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Vaccinia Virus Protein A49 Is an Unexpected Member of the B-cell Lymphoma (Bcl)-2 Protein Family*

Open AccessPublished:January 20, 2015DOI:https://doi.org/10.1074/jbc.M114.624650
      Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κB (NF-κB). VACV protein A49 prevents translocation of NF-κB to the nucleus by sequestering cellular β-TrCP, a protein required for the degradation of the inhibitor of κB. A49 does not share overall sequence similarity with any protein of known structure or function. We solved the crystal structure of A49 from VACV Western Reserve to 1.8 Å resolution and showed, surprisingly, that A49 has the same three-dimensional fold as Bcl-2 family proteins despite lacking identifiable sequence similarity. Whereas Bcl-2 family members characteristically modulate cellular apoptosis, A49 lacks a surface groove suitable for binding BH3 peptides and does not bind proapoptotic Bcl-2 family proteins Bax or Bak. The N-terminal 17 residues of A49 do not adopt a single well ordered conformation, consistent with their proposed role in binding β-TrCP. Whereas pairs of A49 molecules interact symmetrically via a large hydrophobic surface in crystallo, A49 does not dimerize in solution or in cells, and we propose that this hydrophobic interaction surface may mediate binding to a yet undefined cellular partner. A49 represents the eleventh VACV Bcl-2 family protein and, despite these proteins sharing very low sequence identity, structure-based phylogenetic analysis shows that all poxvirus Bcl-2 proteins are structurally more similar to each other than they are to any cellular or herpesvirus Bcl-2 proteins. This is consistent with duplication and diversification of a single BCL2 family gene acquired by an ancestral poxvirus.

      Introduction

      Vaccinia virus (VACV),
      The abbreviations used are: VACV
      vaccinia virus
      IKKβ
      IκB kinase β
      MALS
      multiangle light scattering
      MYXV
      myxoma virus
      SEC
      size exclusion chromatography
      WR
      Western Reserve.
      the vaccine used to eradicate smallpox, has a large dsDNA genome encoding ∼200 ORFs (
      • Moss B.
      ). About half of these genes are not essential for virus replication but rather affect the virulence and host range of the virus by counteracting the host immune response to infection (
      • Gubser C.
      • Hué S.
      • Kellam P.
      • Smith G.L.
      Poxvirus genomes: a phylogenetic analysis.
      ). Understanding how VACV modulates the host immune response can yield unexpected insights into cellular innate immunity (
      • Ferguson B.J.
      • Mansur D.S.
      • Peters N.E.
      • Ren H.
      • Smith G.L.
      DNA-PK is a DNA sensor for IRF-3-dependent innate immunity.
      ) and is essential to fully exploit the promise of VACV as a safe vaccine vector (
      • Walsh S.R.
      • Dolin R.
      Vaccinia viruses: vaccines against smallpox and vectors against infectious diseases and tumors.
      ).
      VACV A49 is a virulence factor that inhibits the activation of the host transcription factor NF-κB (
      • Mansur D.S.
      • Maluquer de Motes C.
      • Unterholzner L.
      • Sumner R.P.
      • Ferguson B.J.
      • Ren H.
      • Strnadova P.
      • Bowie A.G.
      • Smith G.L.
      Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.
      ). NF-κB activity is stimulated by extrinsic proinflammatory signals, such as interleukin 1 or tumor necrosis factor α, and by intracellular signals, such as the recognition of foreign cytoplasmic RNA by retinoic acid-inducible gene I-like helicases (
      • Oeckinghaus A.
      • Ghosh S.
      The NF-κB family of transcription factors and its regulation.
      ). NF-κB is usually retained, inactive, in the cytoplasm via an interaction with the inhibitor of κB (IκBα). Upon proinflammatory stimulation, IκBα is phosphorylated by IκB kinases and subsequently ubiquitinated and degraded. This allows NF-κB to translocate to the nucleus and promote transcription of cytokines and chemokines, including interferon β, that promote inflammation and an anti-viral state in surrounding cells (
      • Balachandran S.
