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. Author manuscript; available in PMC: 2014 Jul 17.
Published in final edited form as: Cell Host Microbe. 2013 Jul 17;14(1):10.1016/j.chom.2013.06.010. doi: 10.1016/j.chom.2013.06.010

Figure 1. VP35 Inhibits PACT Activation of RIG-I Signaling.

Figure 1

(A) IFN-β promoter reporter gene activity in the presence of RIG-I, PACT, or VP35 and following mock transfection, transfection with low-molecular-weight (LMW) poly(I:C), or infection with Sendai virus (SeV), as indicated. IFN-β-firefly luciferase activities were normalized to Renilla luciferase activity expressed from a cotransfected constitutive expression plasmid. Fold induction was determined by setting the vector (mock) transfection to a value of 1. The error bars indicate standard deviation of three independent replicates (*p < 0.001, **p < 0.001).

(B) Endogenous IFN-β mRNA levels as determined by quantitative RT-PCR. Transfections were performed as in (A) except that reporter plasmids were omitted. IFN-β mRNA levels were normalized to β-actin mRNA levels and are represented as relative copy number. The data are the average of three independent experiments, with error bars representing standard deviation (*p = 0.02, **p = 0.01 by Student’s t test).

(C) An IFN bioassay was performed to assess the amount of IFN-α/β in cell supernatants. The data in the chart represent the mean ± SD of three independent experiments; *p = 0.02, **p = 0.01, by Student’s t test.

(D) SeV-induced IRF-3 (S396) phosphorylation in the presence of transfected empty vector (vector), RIG-I, PACT, or VP35 expression plasmid with or without IRF-3 expression plasmid under mock-infected or SeV-infected conditions. Lysates were analyzed by western blotting for phospho-S396 IRF-3 (P-IRF-3), total IRF-3, VP35, HA-PACT, and Flag-RIG-I. Blots are representative of three independent experiments.

(E) ISG54 promoter luciferase reporter gene expression in the presence of RIG-I, PACT, or VP35. Methods were analogous to those described in (A), except a reporter plasmid expressing a firefly luciferase under the control of tandem ISRE elements from the ISG54 promoter was used. Fold luciferase activity was determined relative to the mock-treated, empty vector samples. The error bars indicate standard deviation from three independent replicates, *p = 0.007, **p = 0.01.