title: Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent, dynamin and caveola-mediated endocytosis pathway.
abstract: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.
authors.affiliation: Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai, China.
authors.forename: Yong-Zhe
authors.initials: YZ
authors.lastname: Zhu
authors.forename: Qing-Qiang
authors.initials: QQ
authors.lastname: Xu
authors.forename: Da-Ge
authors.initials: DG
authors.lastname: Wu
authors.forename: Hao
authors.initials: H
authors.lastname: Ren
authors.forename: Ping
authors.initials: P
authors.lastname: Zhao
authors.forename: Wen-Guang
authors.initials: WG
authors.lastname: Lao
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Qing-Yuan
authors.initials: QY
authors.lastname: Tao
authors.forename: Xi-Jing
authors.initials: XJ
authors.lastname: Qian
authors.forename: You-Heng
authors.initials: YH
authors.lastname: Wei
authors.forename: Ming-Mei
authors.initials: MM
authors.lastname: Cao
authors.forename: Zhong-Tian
authors.initials: ZT
authors.lastname: Qi
mesh_terms: D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D002966:Clathrin / Q000502:physiology; D017931:DNA Primers; D034281:Dynamins / Q000502:physiology*; D004664:Encephalitis Virus, Japanese / Q000502:physiology*; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D009447:Neuroblastoma / Q000473:pathology / Q000821:virology*; D034741:RNA, Small Interfering; D051381:Rats; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics
chemical_list: D002966:Clathrin; D017931:DNA Primers; D034741:RNA, Small Interfering; D025262:Vacuolar Proton-Translocating ATPases; D034281:Dynamins