      • Beg A.A.
      Defining emerging roles for NF-kappaB in antivirus responses: revisiting the interferon-β enhanceosome paradigm.
      ). A49 inhibits the ubiquitination of IκBα by binding β-TrCP, a component of the Skp1·Cullin1·F-box protein (SCF)β-TrCP ubiquitin E3 ligase complex. Mutational analysis suggested that it does so via an N-terminal peptide that binds the β-propeller domain of β-TrCP in an extended fashion, similar to the binding mode observed for β-catenin (
      • Wu G.
      • Xu G.
      • Schulman B.A.
      • Jeffrey P.D.
      • Harper J.W.
      • Pavletich N.P.
      Structure of a β-TrCP1-Skp1-β-catenin complex: destruction motif binding and lysine specificity of the SCFβ-TrCP1 ubiquitin ligase.
      ), and thereby blocks binding of IκBα to the same site (
      • Mansur D.S.
      • Maluquer de Motes C.
      • Unterholzner L.
      • Sumner R.P.
      • Ferguson B.J.
      • Ren H.
      • Strnadova P.
      • Bowie A.G.
      • Smith G.L.
      Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.
      ). The importance of inhibiting NF-κB activation to the virus is underscored by the fact that VACV has at least 10 proteins that interrupt this pathway and deletion of any of these genes attenuates the virus (
      • Smith G.L.
      • Benfield C.T.
      • Maluquer de Motes C.
      • Mazzon M.
      • Ember S.W.
      • Ferguson B.J.
      • Sumner R.P.
      Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity.
      ). Inhibiting the degradation of IκBα appears to be of particular importance for poxvirus virulence; ectromelia virus lacks an A49 homologue but it contains EVM005, an ankyrin family protein that contains an F-box domain and is thought to compete with the F-box region of β-TrCP for binding to Skp1, thus preventing IκBα ubiquitination and degradation (
      • van Buuren N.
      • Burles K.
      • Schriewer J.
      • Mehta N.
      • Parker S.
      • Buller R.M.
      • Barry M.
      EVM005: an ectromelia-encoded protein with dual roles in NF-κB inhibition and virulence.
      ).
      Previous studies have identified that many poxvirus immunomodulatory proteins, including 10 VACV proteins, share structural similarity with the Bcl-2 (B-cell lymphoma 2) family of cellular proteins despite lacking identifiable sequence similarity (
      • Aoyagi M.
      • Zhai D.
      • Jin C.
      • Aleshin A.E.
      • Stec B.
      • Reed J.C.
      • Liddington R.C.
      Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein.
      ,
      • Cooray S.
      • Bahar M.W.
      • Abrescia N.G.
      • McVey C.E.
      • Bartlett N.W.
      • Chen R.A.
      • Stuart D.I.
      • Grimes J.M.
      • Smith G.L.
      Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein.
      ,
      • Graham S.C.
      • Bahar M.W.
      • Cooray S.
      • Chen R.A.
      • Whalen D.M.
      • Abrescia N.G.
      • Alderton D.
      • Owens R.J.
      • Stuart D.I.
      • Smith G.L.
      • Grimes J.M.
      Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-κB rather than apoptosis.
      ,
      • Kvansakul M.
      • van Delft M.F.
      • Lee E.F.
      • Gulbis J.M.
      • Fairlie W.D.
      • Huang D.C.
      • Colman P.M.
      A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak.
      ,
      • Kvansakul M.
      • Yang H.
      • Fairlie W.D.
      • Czabotar P.E.
      • Fischer S.F.
      • Perugini M.A.
      • Huang D.C.
      • Colman P.M.
      Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands.
      ,
      • González J.M.
      • Esteban M.
      A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history.
      ). Cellular members of the Bcl-2 family generally share four Bcl-2 homology domains (BH1–BH4) in their primary sequence, and they are key regulators of apoptosis (
      • Czabotar P.E.
