ABSTRACTIn response to viral infection, reactive oxygen species (ROS) mediate innate immune signaling or generate danger signals to activate immune cells. The mechanisms of virally induced ROS are poorly defined, however. We demonstrate that ROS are produced within minutes of adenovirus type 5 (Ad5) infection of macrophages and that oxidative stress supports Ad5-induced cytokine secretion. We show that short hairpin RNA (shRNA) knockdown of TLR9 has no effect on ROS production despite observed decreases in Ad-induced cytokine secretion. A major source of ROS in macrophages is NADPH oxidase. However, shRNA knockdown of the NADPH oxidase subunit NOX2 does not attenuate Ad-induced ROS. Induction of ROS is not observed in cells infected with a temperature-sensitive mutant of Ad2,ts1, which is defective in endosomal membrane penetration during cell entry. Further, Ad5, but notts1, induces the release of lysosomal cathepsin B into the cytoplasm of infected cells. In agreement with this finding, we observe a loss of mitochondrial membrane potential upon Ad infection which requires Ad endosomal membrane penetration and cathepsin B activity. Overexpression of Bcl-2 attenuates Ad5-induced ROS, further supporting the role for mitochondrial membrane destabilization as the source of ROS in response to Ad5 infection. Together, these data suggest that ROS produced in response to Ad5 infection depends on the virally induced endosomal membrane rupture to release lysosomal cathepsins. Furthermore, the release of cathepsins leads to mitochondrial membrane disruption and thus the release of ROS from the mitochondria.