Shang Z, Song H, Shi Y, Qi J, Gao GF. Crystal structure of the capsid protein from Zika virus. J Mol Biol 2018; 430:948–962 [View Article] [PubMed]
    [Google Scholar]
  23. Martins AS, Carvalho FA, Faustino AF, Martins IC, Santos NC. West Nile virus capsid protein interacts with biologically relevant host lipid systems. Front Cell Infect Microbiol 2019; 9:8 [View Article] [PubMed]
    [Google Scholar]
  24. Paul D, Bartenschlager R. Flaviviridae replication organelles: OH, what a Tangled web we weave. Annu Rev Virol 2015; 2:289–310 [View Article] [PubMed]
    [Google Scholar]
  25. Heaton NS, Randall G. Dengue virus-induced autophagy regulates lipid metabolism. Cell Host Microbe 2010; 8:422–432 [View Article] [PubMed]
    [Google Scholar]
  26. Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H et al. Guidelines for the use and interpretation of assays for monitoring autophagy 12, 3rd ed. 2016 pp 1–222
    [Google Scholar]
  27. Calì T, Galli C, Olivari S, Molinari M. Segregation and rapid turnover of EDEM1 by an autophagy-like mechanism modulates standard ERAD and folding activities. Biochem Biophys Res Commun 2008; 371:405–410 [View Article] [PubMed]
    [Google Scholar]
  28. Sharma M, Bhattacharyya S, Nain M, Kaur M, Sood V et al. Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes. Autophagy 2014; 10:1637–1651 [View Article] [PubMed]
    [Google Scholar]
  29. Alirezaei M, Flynn CT, Wood MR, Harkins S, Whitton JL. Coxsackievirus can exploit LC3 in both autophagy-dependent and -independent manners in vivo. Autophagy 2015; 11:1389–1407 [View Article] [PubMed]
    [Google Scholar]
  30. Reggiori F, Monastyrska I, Verheije MH, Calì T, Ulasli M et al. Coronaviruses hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication. Cell Host Microbe 2010; 7:500–508 [View Article] [PubMed]
    [Google Scholar]
  31. Monastyrska I, Ulasli M, Rottier PJ, Guan JL, Reggiori F et al. An autophagy-independent role for LC3 in equine arteritis virus replication. Autophagy 2013; 9:164–174 [View Article] [PubMed]
    [Google Scholar]
  32. Abernathy E, Mateo R, Majzoub K, van Buuren N, Bird SW et al. Differential and convergent utilization of autophagy components by positive-strand RNA viruses. PLoS Biol 2019; 17:e2006926 [View Article] [PubMed]
    [Google Scholar]
  33. Cong YY. Molecular Insights into Viral Respiratory Infections 212 Groningen: University of Groningen; 2019
    [Google Scholar]
  34. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T et al. Lc3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J 2000; 19:5720–5728 [View Article] [PubMed]
    [Google Scholar]
  35. Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H et al. The role of autophagy during the early neonatal starvation period. Nature 2004; 432:1032–1036 [View Article] [PubMed]
    [Google Scholar]
  36. Birgisdottir AB, Lamark T, Johansen T. The LIR motif - crucial for selective autophagy. J Cell Sci 2013; 126:3237–3247 [View Article] [PubMed]
    [Google Scholar]
  37. Johansen T, Lamark T. Selective autophagy: Atg8 family proteins, LIR motifs and cargo receptors. J Mol Biol 2020; 432:80–103 [View Article] [PubMed]
    [Google Scholar]
  38. Shvets E, Fass E, Scherz-Shouval R, Elazar Z. The N-terminus and Phe52 residue of LC3 recruit p62/SQSTM1 into autophagosomes. J Cell Sci 2008; 121:2685–2695 [View Article] [PubMed]
    [Google Scholar]
  39. Engedal N, Seglen PO. Autophagy of cytoplasmic bulk cargo does not require LC3. Autophagy 2016; 12:439–441 [View Article] [PubMed]
    [Google Scholar]
  40. Kanwal A, Kasetti S, Putcha UK, Asthana S, Banerjee SK. Protein kinase C-mediated sodium glucose transporter 1 activation in precondition-induced cardioprotection. Drug Des Devel Ther 2016; 10:2929–2938 [View Article] [PubMed]
    [Google Scholar]
  41. Keown JR, Black MM, Ferron A, Yap M, Barnett MJ et al. A helical LC3-interacting region mediates the interaction between the retroviral restriction factor TRIM5α and mammalian autophagy-related Atg8 proteins. J Biol Chem 2018; 293:18378–18386 [View Article] [PubMed]
    [Google Scholar]
  42. Beale R, Wise H, Stuart A, Ravenhill BJ, Digard P et al. A LC3-interacting motif in the influenza A virus M2 protein is required to subvert autophagy and maintain virion stability. Cell Host Microbe 2014; 15:239–247 [View Article] [PubMed]
    [Google Scholar]
  43. Kwon DH, Kim L, Kim BW, Kim JH, Roh KH et al. A novel conformation of the LC3-interacting region motif revealed by the structure of a complex between LC3B and RavZ. Biochem Biophys Res Commun 2017; 490:1093–1099 [View Article] [PubMed]
