FIG 5.

PB2-588I exacerbates the inhibition of IFN-β. (A to D) The transfection of 293T cells was performed with a luciferase reporter plasmid under the control of an IFN-β promoter and a Renilla control plasmid, PB2 expression plasmids, and plasmids expressing either RIG-I, MDA-5, MAVS, or TBK-1. After 24 h, the cells were lysed, and the luciferase and Renilla activities were measured. The Renilla-adjusted luciferase activity (RLU) in the presence of overexpressed RIG-I, MDA-5, MAVS, or TBK-1 but in the absence of the PB2 protein (empty vector) was set to 100%. The activity in the presence of the PB2 protein was expressed as a percentage of that of the empty vector control. (E and F) The transfection of the 293T cells was performed with a luciferase reporter plasmid under the control of an NF-κB or IRF-3 promoter, a MAVS expression plasmid, and a plasmid expressing the PB2 protein or an empty vector. (G) The transfection of the 293T cells was performed with a luciferase reporter plasmid under the control of an IFN-β promoter, a MAVS expression plasmid, and a plasmid expressing either the wild-type PB2 or the PB2-T588I mutant protein. (H) The lysates from the 293T cells transfected with Flag-MAVS and PCMV-Myc-PB2-588T or PCMV-Myc-PB2-588I and immunoprecipitates were analyzed by Western blotting using anti-Flag and anti-Myc antibodies. (I) Densitometry analysis of the Western blot results of the binding efficiency of coimmunoprecipitate PB2-588I and PB2-588T with Flag-MAVS was done using ImageJ (NIH); the value of PB2-588T was set as the standard, and values shown are ratios of their bands to the standard. The bars represent the standard errors of the means, based on three experiments. *, P < 0.01; **, P < 0.001 (as determined by a two-tailed Student's t test). IP, immunoprecipitation; a-, anti.