      • Lessene G.
      • Strasser A.
      • Adams J.M.
      Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy.
      ). Whereas poxvirus Bcl-2-like proteins F1, M11, and N1 all act to inhibit apoptosis, other poxvirus Bcl-2 family proteins have instead evolved to inhibit activation of the transcription factors NF-κB or IRF3 (
      • Smith G.L.
      • Benfield C.T.
      • Maluquer de Motes C.
      • Mazzon M.
      • Ember S.W.
      • Ferguson B.J.
      • Sumner R.P.
      Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity.
      ,
      • Graham S.C.
      • Bahar M.W.
      • Cooray S.
      • Chen R.A.
      • Whalen D.M.
      • Abrescia N.G.
      • Alderton D.
      • Owens R.J.
      • Stuart D.I.
      • Smith G.L.
      • Grimes J.M.
      Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-κB rather than apoptosis.
      ). Of particular interest is VACV protein N1, which inhibits both apoptosis and NF-κB via two distinct molecular surfaces; inhibition of apoptosis is mediated via a hydrophobic surface groove, whereas inhibition of NF-κB is disrupted by mutating a surface on the opposite face of the protein that mediates N1 dimerization both in solution and in cells (
      • Maluquer de Motes C.
      • Cooray S.
      • Ren H.
      • Almeida G.M.
      • McGourty K.
      • Bahar M.W.
      • Stuart D.I.
      • Grimes J.M.
      • Graham S.C.
      • Smith G.L.
      Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence.
      ).
      Homologues of A49 are found only in a subset of orthopoxviruses (
      • Mansur D.S.
      • Maluquer de Motes C.
      • Unterholzner L.
      • Sumner R.P.
      • Ferguson B.J.
      • Ren H.
      • Strnadova P.
      • Bowie A.G.
      • Smith G.L.
      Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.
      ). Aside from a stretch of six amino acids at its N terminus, the 18.8-kDa A49 protein and its orthologues in other orthopoxviruses share no identifiable sequence identity with any protein of known structure or function. To further investigate the function of A49, we solved its structure to 1.8 Å resolution by x-ray crystallography. Unexpectedly, the structure showed that A49 possesses a Bcl-2-like fold despite not sharing sequence identity with known cellular or poxvirus Bcl-2 family proteins. Structure-based phylogenetic analysis shows that A49 is most closely related to other poxvirus Bcl-2-like proteins, consistent with its evolution from an ancestral poxvirus BCL2 family gene by means of gene duplication and diversification.

      DISCUSSION

      We expressed recombinant VACV A49 and solved its structure to 1.8 Å resolution (Fig. 1). Surprisingly, A49 adopts a Bcl-2-like fold, despite lacking identifiable sequence similarity with other members of the Bcl-2 family. The defining characteristic of cellular Bcl-2 family proteins is their involvement in the regulation of apoptosis. Intrinsic apoptosis is triggered when the proapoptotic Bcl-2 family effector proteins Bak and Bax oligomerize and permeabilize the outer mitochondrial membrane, leading to an irreversible caspase cascade and cell death (
      • Czabotar P.E.
      • Lessene G.
      • Strasser A.
      • Adams J.M.
      Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy.
      ). Antiapoptotic Bcl-2 proteins oppose apoptosis via a surface groove formed by helices α2–α5. This groove binds to the exposed BH3 peptides of activated Bax or Bak, preventing their oligomerization. The groove also binds the BH3 peptides of BH3-only proteins like Bim, sequestering such peptides and thereby preventing them from binding to and activating Bax or Bak (
      • Czabotar P.E.
      • Lessene G.
      • Strasser A.
      • Adams J.M.
      Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy.
      ). MXYV protein M11 and VACV proteins N1 and F1 all adopt the Bcl-2 fold and inhibit apoptosis by binding BH3 peptides via a groove on their surface formed by helices α2–α5 (
      • Cooray S.
