1887

Abstract

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is one of the leading global causes of virus-induced encephalitis. The infectious life-cycle of viruses is heavily dependent on the host membrane trafficking network. Here, we have performed a RNA-interference-based screen using a siRNA panel targeting 136 membrane trafficking proteins to identify the key regulators of JEV infection in HeLa cells. We identified 35 proteins whose siRNA depletion restricts JEV replication by over twofold. We observe that JEV infection in HeLa cells is largely dependent on components of the clathrin-mediated endocytic (CME) pathway. Proteins involved in actin-filament-based processes, specifically CDC42 and members of the ARP2/3 complex are crucial for establishment of infection. Pharmacological pertubations of actin polymerization, a small molecule inhibitor of actin nucleation by the ARP2/3 complex – CK-548 – and the inhibitor of neural Wiskott–Aldrich syndrome proteins– Wiskostatin– inhibited JEV replication, highlighting the important role of the dynamic actin network. Other proteins involved in cargo-recognition for CME and endomembrane system organization were also validated as essential host factors for virus replication.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001182
2018-11-29
2024-03-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/100/2/176.html?itemId=/content/journal/jgv/10.1099/jgv.0.001182&mimeType=html&fmt=ahah

References

  1. Campbell GL, Hills SL, Fischer M, Jacobson JA, Hoke CH et al. Estimated global incidence of Japanese encephalitis: a systematic review. Bull World Health Organ 2011; 89:766-774–774A-774E [View Article][PubMed]
    [Google Scholar]
  2. Daep CA, Muñoz-Jordán JL, Eugenin EA. Flaviviruses, an expanding threat in public health: focus on dengue, West Nile, and Japanese encephalitis virus. J Neurovirol 2014; 20:539–560 [View Article][PubMed]
    [Google Scholar]
  3. Ma L, Li F, Zhang JW, Li W, Zhao DM et al. Host factor SPCS1 regulates the replication of Japanese encephalitis virus through interactions with transmembrane domains of NS2B. J Virol 2018; 92: [View Article][PubMed]
    [Google Scholar]
  4. 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]
  5. Perera-Lecoin M, Meertens L, Carnec X, Amara A. Flavivirus entry receptors: an update. Viruses 2014; 6:69–88 [View Article]
    [Google Scholar]
  6. Taguwa S, Maringer K, Li X, Bernal-Rubio D, Rauch JN et al. Defining hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection. Cell 2015; 163:1108–1123 [View Article][PubMed]
    [Google Scholar]
  7. Wang QY, Shi PY. Flavivirus entry inhibitors. ACS Infect Dis 2015; 1:428–434 [View Article][PubMed]
    [Google Scholar]
  8. Cossart P, Helenius A. Endocytosis of viruses and bacteria. Cold Spring Harb Perspect Biol 2014; 6:a016972 [View Article][PubMed]
    [Google Scholar]
  9. Kalia M, Jameel S. Virus entry paradigms. Amino Acids 2011; 41:1147–1157 [View Article][PubMed]
    [Google Scholar]
  10. Yamauchi Y, Helenius A. Virus entry at a glance. J Cell Sci 2013; 126:1289–1295 [View Article][PubMed]
    [Google Scholar]
  11. Cruz-Oliveira C, Freire JM, Conceição TM, Higa LM, Castanho MA et al. Receptors and routes of dengue virus entry into the host cells. FEMS Microbiol Rev 2015; 39:155–170 [View Article][PubMed]
    [Google Scholar]
  12. de Vries E, Tscherne DM, Wienholts MJ, Cobos-Jiménez V, Scholte F et al. Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway. PLoS Pathog 2011; 7:e1001329 [View Article][PubMed]
    [Google Scholar]
  13. Mercer J, Helenius A. Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells. Science 2008; 320:531–535 [View Article][PubMed]
    [Google Scholar]
  14. Veettil MV, Bandyopadhyay C, Dutta D, Chandran B. Interaction of KSHV with host cell surface receptors and cell entry. Viruses 2014; 6:4024–4046 [View Article][PubMed]
    [Google Scholar]
  15. Kalia M, Khasa R, Sharma M, Nain M, Vrati S. Japanese encephalitis virus infects neuronal cells through a clathrin-independent endocytic mechanism. J Virol 2013; 87:148–162 [View Article][PubMed]
    [Google Scholar]
  16. Nain M, Abdin MZ, Kalia M, Vrati S. Japanese encephalitis virus invasion of cell: allies and alleys. Rev Med Virol 2016; 26:129–141 [View Article][PubMed]
