Comparison of the predicted amino acid sequences of IRF3 from B10.S and IRF3 from SJL. A. Domains of the 419 aa IRF3 include the DNA binding domain (DBD), Nuclear Localization Signal (NLS), Autoinhibitory Domain (ID) 1 and 2, Nuclear Export Signal (NES), IRF association domain (IAD), Apoptosis Activation Domain (AAD), Signal Response Domain (SRD) and the sites phosphorylated by JNK and TBK/IKKε. The 33R(B10.S)>G(SJL/J), 147I>V, and the 169Q>R polymorphisms are noted. B. Alignment of SJL/J IRF3 amino acid sequence with B10.S IRF3 amino acid sequence. Following extraction from agarose and cloning into pTARGET expression vector, the inserted cDNA was sequenced and the predicted amino acid sequence was generated. The DNA binding domain ( ), the autoinhibitory domain 1 ( ), and the signal response/autoinhibitory domain 2 ( ) are noted.

IRF3 protein and IL-23 p19 promoter activity in RAW cells expressing polymorphic IRF3. A. Western blot of IRF3 and tubulin in RAW cells following nucleofection with pCIneo (empty vector), SJL_IRF3pTARGET or B10S_IRF3pTARGET. Transfected RAW264.7 cells were left uninfected or infected with 3 × 10⁵ TMEV PFU. Nine h after infection cells were lysed and 10 ug of protein immunoblotted. Numerical values are ratios of the sum of the densitometric values of three IRF3 bands divided by the densitometric value of the tubulin band. B, C. IL-23 p19 promoter activity in 3 × 10⁵ RAW264.7 cells incubated overnight, were co-transfected with pCIneo (empty vector), SJL_IRF3pTARGET or B10S_IRF3pTARGET plus p19prompGL3 and pRL-SV40 vectors. Transfected RAW264.7 cells were left uninfected or infected with 3 × 10⁵ TMEV PFU. Promoter activity in cells was measured as firefly luciferase-dependent luminescence expressed from p19prompGL3 normalized to Renilla luciferase-dependent luminescence expressed from pRL-SV40 in 24-h cell extracts divided by ratios obtained from uninfected cells. Data are means of 5 samples each of a representative experiment ± standard error; * indicates that the mean fold increase in promoter activity in uninfected cells with SJL_IRF3pTARGET (pSJL-IRF3) or B10S_irf3pTARGET (pB10S-IRF3) is significantly different than promoter activity in uninfected cells with pCIneo control. Brackets indicate significant differences promoter activity in TMEV infected cells.

Effect of SJL-IRF3 and B10.S IRF3 on relative expression of IFNβ, IL-6, ISG56, and TMEV RNA after TMEV infection. Real-time PCR of IFNβ (A), ISG56 (B), IL-6 (C), and TMEV RNA (D) in uninfected or TMEV-infected RAW264.7 cells transfected with pCIneo (control), SJL_IRF3pTARGET (SJL), B10S_IRF3pTARGET (B10.S) as measured after 9 h. Levels of IFNβ, IL-6, ISG56, and TMEV RNA relative to GAPDH RNA were evaluated by real-time PCR. Individual means were evaluated with the Wilcoxon-Mann-Whitney rank-sum U test. Comparisons of means in which p values < 0.05 are bracketed. *P<.05, **P<.01. Data are an average + standard error of 3 replicates per group and two different experiments.

Effect of SJL-IRF3 and B10.S IRF3 on relative expression of IFNβ, ISG56, and IL-6 after polyIC stimulation. Real-time PCR of IFNβ (A), ISG56 (B), and IL-6 (C) in RAW264.7 cells transfected with pCIneo (control), SJL_IRF3pTARGET (SJL), B10S_IRF3pTARGET (B10.S) as measured after 9 h. Levels of IFNβ, IL-6, and ISG56 RNA relative to GAPDH RNA were evaluated by real-time PCR. Individual means were evaluated with the Student t test. Comparisons of means in which p values < 0.05 are bracketed. Data are an average +/- standard error of 3 replicates per group from a representative experiment.

Effect of SJL-IRF3 and B10.S IRF3 on virus induced caspase activation and Bax protein. Caspase activation after 16 h (A) and 35 h (B) in uninfected or TMEV-infected RAW264.7 cells transfected with pCIneo (control), SJL_IRF3pTARGET (SJL), B10S_IRF3pTARGET (B10.S). Levels of caspase activation were evaluated by the release of luciferin from Z-DEVD-luciferin substrate and then measuring luminescence by adding UltraGlo™luciferase from Promega. Individual means of 5 samples each of a representative experiment were evaluated with the Student t test. Comparisons of means in which p values < 0.05 are bracketed. Data are an average ± standard error of 3 replicates per group from a representative experiment. (C) Western blots of Bax and beta-tubulin protein in uninfected or TMEV-infected RAW264.7 cells transfected with pCIneo (Control), SJL_IRF3pTARGET (SJL), or B10SIRF3pTARGET (B10.S). (D) Densitometric ratio of Bax in 9 h TMEV-infected RAW cells with pCIneo (Control), SJL_IRF3pTARGET (SJL-IRF3), or B10SIRF3pTARGET (B10.S-IRF3) with Bax uninfected RAW cells with pCIneo, SJL_IRF3pTARGET, or B10SIRF3pTARGET, respectively.