      • Bahar M.W.
      • Abrescia N.G.
      • McVey C.E.
      • Bartlett N.W.
      • Chen R.A.
      • Stuart D.I.
      • Grimes J.M.
      • Smith G.L.
      Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein.
      ,
      • Kvansakul M.
      • van Delft M.F.
      • Lee E.F.
      • Gulbis J.M.
      • Fairlie W.D.
      • Huang D.C.
      • Colman P.M.
      A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak.
      ,
      • Kvansakul M.
      • Yang H.
      • Fairlie W.D.
      • Czabotar P.E.
      • Fischer S.F.
      • Perugini M.A.
      • Huang D.C.
      • Colman P.M.
      Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands.
      ,
      • Maluquer de Motes C.
      • Cooray S.
      • Ren H.
      • Almeida G.M.
      • McGourty K.
      • Bahar M.W.
      • Stuart D.I.
      • Grimes J.M.
      • Graham S.C.
      • Smith G.L.
      Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence.
      ). The A49 structure shows that A49 lacks a surface groove compatible with binding BH3 peptides and is unable to bind the proapoptotic proteins Bax or Bak (Fig. 2). This indicates that unlike M11, its closest structural relative, A49 does not function to inhibit apoptosis by sequestering effector BH3 peptides.
      A49 inhibits NF-κB activation by inhibiting ubiquitination and subsequent degradation of IκBα (
      • Mansur D.S.
      • Maluquer de Motes C.
      • Unterholzner L.
      • Sumner R.P.
      • Ferguson B.J.
      • Ren H.
      • Strnadova P.
      • Bowie A.G.
      • Smith G.L.
      Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.
      ). A49 achieves this by sequestering the E3 ligase β-TrCP, preventing it from binding the phosphorylated form of IκBα. The interaction with β-TrCP requires A49 residues 6–12 (
      • Mansur D.S.
      • Maluquer de Motes C.
      • Unterholzner L.
      • Sumner R.P.
      • Ferguson B.J.
      • Ren H.
      • Strnadova P.
      • Bowie A.G.
      • Smith G.L.
      Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.
      ), which contain a double serine motif that is likely to be phosphorylated and bind the β-propeller domain of β-TrCP in an extended conformation similar to that observed in the complex of β-TrCP with β-catenin (
      • Mansur D.S.
      • Maluquer de Motes C.
      • Unterholzner L.
      • Sumner R.P.
      • Ferguson B.J.
      • Ren H.
      • Strnadova P.
      • Bowie A.G.
      • Smith G.L.
      Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.
      ,
      • Wu G.
      • Xu G.
      • Schulman B.A.
      • Jeffrey P.D.
      • Harper J.W.
      • Pavletich N.P.
      Structure of a β-TrCP1-Skp1-β-catenin complex: destruction motif binding and lysine specificity of the SCFβ-TrCP1 ubiquitin ligase.
      ). In the structure of full-length A49, we did not observe any interpretable electron density N-terminal to residue 18, nor did we observe “additional” electron density anywhere in the structure that could be interpreted as residues 1–17. Additionally, we observed that A49 crystallized much more readily upon removal of residues 1–12. These observations indicate that the N-terminal β-TrCP-binding residues of A49 lack intrinsic structure and are thus freely available to bind β-TrCP. However, we note that residues Ser7 and Ser12 of full-length A49 produced in E. coli are unlikely to have been phosphorylated, which may possibly influence the conformation of this region.
      The HIV-1 immunomodulatory protein Vpu functions similarly to A49, binding β-TrCP and preventing degradation of IκBα (
      • Bour S.
      • Perrin C.
      • Akari H.
      • Strebel K.
      The human immunodeficiency virus type 1 Vpu protein inhibits NF-κB activation by interfering with β TrCP-mediated degradation of IκB.
