Brian J. Ferguson

DNA-PK is a DNA sensor for IRF-3-dependent innate immunity

Innate immunity is the first immunological defence against pathogens. During virus infection detection of nucleic acids is crucial for the inflammatory response. Here we identify DNA-dependent protein kinase (DNA-PK) as a DNA sensor that activates innate immunity. We show that DNA-PK acts as a pattern recognition receptor, binding cytoplasmic DNA and triggering the transcription of type I interferon (IFN), cytokine and chemokine genes in a manner dependent on IFN regulatory factor 3 (IRF-3), TANK-binding kinase 1 (TBK1) and stimulator of interferon genes (STING). Both cells and mice lacking DNA-PKcs show attenuated cytokine responses to both DNA and DNA viruses but not to RNA or RNA virus infection. DNA-PK has well-established functions in the DNA repair and V(D)J recombination, hence loss of DNA-PK leads to severe combined immunodeficiency (SCID). However, we now define a novel anti-microbial function for DNA-PK, a finding with implications for host defence, vaccine development and autoimmunity. DOI: http://dx.doi.org/10.7554/eLife.00047.001

Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity.

Virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. Poxviruses, such as vaccinia virus (VACV), have large DNA genomes and encode many proteins that are dedicated to host immune evasion. Some of these proteins are secreted from the infected cell, where they bind and neutralize complement factors, interferons, cytokines and chemokines. Other VACV proteins function inside cells to inhibit apoptosis or signalling pathways that lead to the production of interferons and pro-inflammatory cytokines and chemokines. In this review, these VACV immunomodulatory proteins are described and the potential to create more immunogenic VACV strains by manipulation of the gene encoding these proteins is discussed.

New insights into the role of PML in tumour suppression

The PML gene is involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), which generates the oncogenic fusion protein PML (promyelocytic leukaemia protein)-retinoic acid receptor alpha. The PML protein localises to a subnuclear structure called the PML nuclear domain (PML-ND), of which PML is the essential structural component. In APL, PML-NDs are disrupted, thus implicating these structures in the pathogenesis of this leukaemia. Unexpectedly, recent studies indicate that PML and the PML-ND play a tumour suppressive role in several different types of human neoplasms in addition to APL. Because of PMLs extreme versatility and involvement in multiple cellular pathways, understanding the mechanisms underlying its function, and therefore role in tumour suppression, has been a challenging task. In this review, we attempt to critically appraise the more recent advances in this field and propose new avenues of investigation.

AIRE's CARD Revealed, a New Structure for Central Tolerance Provokes Transcriptional Plasticity

Developing T cells encounter peripheral self-antigens in the thymus in order to delete autoreactive clones. It is now known that the autoimmune regulator protein (AIRE), which is expressed in thymic medullary epithelial cells, plays a key role in regulating the thymic transcription of these peripheral tissue-specific antigens. Mutations in the AIRE gene are associated with a severe multiorgan autoimmune syndrome (APECED), and autoimmune reactivities are manifest in AIRE-deficient mice. Functional AIRE protein is expressed as distinct nuclear puncta, although no structural basis existed to explain their relevance to disease. In addressing the cell biologic basis for APECED, we made the unexpected discovery that an AIRE mutation hot spot lies in a caspase recruitment domain. Combined homology modeling and in vitro data now show how APECED mutations influence the activity of this transcriptional regulator. We also provide novel in vivo evidence for AIREs association with a global transcription cofactor, which may underlie AIREs focal, genome-wide, alteration of the transcriptome.

Poxvirus Targeting of E3 Ligase β-TrCP by Molecular Mimicry: A Mechanism to Inhibit NF-κB Activation and Promote Immune Evasion and Virulence

The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKβ causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished β-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

PML depletion disrupts normal mammary gland development and skews the composition of the mammary luminal cell progenitor pool

Nuclear domains of promyelocytic leukemia protein (PML) are known to act as signaling nodes in many cellular processes. Although the impact of PML expression in driving cell fate decisions for injured cells is well established, the function of PML in the context of tissue development is less well understood. Here, the in vivo role of PML in developmental processes in the murine mammary gland has been investigated. Data are presented showing that expression of PML is tightly regulated by three members of the Stat family of transcription factors that orchestrate the functional development of the mammary secretory epithelium during pregnancy. Developmental phenotypes were also discovered in the virgin and pregnant Pml null mouse, typified by aberrant differentiation of mammary epithelia with reduced ductal and alveolar development. PML depletion was also found to disturb the balance of two distinct luminal progenitor populations. Overall, it is shown that PML is required for cell lineage determination in bi-potent luminal progenitor cells and that the precise regulation of PML expression is required for functional differentiation of alveolar cells.

A Mechanism for the Inhibition of DNA-PK-Mediated DNA Sensing by a Virus

The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses.

Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence

Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (vΔN2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the vΔN2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, vΔN2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus.

Mapping the IκB Kinase β (IKKβ)-binding Interface of the B14 Protein, a Vaccinia Virus Inhibitor of IKKβ-mediated Activation of Nuclear Factor κB*

The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKβ subunit of the IKK complex and preventing NF-κB activation in response to pro-inflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKβ, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKβ and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKβ. In contrast, a second monomeric mutant (F130K) neither bound IKKβ nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKβ interaction. Thus, the dimerization and IKKβ-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.

Mapping the iκB kinase beta (ikkβ)-binding interface of B14, a vaccinia virus inhibitor of IKKβ-mediated activation of nuclear factor kappa B

The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKβ subunit of the IKK complex and preventing NF-κB activation in response to pro-inflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKβ, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKβ and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKβ. In contrast, a second monomeric mutant (F130K) neither bound IKKβ nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKβ interaction. Thus, the dimerization and IKKβ-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.