Fig 4. Recombinant viruses have comparable polymerase activity and replicate similarly in cell culture.
(A) HEK293T cells were used for in vivo CAT RNP reconstitution (see Methods), which indicates viral polymerase activity. At 24 h post-reconstitution, CAT protein in total cell extract was analyzed by ELISA. MOCK, plasmids expressing PB1 or NP were omitted. CTRL-CAT indicates CAT accumulation in cells transfected exclusively with pHHNS-CAT plasmid. Three independent experiments were performed; values shown as means (%) ± SD. Significance was determined by two-way ANOVA with Bonferroni post hoc test (ns not significant). (B) Accumulation of PB1 and NP viral proteins was monitored in cell extracts used for CAT analysis, using GADPH as loading control. (C) Cultured A549 cells were infected at 3 pfu/cell with the recombinant viruses indicated in Table 1. At indicated hpi, samples were used to detect the indicated proteins by Western blot. Three independent experiments were performed and one of them is shown as representative. (D) Cultured A549 cells were infected at 10−3 pfu/cell with the recombinant viruses indicated in Table 1. At indicated hpi, supernatants were collected and virus titer determined by plaque assay in MDCK cells. Three independent experiments were performed in triplicate; values shown as means ± SD. Significance was determined by two-way ANOVA with Bonferroni post hoc test (ns not significant).