    [Google Scholar]
  44. Wirth M, Zhang W, Razi M, Nyoni L, Joshi D et al. Molecular determinants regulating selective binding of autophagy adapters and receptors to ATG8 proteins. Nat Commun 2019; 10:2055 [View Article] [PubMed]
    [Google Scholar]
  45. Poonsiri T, Wright GSA, Solomon T, Antonyuk SV. Crystal structure of the Japanese encephalitis virus capsid protein. Viruses 2019; 11:623 [View Article] [PubMed]
    [Google Scholar]
  46. Mackenzie J. Wrapping things up about virus RNA replication. Traffic 2005; 6:967–977 [View Article] [PubMed]
    [Google Scholar]
  47. Mackenzie JM, Kenney MT, Westaway EG. West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis. J Gen Virol 2007; 88:1163–1168 [View Article] [PubMed]
    [Google Scholar]
  48. Miorin L, Maiuri P, Hoenninger VM, Mandl CW, Marcello A. Spatial and temporal organization of tick-borne encephalitis flavivirus replicated RNA in living cells. Virology 2008; 379:64–77 [View Article] [PubMed]
    [Google Scholar]
  49. Gillespie LK, Hoenen A, Morgan G, Mackenzie JM. The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex. J Virol 2010; 84:10438–10447 [View Article] [PubMed]
    [Google Scholar]
  50. Mori Y, Okabayashi T, Yamashita T, Zhao Z, Wakita T et al. Nuclear localization of Japanese encephalitis virus core protein enhances viral replication. J Virol 2005; 79:3448–3458 [View Article] [PubMed]
    [Google Scholar]
  51. Wang SH, Syu WJ, Huang KJ, Lei HY, Yao CW et al. Intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus. J Gen Virol 2002; 83:3093–3102 [View Article] [PubMed]
    [Google Scholar]
  52. Westaway EG, Khromykh AA, Kenney MT, Mackenzie JM, Jones MK. Proteins C and NS4B of the flavivirus Kunjin translocate independently into the nucleus. Virology 1997; 234:31–41 [View Article] [PubMed]
    [Google Scholar]
  53. Lassen S, Grüttner C, Nguyen-Dinh V, Herker E. Perilipin-2 is critical for efficient lipoprotein and hepatitis C virus particle production. J Cell Sci 2019; 132:jcs217042 [View Article] [PubMed]
    [Google Scholar]
  54. Laufman O, Perrino J, Andino R. Viral generated inter-organelle contacts redirect lipid flux for genome replication. Cell 2019; 178:e216275–289 [View Article] [PubMed]
    [Google Scholar]
  55. Galluzzi L, Green DR. Autophagy-Independent functions of the autophagy machinery. Cell 2019; 177:1682–1699 [View Article] [PubMed]
    [Google Scholar]
  56. Leidal AM, Huang HH, Marsh T, Solvik T, Zhang D et al. The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles. Nat Cell Biol 2020; 22:187–199 [View Article] [PubMed]
    [Google Scholar]
  57. Exner T, Beretta CA, Gao Q, Afting C, Romero-Brey I et al. Lipid droplet quantification based on iterative image processing. J Lipid Res 2019; 60:1333–1344 [View Article] [PubMed]
    [Google Scholar]
  58. Sastry GM, Adzhigirey M, Day T, Annabhimoju R, Sherman W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des 2013; 27:221–234 [View Article] [PubMed]
    [Google Scholar]
  59. Mittal L, Srivastava M, Asthana S. Conformational characterization of linker revealed the mechanism of cavity formation by 227G in BVDV RdRP. J Phys Chem B 2019; 123:6150–6160 [View Article] [PubMed]
    [Google Scholar]
  60. Webb B, Sali A. Comparative protein structure modeling using modeller. Curr Protoc Bioinformatics 2016; 54:5 6 1–565 [View Article]
    [Google Scholar]
  61. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM. Aqua and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 1996; 8:477–486 [View Article] [PubMed]
    [Google Scholar]
  62. Srivastava M, Suri C, Singh M, Mathur R, Asthana S. Molecular dynamics simulation reveals the possible druggable hot-spots of USP7. Oncotarget 2018; 9:34289–34305 [View Article] [PubMed]
    [Google Scholar]
  63. Jiménez-García B, Pons C, Fernández-Recio J. pyDockWEB: a web server for rigid-body protein-protein docking using electrostatics and desolvation scoring. Bioinformatics 2013; 29:1698–1699 [View Article] [PubMed]
    [Google Scholar]
  64. Torchala M, Moal IH, Chaleil RAG, Fernandez-Recio J, Bates PA. SwarmDock: a server for flexible protein-protein docking. Bioinformatics 2013; 29:807–809 [View Article] [PubMed]
    [Google Scholar]
  65. Anang S, Kaushik N, Hingane S, Kumari A, Gupta J et al. Potent inhibition of hepatitis E virus release by a cyclic peptide inhibitor of the interaction between viral open reading frame 3 protein and host tumor susceptibility gene 101. J Virol 2018; 92: [View Article] [PubMed]
    [Google Scholar]
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