Autophagy activation is required for influenza A virus-induced apoptosis and replication.

Autophagy and apoptosis are two major interconnected host cell responses to viral infection, including influenza A virus (IAV). Thus, delineating these events could facilitate the development of better treatment options and provide an effective anti-viral strategy for controlling IAV infection. We used A549 cells and mouse embryonic fibroblasts (MEF) to study the role of virus-induced autophagy and apoptosis, the cross-talk between both pathways, and their relation to IAV infection [ATCC strain A/Puerto Rico/8/34(H1N1) (hereafter; PR8)]. PR8-infected and mock-infected cells were analyzed by immunoblotting, immunofluorescence confocal microscopy, electron microscopy and flow cytometry (FACS). We found that PR8 infection simultaneously induced autophagy and apoptosis in A549 cells. Autophagy was associated with Bax and Bak activation, intrinsic caspase cleavage and subsequent PARP-1 and BID cleavage. Both Bax knockout (KO) and Bax/Bak double knockout MEFs displayed inhibition of virus-induced cytopathology and cell death and diminished virus-mediated caspase activation, suggesting that virus-induced apoptosis is Bax/Bak-dependent. Biochemical inhibition of autophagy induction with 3-methyladenine blocked both virus replication and apoptosis pathways. These effects were replicated using autophagy-refractory Atg3 KO and Atg5 KO cells. Taken together, our data indicate that PR8 infection simultaneously induces autophagy and Bax/caspase-dependent apoptosis, with autophagy playing a role to support PR8 replication, in part, by modulating virus-induced apoptosis.