Involvement of ceramide in the propagation of Japanese encephalitis virus

Abstract

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus and one of the most important flaviviruses in the medical and veterinary fields. Although cholesterol has been shown to participate in both the entry and replication steps of JEV, the mechanisms of infection, including the cellular receptors of JEV, remain largely unknown. To clarify the infection mechanisms of JEV, we generated pseudotype (JEVpv) and recombinant (JEVrv) vesicular stomatitis viruses bearing the JEV envelope protein. Both JEVpv and JEVrv exhibited high infectivity for the target cells, and JEVrv was able to propagate and form foci as did authentic JEV. Anti-JEV envelope antibodies neutralized infection of the viruses. Treatment of cells with inhibitors for vacuolar ATPase and clathrin-mediated endocytosis reduced the infectivity of JEVpv, suggesting that JEVpv enters cells via pH- and clathrin-dependent endocytic pathways. Although treatment of the particles of JEVpv, JEVrv, and JEV with cholesterol drastically reduced the infectivity as previously reported, depletion of cholesterol from the particles by treatment with methyl beta-cyclodextrin enhanced infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and propagation of JEVrv, and these enhancements were inhibited by treatment with an SMase inhibitor or C(6)-ceramide. These results suggest that ceramide plays crucial roles in not only entry but also egress processes of JEV, and they should assist in the clarification of JEV propagation and the development of novel therapeutics against diseases caused by infection with flaviviruses.

FIG. 1.

Schematic representation of the genome structures and production of recombinant and pseudotype VSVs. (A) The luciferase, PrME, and E1E2 genes were inserted into the full-length cDNA clone of VSV in place of the G gene and designated ΔG-Luc, ΔG-JEV, and ΔG-HCV, respectively. (B) Recombinant VSVs, JEVrv, HCVrv, and ΔG, bearing the JEV E protein, HCV E1/E2 proteins, and no envelope, respectively, were generated in 293T or Huh7 cells by infection with the respective recombinant VSV after complementation with VSV G protein (*G). (C) Pseudotype VSVs, JEVpv, VSVpv, HCVpv, and MLVpv, were generated by infection with VSVΔG/Luc-*G in 293T or Huh7 cells transiently expressing the respective foreign protein.

FIG. 2.

Characterization of JEVrv and JEVpv. (A) JEV E proteins expressed in cells incorporated into the viral particles were treated with endoglycosidase H (H) or peptide-N-glycosidase F (F) and examined by immunoblotting using anti-E polyclonal antibody. “-” indicates an untreated sample. (B) Infectivities of recombinant viruses (left panel) and pseudotype viruses (right panel) were determined in Huh7, BHK, and Vero cells by a focus-forming assay and measurement of luciferase activity (RLU), respectively. VSV without envelope (ΔG) was used as a negative control. ffu, focus-forming units. (C) Neutralization of JEVrv (left panel) or JEVpv (right panel) infection by anti-E polyclonal antibody. Viruses were incubated with the indicated dilution of antibody for 1 h at room temperature and inoculated into Huh7 cells. Residual infectivities are expressed as percentages. VSV and VSVpv were used as controls. The results shown are from three independent assays, with error bars representing standard deviations.

FIG. 3.

Entry pathways of the pseudotype VSVs. Huh7 cells were pretreated with various concentrations of bafilomycin A₁ (A), chlorpromazine (B), or methyl-β-cyclodextrin (C) for 1 h and inoculated with the pseudotype viruses, JEVpv, HCVpv, VSVpv, and MLVpv. Luciferase activities were determined at 24 h postinfection. The results shown are from three independent assays, with error bars representing standard deviations.

FIG. 4.

Effects of cholesterol on infection with recombinant and pseudotype VSVs. (A) The pseudotype viruses were incubated with various concentrations of cholesterol for 1 h at room temperature and inoculated into Huh7 cells, and luciferase activities were determined at 24 h postinfection. (B) JEV, JEVrv, HCVrv, and VSV were incubated with various concentrations of cholesterol for 1 h at room temperature and inoculated into Huh7 cells, and residual infectivities were determined by focus-forming assay in a culture medium containing 1% methylcellulose at 48 h postinfection for JEV, JEVrv, and HCVrv and at 24 h postinfection for VSV. Foci of infected cells were detected by immunohistochemical staining (lower panel). The rate of focus formation of the viruses was analyzed by counting foci. The results shown are from three independent assays, with error bars representing standard deviations.

FIG. 5.

Effects of SMase and amitriptyline treatment of cells on infection with pseudotype and recombinant VSVs. Huh7 cells were pretreated with various concentrations of SMase for 1 h, and then pseudotype viruses (A) or recombinant viruses (B) were inoculated. The infectivities were determined by luciferase activity measurement or focus-forming assay, and changes in infectivities are expressed as percentages. (C) The purified pseudotype particles were treated with various concentrations of SMase for 1 h and inoculated into Huh7 cells after removal of SMase by ultracentrifugation. Infectivities were determined at 24 h postinfection by measuring luciferase activity, and changes in infectivities are expressed as percentages. (D) Huh7 cells were pretreated with various concentrations of amitriptyline, an inhibitor for the acid SMase, for 1 h, and then pseudotype viruses were inoculated. Infectivities were determined at 24 h postinfection by measuring luciferase activity, and changes in infectivities are expressed as percentages. The results shown are from three independent assays, with error bars representing standard deviations.

FIG. 6.

Effects of SMase on the propagation of JEVrv and JEV. Huh7 cells were pretreated with various concentrations of SMase for 1 h and inoculated with JEVrv or HCVrv (A) or JEV or VSV (B), and infectivities were determined by focus-forming assay in a culture medium containing 1% methylcellulose at 48 h after infection with JEVrv, HCVrv, and JEV and at 24 h after infection with VSV. Titers were determined by counts of foci detected by immunohistochemical staining (lower panels). The results shown are from three independent assays, with error bars representing standard deviations.

FIG. 7.

Involvement of ceramide in infection with JEV. (A) Effects of C₆-ceramide or sphingomyelin in infection with JEVpv. Purified pseudotype viruses were pretreated with various concentrations of C₆-ceramide (upper) or sphingomyelin (lower) for 1 h and then inoculated into Huh7 cells. The infectivities were determined at 24 h postinfection by luciferase activity, and changes in infectivities are expressed as percentages. (B) Effects of C₆-ceramide in infection of JEVrv and JEV. JEV and VSV were pretreated with various concentrations of C₆-ceramide for 1 h, and then the viruses were inoculated into Huh7 cells. At 24 h postinfection, the infectivities were determined by focus-forming assay. (C) Binding of JEV and JEVrv to ceramide beads. Purified viruses were preincubated with (+) or without (−) biotin-ceramide resolved in DMSO and streptavidin-Sepharose 4B. After washing, residual pellets were analyzed by immunoblotting. Inputs are purified viruses. The results shown are from three independent assays, with error bars representing standard deviations.