Effect of V and C proteins on the induction of IFN-β by Sendai virus.

<p>(A) VHS cells were transfected with pIFN-β-luc (350 ng), pJAT-lacZ (200 ng) and either an empty plasmid (300 ng) or an expression plasmid encoding PIV5 V (300 ng), RPV V (300 ng), RPV C (300 ng), PPRV V (300 ng) or PPRV C (300 ng). At least twenty four hours post transfection, cells were infected with 50 HA units of SeV-DI or left uninfected; 7 hours later the cells were lysed and the cells extracts were assayed for luciferase and β-galactosidase activity. RLU were normalised so that the induction seen in cells transfected with empty vector and infected with SeV-DI was set at 100. Error bars represent the standard error of the mean. ANOVA and Tukey pairwise comparison test were performed to determine under which conditions the SeV-DI-treated cells had lower induction than the control (* = p < 0.05). (B) Cell extracts from parallel transfected wells were run on 10% SDS-PAGE gels and Western blotted with anti-V5 or anti-PCNA (loading control). (C) VHS cells were transfected with the reporter plasmids as above, plus empty plasmid or the expression plasmid encoding PPRV V (500 ng), with increasing amounts of the expression plasmid encoding LGP2 (0 ng, 2 ng and 10 ng). The total amount of DNA was kept constant in all samples by adding empty plasmid as required. Twenty four hours post transfection, cells were infected with 50 HA units of SeV-DI or left uninfected. After 7 hours the cells were lysed and the cell extracts were assayed for luciferase and β-galactosidase activity and RLU values calculated and normalised as described above. (C, Inset) The effect of the expression of the V protein at each level of LGP2 is illustrated by replotting the data shown in (C). The normalized RLU for of each amount of LGP2-transfected/SeV-DI infected cells was used as a reference and set at 100. Error bars represent the standard error of the mean (SEM).