      ). However, Vpu also binds the HIV cell surface receptor CD4 and the restriction factor tetherin, promoting their ubiquitination and degradation by bringing them in close proximity to the SCFβ-TrCP E3 ligase complex (
      • Douglas J.L.
      • Viswanathan K.
      • McCarroll M.N.
      • Gustin J.K.
      • Früh K.
      • Moses A.V.
      Vpu directs the degradation of the human immunodeficiency virus restriction factor BST-2/Tetherin via a βTrCP-dependent mechanism.
      ,
      • Margottin F.
      • Bour S.P.
      • Durand H.
      • Selig L.
      • Benichou S.
      • Richard V.
      • Thomas D.
      • Strebel K.
      • Benarous R.
      A novel human WD protein, h-β TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif.
      ). Although a cellular target of A49-mediated proteasomal or lysosomal degradation has yet to be identified, inspection of the A49 crystal structures identified a surface of the protein that is a prime candidate for mediating such interactions. In all crystal forms presented here the two molecules of A49 self-associate via a symmetric interaction between helices α4 and α6 (the 4-6 face). This interaction is similar to the self-association observed in crystals of VACV A46 (
      • Fedosyuk S.
      • Grishkovskaya I.
      • de Almeida Ribeiro Jr., E.
      • Skern T.
      Characterization and structure of the vaccinia virus NF-κB antagonist A46.
      ) but, unlike A46, A49 does not form dimers in solution or in cells (Fig. 3). Inspection of the 4-6 face reveals a hydrophobic surface cleft that would seem ideal for mediating interactions with cellular binding partners (Fig. 3). Dimerization of VACV proteins in crystallo by surfaces that mediate binding to cellular partners has been observed before; the 1-6 face of B14 that mediates its reversible self-association in solution overlaps with its binding site for IKKβ (
      • Benfield C.T.
      • Mansur D.S.
      • McCoy L.E.
      • Ferguson B.J.
      • Bahar M.W.
      • Oldring A.P.
      • Grimes J.M.
      • Stuart D.I.
      • Graham S.C.
      • Smith G.L.
      Mapping the IκB kinase β (IKKβ)-binding interface of the B14 protein, a vaccinia virus inhibitor of IKKβ-mediated activation of nuclear factor κB.
      ), mutations that abolish dimerization of N1 also disrupt its ability to inhibit NF-κB activation (
      • Maluquer de Motes C.
      • Cooray S.
      • Ren H.
      • Almeida G.M.
      • McGourty K.
      • Bahar M.W.
      • Stuart D.I.
      • Grimes J.M.
      • Graham S.C.
      • Smith G.L.
      Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence.
      ), and a residue of A52 required for binding to TRAF6 lies partly buried within the dimerization interface (
      • Stack J.
      • Hurst T.P.
      • Flannery S.M.
      • Brennan K.
      • Rupp S.
      • Oda S.
      • Khan A.R.
      • Bowie A.G.
      Poxviral protein A52 stimulates p38 mitogen-activated protein kinase (MAPK) activation by causing tumor necrosis factor receptor-associated factor 6 (TRAF6) self-association leading to transforming growth factor β-activated kinase 1 (TAK1) recruitment.
      ), although in this case maintaining A52 dimerization seems to be required for efficient TRAF6-mediated stimulation of p38 and subsequent induction of IL-10 expression. It is tempting to speculate that A49 binds other cellular factors via the 4-6 face to promote their β-TrCP-mediated ubiquitination and degradation, although further experiments are required to probe this hypothesis.
      The structure of A49 takes to 11 the number of VACV proteins that have been shown or predicted to share the Bcl-2 fold (Table 3). Although the bulk of poxvirus Bcl-2 proteins share weak but identifiable sequence similarity (
      • González J.M.
      • Esteban M.
      A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history.
      ,
      • Smith G.L.
      • Chan Y.S.
      • Howard S.T.
      Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat.
