Research Article

Caveolin-1-mediated Japanese encephalitis virus entry requires a two-step regulation of actin reorganization

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China

^‡Authors contributed equally

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Mingmei Cao^‡

Department of Medical Microbiology & Parasitology, Second Military Medical University, Shanghai 200433, China

^‡Authors contributed equally

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Hongyuan Song^‡

Department of Ophthalmology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China

^‡Authors contributed equally

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Shenglin Chen

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China

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Xijing Qian

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China

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Ping Zhao

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China

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Hao Ren

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China

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Hailin Tang

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China

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Yan Wang

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China

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Youheng Wei

State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China

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Yongzhe Zhu

*Author for correspondence:

E-mail Address: zhuyongzhe1984@sina.com

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China

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Zhongtian Qi

**Author for correspondence:

E-mail Address: qizt@smmu.edu.cn

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China

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Published Online:17 Mar 2016https://doi.org/10.2217/fmb-2016-0002

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Abstract

Aim: To investigate the detailed mechanism of Japanese encephalitis virus (JEV) cell entry. Materials & methods: Utilize a siRNA library targeting cellular membrane trafficking genes to identify key molecules that mediate JEV entry into human neuronal cells. Results: JEV enters human neuronal cells by caveolin-1-mediated endocytosis, which depends on a two-step regulation of actin cytoskeleton remodeling triggered by RhoA and Rac1: RhoA activation promoted the phosphorylation of caveolin-1, and then Rac1 activation facilitated caveolin-associated viral internalization. Specifically, virus attachment activates the EGFR–PI3K signaling pathway, thereby leading to RhoA activation. Conclusion: This work provides a detailed picture of the entry route and intricate cellular events following the entry of JEV into human neuronal cells, and promotes a better understanding of JEV entry.

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Papers of special note have been highlighted as: • of interest; •• of considerable interest