    [Google Scholar]
  17. Xu Q, Cao M, Song H, Chen S, Qian X et al. Caveolin-1-mediated Japanese encephalitis virus entry requires a two-step regulation of actin reorganization. Future Microbiol 2016; 11:1227–1248 [View Article][PubMed]
    [Google Scholar]
  18. Yang S, He M, Liu X, Li X, Fan B et al. Japanese encephalitis virus infects porcine kidney epithelial PK15 cells via clathrin- and cholesterol-dependent endocytosis. Virol J 2013; 10:258 [View Article][PubMed]
    [Google Scholar]
  19. Zhu YZ, Xu QQ, Wu DG, Ren H, Zhao P et al. Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent, dynamin and caveola-mediated endocytosis pathway. J Virol 2012; 86:13407–13422 [View Article][PubMed]
    [Google Scholar]
  20. Liu CC, Zhang YN, Li ZY, Hou JX, Zhou J et al. Rab5 and rab11 are required for clathrin-dependent endocytosis of japanese encephalitis virus in bhk-21 cells. J Virol 2017; 91: [View Article][PubMed]
    [Google Scholar]
  21. Nawa M. Japanese encephalitis virus infection in Vero cells: the involvement of intracellular acidic vesicles in the early phase of viral infection was observed with the treatment of a specific vacuolar type H+-ATPase inhibitor, bafilomycin A1. Microbiol Immunol 1997; 41:537–543 [View Article][PubMed]
    [Google Scholar]
  22. Taniguchi M, Tasaki T, Ninomiya H, Ueda Y, Kuremoto KI et al. Sphingomyelin generated by sphingomyelin synthase 1 is involved in attachment and infection with Japanese encephalitis virus. Sci Rep 2016; 6:37829 [View Article][PubMed]
    [Google Scholar]
  23. Nawa M, Takasaki T, Yamada K, Kurane I, Akatsuka T. Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug, chlorpromazine. J Gen Virol 2003; 84:1737–1741 [View Article][PubMed]
    [Google Scholar]
  24. Tani H, Shiokawa M, Kaname Y, Kambara H, Mori Y et al. Involvement of ceramide in the propagation of Japanese encephalitis virus. J Virol 2010; 84:2798–2807 [View Article][PubMed]
    [Google Scholar]
  25. Tripathi S, Pohl MO, Zhou Y, Rodriguez-Frandsen A, Wang G et al. Meta- and orthogonal integration of influenza "omics" data defines a role for ubr4 in virus budding. Cell Host Microbe 2015; 18:723–735 [View Article][PubMed]
    [Google Scholar]
  26. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 2015; 43:D447–D452 [View Article][PubMed]
    [Google Scholar]
  27. Kaksonen M, Toret CP, Drubin DG. Harnessing actin dynamics for clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 2006; 7:404–414 [View Article][PubMed]
    [Google Scholar]
  28. Ridley AJ. Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol 2006; 16:522–529 [View Article][PubMed]
    [Google Scholar]
  29. Merrifield CJ, Qualmann B, Kessels MM, Almers W. Neural wiskott aldrich syndrome protein (n-wasp) and the arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts. Eur J Cell Biol 2004; 83:13–18 [View Article][PubMed]
    [Google Scholar]
  30. Pizarro-Cerdá J, Chorev DS, Geiger B, Cossart P. The diverse family of arp2/3 complexes. Trends Cell Biol 2017; 27:93–100 [View Article][PubMed]
    [Google Scholar]
  31. Rotty JD, Wu C, Bear JE. New insights into the regulation and cellular functions of the ARP2/3 complex. Nat Rev Mol Cell Biol 2013; 14:7–12 [View Article][PubMed]
    [Google Scholar]
  32. Votteler J, Sundquist WI. Virus budding and the ESCRT pathway. Cell Host Microbe 2013; 14:232–241 [View Article][PubMed]
    [Google Scholar]
  33. Quan A, Robinson PJ. Syndapin-a membrane remodelling and endocytic F-BAR protein. FEBS J 2013; 280:5198–5212 [View Article][PubMed]
    [Google Scholar]
  34. Pan S, Wang R, Zhou X, He G, Koomen J et al. Involvement of the conserved adaptor protein Alix in actin cytoskeleton assembly. J Biol Chem 2006; 281:34640–34650 [View Article][PubMed]
    [Google Scholar]
  35. Goddette DW, Frieden C. Actin polymerization. The mechanism of action of cytochalasin D. J Biol Chem 1986; 261:15974–15980[PubMed]
    [Google Scholar]
  36. Spector I, Shochet NR, Kashman Y, Groweiss A. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science 1983; 219:493–495 [View Article][PubMed]
    [Google Scholar]
