The Japanese encephalitis virus NS1 protein concentrates ER membranes in a cytoskeleton-independent manner to facilitate viral replication.

Orthoflaviviruses remodel the endoplasmic reticulum (ER) network to construct replication organelles (ROs) for RNA replication. In this study, we demonstrate that the Japanese encephalitis virus (JEV) NS1 protein concentrates ER membranes in the perinuclear region, which provides a substantial membrane source for viral replication. Subsequently, the virus forms main replication organelles within this membrane-concentrated area to facilitate efficient replication. This process relies on the ER localization signal, glycosylation, dimerization, and membrane-binding sites of the NS1 protein. In conclusion, our study highlights the role of the NS1 protein in the formation of the ROs by JEV, providing new insights into orthoflavivirus replication.IMPORTANCEOrthoflaviviruses use the endoplasmic reticulum (ER) membranes for replication by forming invaginations to assemble the replication organelles. Here, we found that Japanese encephalitis virus (JEV) utilizes the NS1 protein to concentrate a significant number of ER membranes in the perinuclear area, thereby providing a membrane source for viral replication and facilitating the formation of main replication organelles (MROs). This process depends on the ER localization signals of NS1, as well as its glycosylation, dimerization, and membrane-binding sites, but not on the cytoskeleton. In summary, our study highlights how NS1 remodels ER membranes to facilitate the formation of MROs for JEV, thereby accelerating viral replication.