Development and Applications of VSV Vectors Based on Cell Tropism

Tani, Hideki; Morikawa, Shigeru; Matsuura, Yoshiharu

Japanese encephalitis virus, a mosquito-borne zoonotic pathogen, is the leading cause of viral encephalitis in humans, with ∼50,000 cases reported annually worldwide. JEV is an enveloped virus belonging to the family Flaviviridae and the genus Flavivirus, which also includes Dengue virus, West Nile virus, Yellow fever virus, and Tick-borne encephalitis virus (Gubler et al., 2007) . The genome consists of a single-stranded positive-sense RNA of approximately 11 kb, encoding a single large polyprotein, which is cleaved by host-and virus-encoded proteases into three structural (C, PrM, and E) and non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The envelope protein (E) is a 53-kDa glycoprotein, which is a major component of the virion surface and has been found to be associated with all the biological properties of the virus, such as attachment to cellular receptors, penetration, fusion with the endosomal membrane, host cell range and cell tropism, and neutralization to antibodies. Although a number of cellular components that interacted with E protein, such as heat shock cognate protein 70 (Ren et al., 2007) , heat shock protein 70 (Das et al., 2009) , vimentin (Das et al., 2011) , glycosaminoglycans (Su et al., 2001; Lee and Lobigs, 2002) , and laminin (Boonsanay and Smith, 2007) , have been shown to participate in JEV binding or penetration, the precise mechanisms remain largely unknown. The pseudotype and recombinant VSV systems have offered us useful tools to focus on the study of entry mechanisms of JEV E proteins by using control viruses harboring an appropriate protein on identical particles. Both pseudotype (JEVpv) and recombinant (JEVrv) VSV bearing the JEV E protein exhibited high infectivity for the target cells, and JEVrv, but not JEVpv, was able to propagate and form foci, as did authentic JEV . Both JEVpv and JEVrv were neutralized by anti-JEV E antibodies. 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. Treatment of the JEVpv and JEVrv with cholesterol drastically reduced the infectivity, as previously reported on authentic JEV (Lee et al., 2008) . In contrast, depletion of cholesterol from the viruses by treatment with methyl β-cyclodextrin enhanced the infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and JEVrv . These enhancements were inhibited by treatment with an SMase inhibitor or C 6 -ceramide. Involvement of ceramide in the entry of JEV was confirmed by co-precipitation of the JEV E protein with labeled-ceramide . In our study, it was demonstrated that cellular lipid components such as cholesterol and ceramide play crucial roles in the entry of JEV. Modification of sphingolipids on the plasma membrane of the cells might be a novel target for the development of antivirals against JEV infection.