  37. Bubb MR, Senderowicz AM, Sausville EA, Duncan KL, Korn ED. Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J Biol Chem 1994; 269:14869–14871[PubMed]
    [Google Scholar]
  38. Nolen BJ, Tomasevic N, Russell A, Pierce DW, Jia Z et al. Characterization of two classes of small molecule inhibitors of Arp2/3 complex. Nature 2009; 460:1031–1034 [View Article][PubMed]
    [Google Scholar]
  39. Peterson JR, Lokey RS, Mitchison TJ, Kirschner MW. A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions. Proc Natl Acad Sci USA 2001; 98:10624–10629 [View Article][PubMed]
    [Google Scholar]
  40. Kirchhausen T, Owen D, Harrison SC. Molecular structure, function, and dynamics of clathrin-mediated membrane traffic. Cold Spring Harb Perspect Biol 2014; 6:a016725 [View Article][PubMed]
    [Google Scholar]
  41. Yarar D, Waterman-Storer CM, Schmid SL. A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis. Mol Biol Cell 2005; 16:964–975 [View Article][PubMed]
    [Google Scholar]
  42. Wolfe BL, Trejo J. Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis. Traffic 2007; 8:462–470 [View Article][PubMed]
    [Google Scholar]
  43. Aghamohammadzadeh S, Ayscough KR. Differential requirements for actin during yeast and mammalian endocytosis. Nat Cell Biol 2009; 11:1039–1042 [View Article][PubMed]
    [Google Scholar]
  44. Boulant S, Kural C, Zeeh JC, Ubelmann F, Kirchhausen T. Actin dynamics counteract membrane tension during clathrin-mediated endocytosis. Nat Cell Biol 2011; 13:1124–1131 [View Article][PubMed]
    [Google Scholar]
  45. Skruzny M, Brach T, Ciuffa R, Rybina S, Wachsmuth M et al. Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis. Proc Natl Acad Sci USA 2012; 109:E2533E2542 [View Article][PubMed]
    [Google Scholar]
  46. Humphries AC, Way M. The non-canonical roles of clathrin and actin in pathogen internalization, egress and spread. Nat Rev Microbiol 2013; 11:551–560 [View Article][PubMed]
    [Google Scholar]
  47. Piccinotti S, Kirchhausen T, Whelan SP. Uptake of rabies virus into epithelial cells by clathrin-mediated endocytosis depends upon actin. J Virol 2013; 87:11637–11647 [View Article][PubMed]
    [Google Scholar]
  48. Taylor MP, Koyuncu OO, Enquist LW. Subversion of the actin cytoskeleton during viral infection. Nat Rev Microbiol 2011; 9:427–439 [View Article][PubMed]
    [Google Scholar]
  49. Cureton DK, Massol RH, Whelan SP, Kirchhausen T. The length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis. PLoS Pathog 2010; 6:e1001127 [View Article][PubMed]
    [Google Scholar]
  50. Hackett BA, Cherry S. Flavivirus internalization is regulated by a size-dependent endocytic pathway. Proc Natl Acad Sci USA 2018; 115:4246–4251 [View Article][PubMed]
    [Google Scholar]
  51. Swaine T, Dittmar MT. CDC42 use in viral cell entry processes by RNA Viruses. Viruses 2015; 7:6526–6536 [View Article][PubMed]
    [Google Scholar]
  52. Pollard TD. Regulation of actin filament assembly by Arp2/3 complex and formins. Annu Rev Biophys Biomol Struct 2007; 36:451–477 [View Article][PubMed]
    [Google Scholar]
  53. Pollard TD, Beltzner CC. Structure and function of the Arp2/3 complex. Curr Opin Struct Biol 2002; 12:768–774 [View Article][PubMed]
    [Google Scholar]
  54. Welch MD, Rosenblatt J, Skoble J, Portnoy DA, Mitchison TJ. Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science 1998; 281:105–108 [View Article][PubMed]
    [Google Scholar]
  55. Cossart P. Actin-based motility of pathogens: the Arp2/3 complex is a central player. Cell Microbiol 2000; 2:195–205 [View Article][PubMed]
    [Google Scholar]
  56. Cudmore S, Cossart P, Griffiths G, Way M. Actin-based motility of vaccinia virus. Nature 1995; 378:636–638 [View Article][PubMed]
    [Google Scholar]
  57. Guerriero CJ, Weisz OA. N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels. Am J Physiol Cell Physiol 2007; 292:C1562–C1566 [View Article][PubMed]
    [Google Scholar]
  58. Vrati S, Agarwal V, Malik P, Wani SA, Saini M. Molecular characterization of an Indian isolate of Japanese encephalitis virus that shows an extended lag phase during growth. J Gen Virol 1999; 80:1665–1671 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001182
Loading
/content/journal/jgv/10.1099/jgv.0.001182
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error