Journal: Journal of Virology

Article Title: Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2

doi: 10.1128/JVI.00742-11

Figure Lengend Snippet: chMDA5, chCARDIF, and chIRF3 are required to sense viral danger signals in chicken cells. (A to D) The influence of the shRNA-mediated silencing of chMDA5, chCARDIF, and chIRF3 on the induction of the chIFN-β promoter reporter was analyzed in DF-1 cells. (E and F) The effects of siRNA-based silencing of chMDA5 and chCARDIF on AIV-induced chIFN-β mRNA induction were examined in DF-1 and HD-11 cells. (A to D) The cells were cotransfected with pRFPRNAiC-derived plasmids (at 1 μg/ml) for shRNA transcription as indicated and with reporter plasmids for chIFN-β promoter activity. Note that the shRNA directed against chMDA5 is targeted to the 5′-terminal region of the mRNA. The cells were stimulated with the expression of N-chMDA5 (100 ng/ml), chCARDIF (200 ng/ml), or chIRF3 (200 ng/ml) using pcDNA6-derived constructs (A); with t-p(I:C) (1.5 μg/ml) (B); with t-IVT-RNA (0.5 μg/ml) or with VSV*MQΔG infection (MOI, 10) (C); and with Vac-ΔNS1 infection (MOI, 10) (D). After 18 h, a dual-luciferase assay was performed. Results were obtained from four (A and C) or six (B and D) experiments. The data were expressed relative to the luciferase activity in DF-1 cells transfected with nontargeting pRFPRNAiC-CTRL-sh set to 100%. Error bars indicate standard deviations. In cells transfected with pRFPRNAiC CTRL-sh plasmid, the fold induction above that of unstimulated cells with t-p(I:C), t-IVT RNA, VSV replicon, and Vac-ΔNS1 were 57 (±5.3 SD), 69.9 (±23.1), 9.5 (±3.4), and 76 (±15.1), respectively. (E and F) DF-1 and HD-11 cells were transfected with specific siRNA targeting chMDA5 or chCARDIF or with random control siRNA (CTRL-siRNA) as indicated. The cells then were infected with Vac-ΔNS1 or mock infected. Relative chIFN-β mRNA levels were assessed by quantitative real-time RT-PCR analysis 6 h after infection. Average mRNA levels derived from infected cells treated with CTRL-siRNA were set to 100%. Experiments were performed in quadruplicate. Error bars indicate standard deviations. The differences between the effects observed from targeting siRNA and CTRL-siRNA were statistically significant (t test).

Article Snippet: The control short hairpin RNA (shRNA) (CTRL-sh) sequence was derived from a random short interfering RNA (siRNA) designed by Microsynth (Balgach, Switzerland).

Techniques: shRNA, Derivative Assay, Activity Assay, Expressing, Construct, Infection, Luciferase, Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2

doi: 10.1128/JVI.00742-11

Figure Lengend Snippet: Validation of shRNA- or siRNA-mediated silencing of chMDA5 and chCARDIF gene expression. Specific shRNA-mediated (A and B) and siRNA-mediated (C to E) knockdowns of target gene expression were investigated in chicken DF-1 cells. (A and B) The cells were cotransfected with control vector or with plasmids expressing either C-terminal V5-tagged chMDA5 (chMDA5-V5) (A) or N-terminal HT7-tagged chCARDIF (HT7-chCARDIF) (B) together with the indicated pRFPRNAiC-derived plasmids for the transcription of specific or random control shRNAs (all plasmids were at 0.5 μg/ml). After 2 days, the expression levels of tagged chMDA5 and chCARDIF were quantified by Western blotting and immunodetection using anti-V5 antibody or anti-HT7 polyclonal serum. For normalization purposes, the β-actin content was determined with anti-β-actin MAb. The relative signal intensity measured in the presence of gene-specific shRNA is indicated as a percentage of the signal measured with CTRL-shRNA. (C) Cells were transfected with chMDA5-V5 or the vector control (at 0.5 μg/ml) for 6 h, followed by siRNA transfections as indicated. Two days later, the relative protein expression level of chMDA5-V5 was quantified by Western blot analysis as described for panels A and B. (D and E) Cells were transfected with specific siRNA targeting chMDA5 or chCARDIF or with random control siRNA (CTRL-siRNA) as indicated. Two days after transfection, the endogenous chMDA5 (D) or chCARDIF (E) mRNA levels were assessed by quantitative real-time RT-PCR and normalized to the 18S RNA content. The average normalized mRNA levels measured in cells treated with CTRL-siRNA were set to 100%. Transfections were performed in quadruplicate. Error bars indicate standard deviations. The differences between the effects observed with siRNA and CTRL-siRNA were statistically significant (t test).

Article Snippet: The control short hairpin RNA (shRNA) (CTRL-sh) sequence was derived from a random short interfering RNA (siRNA) designed by Microsynth (Balgach, Switzerland).

Techniques: shRNA, Expressing, Plasmid Preparation, Derivative Assay, Western Blot, Immunodetection, Transfection, Quantitative RT-PCR