      ), A49 could not be identified as a Bcl-2 family protein based on sequence alone. Nonetheless, structure-based phylogenetic analysis shows A49 to be more closely related to poxvirus than herpesvirus or cellular Bcl-2 proteins (Fig. 4). This is consistent with poxvirus Bcl-2 family proteins having arisen from gene duplication and divergence following a single gene acquisition event, structural similarity having been conserved despite vast sequence divergence. The terminal regions of the poxvirus genome are highly variable, containing non-essential genes that act to determine host range and inhibit the host immune response (
      • Gubser C.
      • Hué S.
      • Kellam P.
      • Smith G.L.
      Poxvirus genomes: a phylogenetic analysis.
      ). In addition to terminal transpositions, whereby genes from one end of the linear genome are duplicated at the other end (
      • Smith G.L.
      • Chan Y.S.
      • Howard S.T.
      Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat.
      ,
      • Kotwal G.J.
      • Moss B.
      Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant.
      ,
      • Pickup D.J.
      • Ink B.S.
      • Parsons B.L.
      • Hu W.
      • Joklik W.K.
      Spontaneous deletions and duplications of sequences in the genome of cowpox virus.
      ), a recent study showed that poxviruses deploy “genomic accordions” when under selective pressure, their genomes rapidly expanding to incorporate multiple copies of genes near points of genomic instability (
      • Elde N.C.
      • Child S.J.
      • Eickbush M.T.
      • Kitzman J.O.
      • Rogers K.S.
      • Shendure J.
      • Geballe A.P.
      • Malik H.S.
      Poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses.
      ). This expansion increases the probability that duplicated genes will acquire “advantageous” mutations, potentially conferring a divergent function upon the mutated gene. The host innate immune response places large DNA viruses under significant selective pressure (
      • French A.R.
      • Pingel J.T.
      • Wagner M.
      • Bubic I.
      • Yang L.
      • Kim S.
      • Koszinowski U.
      • Jonjic S.
      • Yokoyama W.M.
      Escape of mutant double-stranded DNA virus from innate immune control.
      ), and poxvirus Bcl-2 family proteins all act to inhibit the cellular responses to infection. Inspection of the low frequency duplications observed in the Copenhagen strain of VACV (or strains derived therefrom) (
      • Elde N.C.
      • Child S.J.
      • Eickbush M.T.
      • Kitzman J.O.
      • Rogers K.S.
      • Shendure J.
      • Geballe A.P.
      • Malik H.S.
      Poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses.
      ) show independent gene duplications encompassing several Bcl-2 family proteins: the duplicated VACV Copenhagen region spanning nucleotides 143,153–156,405 contains A46 and A49; the duplicated region spanning nucleotides 22,298–29,837 contains C1, N1, and N2; and the duplicated regions spanning nucleotides 24,975–47,387 or 25,066–47,467 contain N1, K7, and F1. This is consistent with the gene duplication and differentiation events that gave rise to the 11 VACV Bcl-2 family immunomodulatory proteins having arisen as a result of ancestral poxviruses deploying their genomic accordions in response to selective pressure generated by adaptation in the host or changes in host range. Although A49 has high sequence divergence from the other poxvirus Bcl-2 proteins, its absence from Yata-, Lepri-, Sui-, Cervid-, or Capripoxviruses, all of which have multiple Bcl-2 like proteins, makes it a poor candidate for being the BCL2 gene acquired originally by the ancestral poxvirus.
      TABLE 3VACV proteins with a Bcl-2-like fold
      Protein
      Protein names are for VACV strain Copenhagen except as noted.
      FunctionReferences
      C16/B22Unknown
      • Graham S.C.
      • Bahar M.W.
      • Cooray S.
      • Chen R.A.
      • Whalen D.M.
      • Abrescia N.G.
      • Alderton D.
      • Owens R.J.
      • Stuart D.I.
      • Smith G.L.
      • Grimes J.M.
      Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-κB rather than apoptosis.
      C6Inhibition of IRF3 and IRF7
      • Graham S.C.
      • Bahar M.W.
      • Cooray S.
