FIG 4.

Mutation of putative N-glycosylation sites on DENV NS4B affected infectious virus production. (A and C) Detection of viral production efficiency in mammalian BHK-21 cells (A) and mosquito C6/36 cells (C). FL RNA (10 μg for BHK-21 cells or 15 μg for C6/36) encoding WT, N58Q, N62Q, or N58QN62Q NS4B was electroporated into BHK-21 cells (A) or C6/36 cells (C). For panel A, culture fluid (extracellular fraction) and cell lysate (intracellular fraction) were collected at 144 hpe. For panel C, culture fluid was collected at 7 dpe and 14 dpe (days postelectroporation). Viral titer was determined by focus-forming assay in naive BHK-21 cells. Focus-forming assays were performed in triplicate with one dilution to determine the mean and standard deviation. The experiment was performed independently in triplicate, and the data shown are representative of one independent electroporation experiment. Asterisks (**, P < 0.005; ***, P < 0.0005) indicate results significantly different from the WT. (B and D) Characterization of NS4B N-glycosylation mutant viruses derived from WT or NS4B mutant FL RNA-electroporated BHK-21 cells (B) and C6/36 cells (D). The culture fluids from 144 hpe of BHK-21 cells (B) and 14 dpe of C6/36 cells (D) were subjected to plaque assay in naive BHK-21 cells, and plaques were fixed after 12 days postinfection (dpi).