References

1 Unni SK, Růžek D, Chhatbar C, Mishra R, Johri MK, Singh SK. Japanese encephalitis virus: from genome to infectome. Microb. Infect. 13(4), 312–321 (2011). • Comprehensive review on the Japanese Encephalitis Virus (JEV) life cycle, pathogenesis and host immune responses to JEV infection.
Medline CASGoogle Scholar
2 Yun S-I, Lee Y-M. Japanese encephalitis: the virus and vaccines. Hum. Vaccin. Immunother. 10(2), 263–279 (2014).
Medline CASGoogle Scholar
3 Hotez PJ, Bottazzi ME, Strych U et al. Neglected tropical diseases among the Association of Southeast Asian Nations (ASEAN): overview and update. PLoS Negl. Trop. Dis. 9(4), e0003575 (2015).
MedlineGoogle Scholar
4 Campbell GL, Hills SL, Fischer M et al. Estimated global incidence of Japanese encephalitis: a systematic review. Bull. World Health Organ. 89(10), 766–774 (2011).
MedlineGoogle Scholar
5 Misra UK, Kalita J. Overview: Japanese encephalitis. Prog. Neurobiol. 91(2), 108–120 (2010).
Medline CASGoogle Scholar
6 Schelhaas M. Come in and take your coat off – how host cells provide endocytosis for virus entry. Cell. Microbiol. 12(10), 1378–1388 (2010).
Medline CASGoogle Scholar
7 Acosta EG, Castilla V, Damonte EB. Alternative infectious entry pathways for dengue virus serotypes into mammalian cells. Cell. Microbiol. 11(10), 1533–1549 (2009).
Medline CASGoogle Scholar
8 Chu J, Leong P, Ng M. Analysis of the endocytic pathway mediating the infectious entry of mosquito-borne flavivirus west Nile into Aedes albopictus mosquito (C6/36) cells. Virology 349(2), 463–475 (2006).
Medline CASGoogle Scholar
9 Nawa M, Takasaki T, Yamada K-I, Kurane I, Akatsuka T. Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug, chlorpromazine. J. Gen. Virol. 84(7), 1737–1741 (2003).
Medline CASGoogle Scholar
10 Chuang C-K, Yang T-H, Chen T-H, Yang C-F, Chen W-J. Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells. J. Gen. Virol. 96(Pt 4), 793–803 (2015).
Medline CASGoogle Scholar
11 Das S, Chakraborty S, Basu A. Critical role of lipid rafts in virus entry and activation of phosphoinositide 3′ kinase/Akt signaling during early stages of Japanese encephalitis virus infection in neural stem/progenitor cells. J. Neurochem. 115(2), 537–549 (2010).
Medline CASGoogle Scholar
12 Kalia M, Khasa R, Sharma M, Nain M, Vrati S. Japanese encephalitis virus infects neuronal cells through a clathrin-independent endocytic mechanism. J. Virol. 87(1), 148–162 (2013).
Medline CASGoogle Scholar
13 Zhu Y-Z, Xu Q-Q, Wu D-G et al. Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent, dynamin and caveola-mediated endocytosis pathway. J. Virol. 86(24), 13407–13422 (2012).
Medline CASGoogle Scholar
14 Taylor MP, Koyuncu OO, Enquist LW. Subversion of the actin cytoskeleton during viral infection. Nat. Rev. Microbiol. 9(6), 427–439 (2011). •• Describe how the interaction of viral proteins with the cell modulates the structure and function of the actin cytoskeleton to initiate, sustain and spread infections.
Medline CASGoogle Scholar
15 Lehmann MJ, Sherer NM, Marks CB, Pypaert M, Mothes W. Actin-and myosin-driven movement of viruses along filopodia precedes their entry into cells. J. Cell Biol. 170(2), 317–325 (2005).
Medline CASGoogle Scholar
16 Mercer J, Schelhaas M, Helenius A. Virus entry by endocytosis. Annu. Rev. Biochem. 79(79), 803–833 (2010). • Comprehensive review on the cell biology of virus entry and the different strategies and endocytic mechanisms used by animal viruses.
Medline CASGoogle Scholar
17 Wurth MA, Schowalter RM, Smith EC, Moncman CL, Dutch RE, Mccann RO. The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell–cell fusion. Virology 404(1), 117–126 (2010).
Medline CASGoogle Scholar
18 Brazzoli M, Bianchi A, Filippini S et al. CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes. J. Virol. 82(17), 8316–8329 (2008).
Medline CASGoogle Scholar
19 Cureton DK, Massol RH, Saffarian S, Kirchhausen TL, Whelan SP. Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization. PLoS Pathog. 5(4), e1000394–e1000394 (2009).
MedlineGoogle Scholar
20 Sum H, Sia M. The involvement of microtubules and actin during the infection of Japanese encephalitis virus in neuroblastoma cell line, IMR32. BioMed. Res. Int. 2015, 695283 (2015).
MedlineGoogle Scholar
21 Hall A. Rho GTPases and the actin cytoskeleton. Science 279(5350), 509–514 (1998).
Medline CASGoogle Scholar
22 Freeman MC, Peek CT, Becker MM, Smith EC, Denison MR. Coronaviruses induce entry-independent, continuous macropinocytosis. MBio 5(4), e01340–e01314 (2014).
Medline CASGoogle Scholar
23 Munter S, Way M, Frischknecht F. Signaling during pathogen infection. Sci. Signal. 2006(335), re5 (2006).
Google Scholar
24 Cao H, Thompson H, Krueger E, Mcniven M. Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin. J. Cell Sci. 113(11), 1993–2002 (2000).
Medline CASGoogle Scholar
25 Benmerah A, Bayrou M, Cerf-Bensussan N, Dautry-Varsat A. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Sci. 112(9), 1303–1311 (1999).
Medline CASGoogle Scholar
26 Coyne CB, Bergelson JM. Virus-induced Abl and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions. Cell 124(1), 119–131 (2006). •• Extensive investigation revealing the tyrosine kinase activity and the distinct intracellular signals were triggered by viruses during the entry process.
Medline CASGoogle Scholar