      • Chen R.A.
      • Whalen D.M.
      • Abrescia N.G.
      • Alderton D.
      • Owens R.J.
      • Stuart D.I.
      • Smith G.L.
      • Grimes J.M.
      Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-κB rather than apoptosis.
      ,
      • Unterholzner L.
      • Sumner R.P.
      • Baran M.
      • Ren H.
      • Mansur D.S.
      • Bourke N.M.
      • Randow F.
      • Smith G.L.
      • Bowie A.G.
      Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.
      C1Unknown
      • González J.M.
      • Esteban M.
      A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history.
      N1Inhibition of NF-κB and apoptosis
      • Aoyagi M.
      • Zhai D.
      • Jin C.
      • Aleshin A.E.
      • Stec B.
      • Reed J.C.
      • Liddington R.C.
      Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein.
      ,
      • Cooray S.
      • Bahar M.W.
      • Abrescia N.G.
      • McVey C.E.
      • Bartlett N.W.
      • Chen R.A.
      • Stuart D.I.
      • Grimes J.M.
      • Smith G.L.
      Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein.
      ,
      • DiPerna G.
      • Stack J.
      • Bowie A.G.
      • Boyd A.
      • Kotwal G.
      • Zhang Z.
      • Arvikar S.
      • Latz E.
      • Fitzgerald K.A.
      • Marshall W.L.
      Poxvirus protein N1L targets the I-κB kinase complex, inhibits signaling to NF-κB by the tumor necrosis factor superfamily of receptors, and inhibits NF-κB and IRF3 signaling by Toll-like receptors.
      N2Inhibition of IRF3
      • González J.M.
      • Esteban M.
      A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history.
      ,
      • Ferguson B.J.
      • Benfield C.T.
      • Ren H.
      • Lee V.H.
      • Frazer G.L.
      • Strnadova P.
      • Sumner R.P.
      • Smith G.L.
      Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence.
      K7Inhibition of NF-κB and IRF3
      • Graham S.C.
      • Bahar M.W.
      • Cooray S.
      • Chen R.A.
      • Whalen D.M.
      • Abrescia N.G.
      • Alderton D.
      • Owens R.J.
      • Stuart D.I.
      • Smith G.L.
      • Grimes J.M.
      Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-κB rather than apoptosis.
      ,
      • Schröder M.
      • Baran M.
      • Bowie A.G.
      Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKϵ-mediated IRF activation.
      ,
      • Kalverda A.P.
      • Thompson G.S.
      • Vogel A.
      • Schröder M.
      • Bowie A.G.
      • Khan A.R.
      • Homans S.W.
      Poxvirus K7 protein adopts a Bcl-2 fold: biochemical mapping of its interactions with human DEAD box RNA helicase DDX3.
      F1Inhibition of apoptosis and IL-1β production
      • Kvansakul M.
      • Yang H.
      • Fairlie W.D.
      • Czabotar P.E.
      • Fischer S.F.
      • Perugini M.A.
      • Huang D.C.
      • Colman P.M.
      Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands.
      ,
      • Gerlic M.
      • Faustin B.
      • Postigo A.
      • Yu E.C.
      • Proell M.
      • Gombosuren N.
      • Krajewska M.
      • Flynn R.
      • Croft M.
      • Way M.
      • Satterthwait A.
      • Liddington R.C.
      • Salek-Ardakani S.
      • Matsuzawa S.
      • Reed J.C.
      Vaccinia virus F1L protein promotes virulence by inhibiting inflammasome activation.
      ,
      • Postigo A.
      • Cross J.R.
      • Downward J.
      • Way M.
      Interaction of F1L with the BH3 domain of Bak is responsible for inhibiting vaccinia-induced apoptosis.
      ,
      • Wasilenko S.T.
      • Stewart T.L.
      • Meyers A.F.
      • Barry M.
      Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis.
      A46Inhibition of MAPKs, NF-κB and IRF3
      • Graham S.C.
