Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway

Abstract

bJapanese 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.

Entry of Classical Swine Fever Virus into PK-15 Cells via a pH-, Dynamin-, and Cholesterol-Dependent, Clathrin-Mediated Endocytic Pathway That Requires Rab5 and Rab7

“…Following pretreatment with 25 M chloroquine or 10 mM NH 4 Cl, viral RNA copy numbers of CSFV were reduced to 6.6% or 31.6% of those in untreated cells, whereas viral RNA copy numbers of JEV, another member of the Flaviviridae, used as a control, remained unchanged. In contrast, JEV infection was significantly inhibited by chloroquine or NH 4 Cl treatment in Vero cells (data not shown), consistent with data from a previous report (33). Taken together, the results indicated that CSFV infection required low-pH environments in the endosomes.…”

“…More than 70% inhibition of CSFV infection with 160 M dynasore was observed. Consistent with previously reported results (33), JEV infection was effectively blocked by dynasore treatment at 160 M (Fig. 2C).…”

“…The lipid raft is readily disrupted if membrane cholesterol is selectively extracted by pharmacological agents such as M␤CD or filipin (52,53). Depletion of cholesterol inhibits JEV during the early stages of infection (33,38,54). We conducted a series of experiments to determine if cholesterol is involved in CSFV infection.…”

“…Dynamin is a 100-kDa GTPase that plays an essential role in cellular membrane fission during vesicle formation and therefore is required for clathrinand caveola-mediated endocytosis (33,(39)(40)(41)(42). Dynamin has been shown to be required for the internalization of numerous viruses (43)(44)(45)(46).…”

“…PK-15 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 g/ml streptomycin, and 100 U/ml penicillin (Gibco, Invitrogen). Cells of the rat neuroblastoma line B104, a gift from Zhong-Tian Qi (33), were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS with 100 g/ml streptomycin and 100 U/ml penicillin. The virulent CSFV Shimen strain (GenBank accession number AF092448) was obtained from the National Institute of Veterinary Drug Control (Beijing, China).…”