27 Patel KP, Coyne CB, Bergelson JM. Dynamin-and lipid raft-dependent entry of decay-accelerating factor (DAF)-binding and non-DAF-binding coxsackieviruses into nonpolarized cells. J. Virol. 83(21), 11064–11077 (2009).
Medline CASGoogle Scholar
28 Subauste MC, Von Herrath M, Benard V et al. Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas. J. Biol. Chem. 275(13), 9725–9733 (2000).
Medline CASGoogle Scholar
29 Leong KLJ, Ng MM-L, Chu JJH. The essential role of clathrin-mediated endocytosis in the infectious entry of human enterovirus 71. J. Biol. Chem. 286(1), 309–321 (2011).
MedlineGoogle Scholar
30 Zhang J-H, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4(2), 67–73 (1999).
Medline CASGoogle Scholar
31 Tani H, Shiokawa M, Kaname Y et al. Involvement of ceramide in the propagation of Japanese encephalitis virus. J. Virol. 84(6), 2798–2807 (2010).
Medline CASGoogle Scholar
32 Aoki T, Nomura R, Fujimoto T. Tyrosine phosphorylation of caveolin-1 in the endothelium. Exp. Cell Res. 253(2), 629–636 (1999).
Medline CASGoogle Scholar
33 Bernstein BW, Bamburg JR. ADF/cofilin: a functional node in cell biology. Trends Cell Biol. 20(4), 187–195 (2010).
Medline CASGoogle Scholar
34 Niwa R, Nagata-Ohashi K, Takeichi M, Mizuno K, Uemura T. Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108(2), 233–246 (2002).
Medline CASGoogle Scholar
35 Spiering D, Hodgson L. Dynamics of the Rho-family small GTPases in actin regulation and motility. Cell Adh. Migr. 5(2), 170–180 (2011). • Describe the Rho family GTPase biology and the dynamics of these important signaling proteins that ultimately coordinate the actin cytoskeleton rearrangements.
MedlineGoogle Scholar
36 Pelkmans L, Fava E, Grabner H et al. Genome-wide analysis of human kinases in clathrin-and caveolae/raft-mediated endocytosis. Nature 436(7047), 78–86 (2005).
Medline CASGoogle Scholar
37 Prenzel N, Fischer O, Streit S, Hart S, Ullrich A. The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr. Relat. Cancer 8(1), 11–31 (2001).
Medline CASGoogle Scholar
38 Marsh M, Helenius A. Virus entry: open sesame. Cell 124(4), 729–740 (2006).
Medline CASGoogle Scholar
39 Echarri A, Muriel O, Pavón DM et al. Caveolar domain organization and trafficking is regulated by Abl kinases and mDia1. J. Cell Sci. 125(13), 3097–3113 (2012).
Medline CASGoogle Scholar
40 Muriel O, Echarri A, Hellriegel C, Pavón DM, Beccari L, Del Pozo MA. Phosphorylated filamin A regulates actin-linked caveolae dynamics. J. Cell Sci. 124(16), 2763–2776 (2011).
Medline CASGoogle Scholar
41 Sverdlov M, Shajahan AN, Minshall RD. Tyrosine phosphorylation-dependence of caveolae-mediated endocytosis. J. Cell. Mol. Med. 11(6), 1239–1250 (2007).
Medline CASGoogle Scholar
42 Pelkmans L, Püntener D, Helenius A. Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 296(5567), 535–539 (2002).
Medline CASGoogle Scholar
43 Favoreel HW, Enquist L, Feierbach B. Actin and Rho GTPases in herpesvirus biology. Trends Microbiol. 15(9), 426–433 (2007).
Medline CASGoogle Scholar
44 Hoppe S, Schelhaas M, Jaeger V, Liebig T, Petermann P, Knebel-Mörsdorf D. Early herpes simplex virus type 1 infection is dependent on regulated Rac1/Cdc42 signalling in epithelial MDCKII cells. J. Gen. Virol. 87(12), 3483–3494 (2006).
Medline CASGoogle Scholar
45 Zheng K, Xiang Y, Wang X et al. Epidermal growth factor receptor–PI3K signaling controls cofilin activity to facilitate herpes simplex virus 1 entry into neuronal cells. MBio 5(1), e00958–e00913 (2014).
MedlineGoogle Scholar
46 Huang WR, Wang YC, Chi PI et al. Cell entry of avian reovirus follows a caveolin-1-mediated and dynamin-2-dependent endocytic pathway that requires activation of p38 mitogen-activated protein kinase (MAPK) and Src signaling pathways as well as microtubules and small GTPase Rab5 protein. J. Biol. Chem. 286(35), 30780–30794 (2011).
Medline CASGoogle Scholar
47 Vehlow A, Soong D, Vizcay-Barrena G et al. Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis. EMBO J. 32(20), 2722–2734 (2013).
Medline CASGoogle Scholar
48 Zheng K, Kitazato K, Wang Y. Viruses exploit the function of epidermal growth factor receptor. Rev. Med. Virol. 24(4), 274–286 (2014). • Overview of the mechanisms by which viruses alter EGFR signalling in favor of their entry, replication, inflammation and viral antagonism to the host antiviral system.
Medline CASGoogle Scholar
49 Zona L, Lupberger J, Sidahmed-Adrar N et al. HRas signal transduction promotes hepatitis C virus cell entry by triggering assembly of the host tetraspanin receptor complex. Cell Host Microbe 13(3), 302–313 (2013).
Medline CASGoogle Scholar

Vol. 11, No. 10

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Published online 17 March 2016

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Acknowledgements

The authors are grateful to Yongxin Yu (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) for the JEV SA14 strain.

Financial & competing interests disclosure

This work was funded by the National Natural Science Foundation of China (31370196, 81221061, 81273557 and 81302812), the Military S&T Project (AWS11C001), Medical Youth Science Program (13QNP100), Shanghai Municipal Natural Science Foundation (13ZR1449300) and the National S&T Major Project for Infectious Diseases Control (2013ZX10004104). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Conflict of interest

There are no conflicts of interest to disclose for all authors.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

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