The nucleic acid sensors MDA-5, RIG-I and DAI/Zbp1 are not critical for Ad-induced type I IFN production Next, we investigated whether the cytoplasmic RNA sensors MDA-5 or RIG-I, or the putative DNA sensor DAI/Zbp1 may play a major role in the induction of the IFN-ab response to Ad. The possible involvement of MDA-5 was tested using BMDCs and BMMs from MDA-5 deficient mice. Normal IFN-ab responses to Ad were obtained in MDA-5-deficient BMDCs cells (Fig. 8A ) and also in BMMs (not shown), whereas the responses to the known MDA-5 ligand Poly I:C were abrogated (Fig. 8A) . The possible involvement of RIG-I was checked using a dominant negative form of RIG-I (RIG-IC) [64] . BMDCs from IRF-3 2/2 mice were transfected with a GFP-expressing plasmid (transfection control) with or without RIG-IC and subsequently stimulated with Ad or control leader RNA. We used IRF-3 2/2 cells to avoid any induction of IFN-ab by the plasmid itself, which is, contrary to that by Ad, strictly IRF-3 dependent. The induction of IFN-b mRNA was analyzed in sorted GFP-positive cells. As shown in Fig. 8B , left, the transfection of RIG-IC prevented induction of IFN-b mRNA by the leader RNA, but not by Ad. The role of Zbp1/DAI was analyzed using siRNA-mediated knockdown of DAI/Zbp1 in BMDCs. For this purpose, cells from IRF-32/2 mice were co-transfected with DAI/Zbp1 targeting siRNAs and a GFP expressing plasmid and subsequently stimulated with Ad. As shown in Fig. 8B , right, the transfection of BMDCs resulted in strongly reduced DAI/Zbp1 expression but not in a reduced IFNb mRNA induction by Ad in GFP-positive cells. Control experiments with L-929 cells showed that transfection of the gene-specific siRNA downregulated the expression of DAI/Zbp1 on both the mRNA and protein levels and efficiently inhibited the IFN-b response to B-DNA ( Fig. S10A-C) . Collectively, our results indicate that known nucleic acid sensors such as MDA-5, RIG-I and Zbp1/DAI are not involved in the Ad-induced type I IFN production.