N-linked glycosylation of the E protein is essential for the release of sub-viral particles (SVPs).

(A) Primary structure of the JEV polyprotein and the domain arrangement of its E protein (upper left), N-glycosylation site mutations in the JEprME-HiBiT construct (lower left), and membrane topology and cleavage site of the prME-HiBiT structural proteins on the endoplasmic reticulum membrane (lower right). The host signal peptidase cleaves at the signal peptide-prM and -prM-E junctions (black arrow), and the host furin protease cleaves at the pr-M junction (white arrow). (B) Schematic drawing of the experimental setup. (C) Immunoblotting analysis of JEV SVP release. 293T cells transfected with the indicated prME-HiBiT constructs were cultured for 48 hours, and then cells and culture supernatant were harvested. The amounts of E protein, prM protein, and alpha-tubulin in the cell lysate (cell) and in the 100,000 xg precipitate fraction of culture supernatant (100K pellet) were analyzed via immunoblotting using anti-E protein (second and fifth), anti-prM protein (third and sixth), and anti-alpha-tubulin antibodies (bottom), respectively. E protein was also detected with HiBiT blotting (top, forth). (D and E) Quantification of E (D) or prM (E) proteins in the 100K pellet (left) and cell (middle). Graphs show quantification of the immunoblots in (C). Secretion rates of E and prM proteins (right) were calculated by dividing the amount of the 100K pellet by the total E and prM amount (100K pellet + cell), respectively. (F) HiBiT-dependent NanoLuc luciferase activity (HiBiT activity) measurement analysis of JEV SVP release. HiBiT activity in both cell lysate (middle) and the 500 xg centrifugation fraction of culture supernatants (left) in (C) was measured. Secretion rates were determined by dividing the HiBiT value in culture supernatants by the total HiBiT value in the cell culture (sup + cell). Standard deviation is represented by error bars. *p < 0.05; **p < 0.01; ***p < 0.0001; NS, not significant. (G) Sucrose density gradient fractionation analysis of released SVPs. Culture supernatants of 293T cells expressing prME-HiBiT proteins were treated with (left panel, dotted line) or without (left panel, solid line, and right panel) 1% Triton X-100 and then subjected to 10–40% sucrose density gradient ultracentrifugation. HiBiT activities in each fraction were measured. Error bars represent standard deviation. Means and SDs from three independent experiments are presented.