Fig. 6.

N-S194L enhances N protein expression. (A) Expression of N protein. HEK-293T cells were transfected with plasmids encoding Flag-N, Flag-N-S194L, or vector, and cell lysates were subject to analysis by western blotting at 24 h posttransfection. (B) Vero E6 or Calu-3 cells were inoculated with rWuH-1, rWuH-1_Nsp3-S676T, rWuH-1_{Nsp3-S676T;N-S194L}, or rWuH-1_N-S194L at MOI = 8. Supernatants were collected from cells at 8 and 24 hpi to determine virus titers by plaque assay (n = 4 replicates). (C) Vero E6 or Calu-3 cells infected with rWuH-1, rWuH-1_Nsp3-S676T, rWuH-1_{Nsp3-S676T;N-S194L}, or rWuH-1_N-S194L were lysed at 8 and 24 hpi and then subject to western blotting to detect N protein. (D) Vero E6 cells were transfected with NC siRNA or siRNA targeting eIF4G or eIF4E; after 48 h, the transfected cells were infected with 0.05 PFU/cell rWuH-1, rWuH-1_Nsp3-S676T, rWuH-1_{Nsp3-S676T;N-S194L}, or rWuH-1_N-S194L. Supernatants were harvested at 24 h to measure virus titers by plaque assay, and cell lysates were harvested for detecting viral N gene expression by qRT-PCR (n = 3 replicates). A one-way ANOVA test with Tukey’s multiple comparisons test was used for assessing statistical significances in (B) and (D). (A–D) Representative of two to five independent experiments.