Generation and Characterization of a Mammalian Cell Line Continuously Expressing Japanese Encephalitis Virus Subviral Particles

Abstract

We have generated a cell line (F cells) producing a secreted form of Japanese encephalitis virus (JEV) subviral particle (extracellular particles [EPs]) that contains the JEV envelope glycoprotein (E) and a precursor (prM) of the virion membrane protein (M). The F cells were engineered to synthesize these JEV products from a cDNA encoding a mutated (furin proteinase resistant) form of prM, since stable cell lines expressing E and the authentic form of prM could not be obtained, due (in part) to the cell-fusing ability of EPs containing E and M. Our biochemical alteration of the prM protein was critical for the successful production of EP-producing cell lines. EPs produced by F cells share the biochemical properties of empty viral particles produced by JEV-infected cells, except that the F-cell EPs lack hemagglutinating activity and M. F-cell EPs were recognized by a panel of monoclonal antibodies to E, and EPs were shown to be useful as vaccine candidates in mice and as diagnostic reagents in evaluating human immune responses to JE vaccination. The amounts of E antigen released into the culture fluid of F cells were similar to those found in virion fractions of JEV-infected cell culture fluids or JEV-infected weanling mouse brains (the current source of antigen used to produce human vaccines for JE). Thus, the F-cell line would appear to be a useful source of antigen for JE vaccines and diagnostics.

Production of Japanese encephalitis virus-like particles in insect cells

“…During flavivirus virion maturation, the cleavage from prM to M causes the rearrangement of E proteins on virus particles, leading to the formation of mature virions that can show cell-fusion activity. As reported previously for mammalian cells, 5,6 the use of the gene encoding a mutated (furin proteinase resistant) form of prM was effective in generating highly productive recombinant insect cells. Finally, suspension culture was useful in improving productivity.…”

“…VLPs are a highly effective type of subunit vaccine that can elicit strong immune responses because of their repeated dense display of viral antigens in an authentic conformation. [3][4][5][6][7] Therefore, VLPs offer a promising approach to the development of safe and efficacious vaccines and diagnostic antigens.…”

“…21 The virion of flaviviruses, such as JE, dengue, West Nile, and yellow fever viruses, consists of a nucleocapsid structure surrounded by a lipid bilayer inserted with an envelope (E) glycoprotein and a membrane (M) protein. 5,6,[22][23][24][25] The E protein is the major surface protein with a role in cellular receptor binding and membrane fusion and induces neutralizing antibodies that protect hosts against disease. The M protein is synthesized as the precursor membrane (prM) protein in infected cells, which is then cleaved to M by a cellular protease, furin, during virion maturation.…”

N-glycosylation of the premembrane protein of Japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles

“…Previous studies have shown that high level expression of VLPs could result in fusion, apoptosis, and death in some JEV susceptible cells (Kojima et al, 2003;Konishi et al, 2001). In this study, apoptosis induced by JEV VLPs was found in pCprME transfected 293FT cells, which suggested that the VLPs were also toxic for this cell line.…”

Hepatitis C virus ultrastructure and morphogenesis

“…2A) and the envelope (E1-E2) sequences separatly. Although some subviral enveloped particles lacking cores have been described for other Flaviviridae such as the Japanese encephalitis virus (Konishi et al, 2001), HCV E1-E2 production did not result in the detection of particles by EM (unpublished data). However, production of the core protein alone resulted in the budding of VLPs towards the ER lumen, and the detection of electrondense areas lining the cytosolic side of the ER lumen (Blanchard et al, 2003b).…”