Various states of the capsid proteins released from Japanese encephalitis virus-infected cells.

In orthoflaviviruses, the viral capsid protein plays a crucial role in genome packaging and formation of infectious viral particles. However, their functions are believed to be diverse owing to their unique properties. In this study, we investigated the secretion of a capsid protein, independent of its role in viral particle assembly, using a recombinant Japanese encephalitis virus (JEV) expressing a capsid protein fused with a HiBiT tag (JEV C-HiBiT), a highly sensitive reporter tag. JEV C-HiBiT exhibited a growth rate similar to that of JEV WT, although the infected cells showed strong HiBiT-dependent NanoLuc luciferase activity. Sucrose density gradient fractionation analysis of the culture supernatants from JEV C-HiBiT-infected 293T cells revealed that the capsid was released in two distinct states. Studies on secondary infection and comparisons with transiently expressing cells indicated that the heavier peaks corresponded to virions, whereas the lighter peaks corresponded to free capsid proteins. Additionally, when SH-SY5Y, K562, and C6/36 cells were used as host cells, additional capsid protein peaks corresponding to subviral particles and/or membrane vesicles were detected. Treatment with Bafilomycin A1 enhanced free capsid protein secretion, and capsid proteins were localized within the lysosomes, suggesting that the free capsids were released by the lysosome-mediated secretion pathway. These findings indicate that the capsid protein is not merely a structural factor required for genome packaging but may also play multiple roles in viral propagation.