Viral infection is a stimulus for apoptosis, and in order to sustain viral replication many viruses are known to carry genes encoding apoptosis inhibitors. F1L, encoded by the orthopoxvirus modified vaccinia virus Ankara (MVA) has a Bcl-2-like structure. An MVA mutant lacking F1L (MVAΔF1L) induces apoptosis, indicating that MVA infection activates and F1L functions to inhibit the apoptotic pathway. In this study we investigated the events leading to apoptosis upon infection by MVAΔF1L. Apoptosis largely proceeded through the pro-apoptotic Bcl-2 family protein Bak with some contribution from Bax. Of the family of pro-apoptotic BH3-only proteins, only the loss of Noxa provided substantial protection, while the loss of Bim had a minor effect. In mice, MVA preferentially infected macrophages and DCs in vivo. In both cell types wt MVA induced apoptosis albeit more weakly than MVAΔF1L. The loss of Noxa had a significant protective effect in macrophages, DC and primary lymphocytes, and the combined loss of Bim and Noxa provided strong protection. Noxa protein was induced during infection, and the induction of Noxa protein and apoptosis induction required transcription factor IRF3 and type I interferon signalling. We further observed that helicases RIG-I and MDA5 and their signalling adapter MAVS contribute to Noxa induction and apoptosis in response to MVA infection. RNA isolated from MVA-infected cells induced Noxa expression and apoptosis when transfected in the absence of viral infection. We thus here describe a pathway leading from the detection of viral RNA during MVA infection by the cytosolic helicase-pathway, to the up-regulation of Noxa and apoptosis via IRF3 and type I IFN signalling.

BackgroundOne of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to permeabilize the mitochondrial outer membrane during apoptosis. While Bax is mostly cytosolic and translocates to mitochondria following an apoptotic stimulus, Bak is constitutively integrated within the outer membrane. Membrane anchorage occurs via a C-terminal transmembrane domain that has been studied in Bax but not in Bak, therefore what governs their distinct subcellular distribution is uncertain. In addition, whether the distinct subcellular distributions of Bak and Bax contributes to their differential regulation during apoptosis remains unclear.Methodology/Principal FindingsTo gain insight into Bak and Bax targeting to mitochondria, elements of the Bak C-terminus were mutated, or swapped with those of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of three basic residues within the C-segment destabilized Bak. Replacing the Bak C-segment with that from Bax rescued stability and function, but unexpectedly resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS translocated to the mitochondrial outer membrane, inserted its transmembrane domain, oligomerized, and released cytochrome c. Despite this Bax-like subcellular distribution, Bak/BaxCS retained Bak-like regulation following targeting of Mcl-1.Conclusions/SignificanceResidues in the C-segment of Bak and of Bax contribute to their distinct subcellular localizations. That a semi-cytosolic form of Bak, Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates that Bak and Bax share a conserved mode of activation. In addition, the differential regulation of Bak and Bax by Mcl-1 is predominantly independent of the initial subcellular localizations of Bak and Bax.

Many viruses regulate a crucial point in the apoptotic pathway by expressing viral Bcl-2 homologues, which have become useful tools to investigate the mechanisms behind the control of the mitochondrial checkpoint of apoptosis. Concurrently, a number of viral inhibitors of innate immune signalling have been instrumental tools in the discovery of key host pathways. Here we discuss how viral inhibitors of the apoptotic and innate signalling pathways have further enhanced the understanding of both research fields and are beginning to shed light on how these two pathways converge.