Summary Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP-1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation, and implicate DAI as a sensor of RNA viruses.

The immune system detects viral infections and mutations in parenchymal cells when antigens from these cells are crosspresented on MHC class I molecules of professional antigen-presenting cells (APC). Exogenous antigens are crosspresented through TAP-dependent (cytosolic) or poorly understood TAP-independent (vacuolar) pathways. The TAP-independent pathway is blocked by the cysteine protease inhibitor, leupeptin, but not by proteasome inhibitors, which is opposite to the effects of these agents on the TAP-dependent pathway. Dendritic cells lacking the cysteine protease cathepsin S lack the TAP-independent pathway. Mice whose APC lack cathepsin S have reduced crosspriming to particulate and cell-associated antigens, as well as to influenza virus. Cathepsin S-deficient phagosomes generate a class I-presented peptide poorly. In contrast, cathepsin S-sufficient phagosomes and recombinant cathepsin S produce the mature epitope. Therefore, cathepsin S plays a major role in generating presented peptides for the vacuolar pathway of crosspresentation, and this mechanism is active in vivo.

Cytotoxic T lymphocytes (CTLs) are thought to detect viral infections by monitoring the surface of all cells for the presence of viral peptides bound to major histocompatibility complex (MHC) class I molecules. In most cells, peptides presented by MHC class I molecules are derived exclusively from proteins synthesized by the antigen-bearing cells 1 . Macrophages and dendritic cells also have an alternative MHC class I pathway that can present peptides derived from extracellular antigens; however, the physiological role of this process is unclear 2 . Here we show that virally infected non-haematopoietic cells are unable to stimulate primary CTL-mediated immunity directly. Instead, bonemarrow-derived cells are required as antigen-presenting cells (APCs) to initiate anti-viral CTL responses. In these APCs, the alternative (exogenous) MHC class I pathway is the obligatory mechanism for the initiation of CTL responses to viruses that infect only non-haematopoietic cells.The 'classical' MHC class I antigen-presentation pathway is thought to be the major mechanism used by the immune system to detect viral infections in all cells. In this pathway, proteins synthesized by a cell are degraded in the cytoplasm into oligopeptides, a fraction of which are transported into the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP). In the ER these peptides bind to new MHC class I molecules and the resulting complexes are transported to the cell surface. As MHC class I molecules must bind peptides in order to be transported to the plasma membrane, TAP is required for normal MHC class I expression at the cell surface and for antigen presentation 1 .To determine whether non-haematopoietic cells can function as APCs to initiate CTL responses to viruses, we constructed bonemarrow chimaeras by lethally irradiating C57Bl/6 mice (B6 mice; MHC class I haplotype H-2 b ) and reconstituting them with bone marrow from TAP 0/0 mice 3 (also H-2 b ; all bone-marrow chimaeras will be referred to as bone-marrow donor → irradiated recipient). As bone-marrow-derived cells in TAP 0=0 → B6 mice cannot transfer peptides from the cytosol to the ER, they are unable to use the classical MHC class I pathway 3,4 .The chimaeric mice were assayed for the generation of CTL responses following infection with wild-type vaccinia virus or with vaccinia-OVA, a recombinant vaccinia virus carrying chicken ovalbumin (OVA) as a full-length protein 5 . Although they have intact MHC class I antigen presentation in non-haematopoietic tissues, TAP 0=0 → B6 mice did not generate CTL responses to vaccinia-viral antigens (Fig. 1a) or to OVA (Fig. 1b). In contrast, robust CTL responses to these antigens were detected in control B6 → B6 mice. These results indicate that the generation of CTL responses to vaccinia virus requires bone-marrow-derived cells with functional TAP molecules.Next, we determined whether the inability of TAP 0=0 → B6 mice to generate CTL responses was due to a defect in CD8 + T cells or to a failure in antigen presenta...

