It should be noted that a further increase in the Ad2Ts1 dose (2.16610 11 particles) resulted in detectable, albeit very low levels of plasma IFN-ab that were released with different kinetics (Fig. 10C) . In this case, IFN-ab was detectable as early as 2 h after infection and, in contrast to the results we obtained with wt viruses ( Figures 1A, 10A and S1A), the levels of IFN-ab did not increase significantly at the later time-points. This already suggests a mechanism for type I IFN induction by Ad2Ts1 that is fundamentally different from the IFN-ab induction seen with wt Ad2. Since Ads are DNA viruses, they can possibly be detected by TLR9. In fact, the innate immune recognition of Ad in pDCs is TLR9 dependent. We therefore repeated the experiment with a high dose of Ad2Ts1 using TLR9 2/2 mice. As shown in Fig. 10C , TLR9 2/2 mice did not produce IFN-ab in response to the mutant Ad2Ts1, quite in contrast to the results obtained with the wt virus (see Fig. 4A ) Likewise, there was no detectable IFN-ab release in Ad2Ts1-infected mice deficient in MyD88, an essential component of TLR9 signaling (not shown). Furthermore, the corresponding amount of empty particles (DNA-free) of Ad2Ts1 elicited no IFN-ab response in wt mice (Fig. 10C) . These data illustrate the critical role of TLR9 in the induction of IFN-ab by means of high doses of Ad2Ts1. To exclude the possibility that contaminating DNA on the surface of the virions was responsible for the TLR9dependent IFN-ab induction, we treated the mutant virions with bensonase, which destroys all kinds of free nucleic acids. Bensonase-treated Ad2Ts1 still induced an IFN-ab response in We also tested the role of endosomal escape of Ad in the in vitro induction in BMDCs of IFN-ab and IL-6. Fig. 10D shows a dose dependent production of IFN-ab by Ad2Ts1 grown at permissive temperature and Figures S12C and D the production of IFN-ab and IL-6 by wt Ad2, but no production of either of the cytokines by Ad2Ts1 grown at the restrictive temperature. Interestingly, liposomal transfection of whole Ad2 Ts1 virions (but not of empty virus particles) in BMDCs resulted in the significant production of IFN-ab that however, was critically dependent on IRF-3 (Fig. 7A) . Similarly, Ad2 Ts1 did not induce IFN-ab production in human monocyte derived DCs (Fig. S12E) . The requirement of low pH for Ad3 infection was also tested using bafilomycin A1, a drug known to inhibit the acidification of endosomes. Experiments shown in Fig. S13A , B revealed that cells treated with this drug produced significantly reduced levels of IFN-ab and IL-6 respectively, in response to Ad3. This is consistent with the notion that Ad3 and Ad7 infection of cultured cells requires low endosomal pH [70, 71] . Similar results were obtained using treatment with ammonium chloride, another acidification inhibitor known to block Ad escape from endosomes (not shown). Collectively, the data in vitro and in vivo provide evidence that the late phase of the Ad infectious entry, in which the virus escapes from the endosome, triggers an innate response characterized by the production of type I IFNs and IL-6.