Fig 4. The non-secretory NS1I273H sustains the role of RNA replication and is compensated for the role of the infectious particle formation by the replication-defective NS1F160D.
(A) An illustration shows the experimental workflow. JEV NS1I273H was infected into Huh7 cells and RNA copies were determined at 12, 24, 48, and 72 hpi. Recovery of infectious particles in the culture supernatants of Huh7 cells expressing JEV NS1 (NS1) upon infection with JEV NS1I273H. (B) Huh7 cells infected with JEV (left) or JEV NS1I273H (right) were fixed at 48 hpi and observed by transmission electron microscopy. Arrowheads and arrows indicate electron-dense virus particles and double membrane-derived vesicles, respectively. Huh7 cells or Huh7 cells expressing either the wild type, F160D, or I273H were infected with JEV NS1I273H (C) or JEV NS1F160D (D) and infectious titers were measured at 96 hpi. (E) Expressions of the wild type and mutant NS1 protein in the culture supernatants (Sup) and cells (Cell) were determined by immunoblotting at 48 hpi of lentiviruses into BHK-21 cells. Statistical significance was assessed using Student’s t-test (right panel of A) and is indicated by asterisks (*) (versus control cells).