Ectromelia virus (ECTV) is an orthopoxvirus (OPV) that causes mousepox, the murine equivalent of human smallpox. C57BL/6 (B6) mice are naturally resistant to mousepox due to the concerted action of innate and adaptive immune responses. Previous studies have shown that natural killer (NK) cells are a component of innate immunity that is essential for the B6 mice resistance to mousepox. However, the mechanism of NK cell–mediated resistance to OPV disease remains undefined. Here we show that B6 mice resistance to mousepox requires the direct cytolytic function of NK cells, as well as their ability to boost the T cell response. Furthermore, we show that the activating receptor NKG2D is required for optimal NK cell–mediated resistance to disease and lethality. Together, our results have important implication towards the understanding of natural resistance to pathogenic viral infections.

It is believed that CD8+ T lymphocytes or Abs can independently clear many primary viral infections, including those caused by Orthopoxviruses (OPV), a genus that includes the human pathogens variola and monkeypox and the vaccine species vaccinia virus. However, most experiments addressing the role of Abs and CD8+ T cells in protection have used viruses that are not specific for the host. In the present study, we used the mouse-specific OPV ectromelia virus and mice deficient in CD40, B cells, or CD8+ T cells and adoptive transfers of CD8+ T or B lymphocytes to show that the protection afforded by CD8+ T cells is incomplete. Despite sustained CD8+ T cell responses, in the absence of Ab responses ectromelia virus persists. This results in delayed disease and inexorably leads to death. Therefore, CD8+ T lymphocytes and Abs are not redundant but complementary and essential to survive infections with a highly pathogenic viruses in the natural host.

Summary It is well established that natural killer (NK) cells confer resistance to many viral diseases, but only in a few instances the molecular mechanisms whereby NK cells recognize virus-infected cells are known. Here we show that CD94, a molecule preferentially expressed by NK cells, is essential for the resistance of C57BL/6 mice to mousepox, a disease caused by the Orthopoxvirus ectromelia virus. Ectromelia virus-infected cells expressing the major histocompatibility complex (MHC) class Ib molecule Qa-1b are specifically recognized by the activating receptor formed by CD94 and NKG2E. Because CD94-NKG2 receptors and their ligands are highly conserved in rodents and humans, a similar mechanism may exist during human infections with the smallpox and monkeypox viruses, which are highly homologous to ectromelia virus.

Nonliving antiviral vaccines traditionally target proteins expressed at the surface of the virion with the hope of inducing neutralizing antibodies. Orthopoxviruses (OPVs), such as the human smallpox virus and the mouse-equivalent ectromelia virus (ECTV; an agent of mousepox), encode immune response modifiers (IRMs) that can increase virulence by decreasing the host immune response. We show that one of these IRMs, the type I interferon (IFN) binding protein (bp) of ECTV, is essential for ECTV virulence and is a natural target of the antibody response. More strikingly, we demonstrate that immunization with recombinant type I IFN bp protects mice from lethal mousepox. Collectively, our experiments have important implications for our understanding of the role of IRMs in OPV virulence and of type I IFNs in OPV infections. Furthermore, our work provides proof of concept that effective antiviral vaccines can be made to prevent disease by targeting virulence factors as an alternative to the traditional approach that attempts to prevent infection by virus neutralization.

Summary Toll Like Receptor 9 (TLR9), its adapter MyD88, the downstream transcription factor interferon regulatory factor 7 (IRF7) and type I interferons (IFN-I) are all required for resistance to infection with ectromelia virus (ECTV). However, it is not known how or in which cells these effectors function to promote survival. Here, we showed that after infection with ECTV, the TLR9-MyD88-IRF7 pathway was necessary in CD11c+ cells for the expression of proinflammatory cytokines and the recruitment of inflammatory monocytes (iMo) to the draining lymph node (D-LN). In the D-LN, the major producers of IFN-I were infected iMo, which used the DNA sensor-adapter STING to activate IRF7 and nuclear factor κB (NF-κB) signaling to induce the expression of IFNα and IFNβ, respectively. Thus, in vivo, two pathways of DNA pathogen sensing act sequentially in two distinct cell types to orchestrate resistance to a viral disease.