Background

The flavivirus, a group of arboviruses including dengue virus, West Nile virus, tick-borne encephalitis virus, Zika virus, yellow fever virus, and Japanese encephalitis virus (JEV), is associated with substantial morbidity and mortality in human populations (Chambers et al., 1990). Flaviviruses have a single ORF genome encoding a long polypeptide, which is post-translationally cleaved into three structural (C, prM and E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins by the host signal peptidase and NS2B-NS3 viral protease (Chambers et al., 1990).

Viral genomic RNA synthesized by the NS5 RNA-dependent RNA polymerase associates with C protein to form the nucleocapsid, which in turn is packaged in the envelope composed of host cell-derived lipid membrane, and viral prM and E transmembrane proteins to form the virion. It has been found that a reduced form of flaviviral genomic RNA, designated as subgenomic replicon, which lacks all structural genes, can replicate itself in host cells (Suzuki et al., 2014), and that the subgenomic RNA is incorporated into the virus-like particle to form single-round infectious particles (SRIPs) when C, prM and E proteins are supplied in trans (Suzuki et al., 2014; Matsuda et al., 2018).

Besides major infectious particles, virus-infected cells often produce minor particles, some of which are hard to detect with immunochemical methods, due to their small amounts. To study typical and atypical particles released from flavivirus-infected cells, we modified a SRIP system (Matsuda et al., 2018) to develop a JEV HiBiT-SRIP assay system. In this system, the C protein is labeled with a highly sensitive HiBiT peptide tag, to enable high-resolution sedimentation profiling of particles produced by the cells expressing viral structural and nonstructural factors (Ishida et al., 2019). Cells transfected with a set of plasmids (referred to as SRIPs-producer cells) produce SRIPs and non-infectious subviral particles containing C-HiBiT, which can be separated by sucrose density gradient centrifugation. SRIPs in sedimentation fractions can be detected by measurement of cellular NanoLuc levels after inoculation of other cells (referred to as target cells) with all the fractions, which allows SRIP-borne NanoLuc expression (Figure 1). It is possible to apply the principle of JEV HiBiT-SRIP assay to other viruses.

Figure 1. Overview of HiBiT-SRIP system. A. HiBiT-SRIP system plasmids. HDV-Rz: self-cleaving HDV Ribozyme; pA: SV40 polyadenylation signal; CMVp: CMV promoter; CAGp: CAG promoter. B. Schematic presentation of HiBiT-SRIP experiments. JErep-nluc: subgenomic replicon with a gene for NanoLuc as the reporter.