N-linked glycosylation of the NS1 protein modulates progeny virion assembly in orthoflaviviruses

Immunofluorescence and coimmunoprecipitation assays further demonstrated that deglycosylated NS1 is largely retained in the ER and enhances its interaction with the E protein. Retention using selective hooks (RUSH)-based live-cell imaging assay revealed that NS1 deglycosylation disrupts the trafficking of E protein from the ER to the Golgi apparatus. In addition, cycloheximide chase analysis showed that NS1 deglycosylation impairs its solubilization and then causes rapid degradation of NS1 and E protein by the host proteasomal pathway.

Notably, the efficient viral assembly through NS1 glycosylation is a common feature among flaviviruses (West Nile virus and Yellow Fever virus). Our results consistently demonstrated that the glycosylation modification process of NS1 is highly synchronized with the maturation and assembly of viral particles during their trafficking from the ER to the Golgi apparatus. Collectively, our study confirms that NS1 glycosylation of orthoflavivirus species regulates progeny virion assembly by modulating the NS1-E interaction.