      • Bahar M.W.
      • Cooray S.
      • Chen R.A.
      • Whalen D.M.
      • Abrescia N.G.
      • Alderton D.
      • Owens R.J.
      • Stuart D.I.
      • Smith G.L.
      • Grimes J.M.
      Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-κB rather than apoptosis.
      ,
      • Fedosyuk S.
      • Grishkovskaya I.
      • de Almeida Ribeiro Jr., E.
      • Skern T.
      Characterization and structure of the vaccinia virus NF-κB antagonist A46.
      ,
      • Stack J.
      • Haga I.R.
      • Schröder M.
      • Bartlett N.W.
      • Maloney G.
      • Reading P.C.
      • Fitzgerald K.A.
      • Smith G.L.
      • Bowie A.G.
      Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence.
      ,
      • Bowie A.
      • Kiss-Toth E.
      • Symons J.A.
      • Smith G.L.
      • Dower S.K.
      • O'Neill L.A.
      A46R and A52R from vaccinia virus are antagonists of host IL-1 and Toll-like receptor signaling.
      A49Inhibition of NF-κB
      • Mansur D.S.
      • Maluquer de Motes C.
      • Unterholzner L.
      • Sumner R.P.
      • Ferguson B.J.
      • Ren H.
      • Strnadova P.
      • Bowie A.G.
      • Smith G.L.
      Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.
      , this study
      A52Inhibition of NF-κB and stimulation of p38 MAPK
      • Graham S.C.
      • Bahar M.W.
      • Cooray S.
      • Chen R.A.
      • Whalen D.M.
      • Abrescia N.G.
      • Alderton D.
      • Owens R.J.
      • Stuart D.I.
      • Smith G.L.
      • Grimes J.M.
      Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-κB rather than apoptosis.
      ,
      • Harte M.T.
      • Haga I.R.
      • Maloney G.
      • Gray P.
      • Reading P.C.
      • Bartlett N.W.
      • Smith G.L.
      • Bowie A.
      • O'Neill L.A.
      The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense.
      ,
      • Stack J.
      • Hurst T.P.
      • Flannery S.M.
      • Brennan K.
      • Rupp S.
      • Oda S.
      • Khan A.R.
      • Bowie A.G.
      Poxviral protein A52 stimulates p38 mitogen-activated protein kinase (MAPK) activation by causing tumor necrosis factor receptor-associated factor 6 (TRAF6) self-association leading to transforming growth factor β-activated kinase 1 (TAK1) recruitment.
      ,
      • Bowie A.
      • Kiss-Toth E.
      • Symons J.A.
      • Smith G.L.
      • Dower S.K.
      • O'Neill L.A.
      A46R and A52R from vaccinia virus are antagonists of host IL-1 and Toll-like receptor signaling.
      B14
      Encoded by VACV WR gene B14R, which is equivalent to VACV Copenhagen gene B15R (22).
      Inhibition of NF-κB
      • Graham S.C.
      • Bahar M.W.
      • Cooray S.
      • Chen R.A.
      • Whalen D.M.
      • Abrescia N.G.
      • Alderton D.
      • Owens R.J.
      • Stuart D.I.
      • Smith G.L.
      • Grimes J.M.
      Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-κB rather than apoptosis.
      ,
      • Chen R.A.
      • Ryzhakov G.
      • Cooray S.
      • Randow F.
      • Smith G.L.
      Inhibition of IκB kinase by vaccinia virus virulence factor B14.
      a Protein names are for VACV strain Copenhagen except as noted.
      b Encoded by VACV WR gene B14R, which is equivalent to VACV Copenhagen gene B15R (
      • Chen R.A.
      • Jacobs N.
      • Smith G.L.
      Vaccinia virus strain Western Reserve protein B14 is an intracellular virulence factor.
      ).
      In summary, we have shown that A49 is an unanticipated eleventh member of the VACV Bcl-2-like immunomodulatory protein family. A49 lacks a BH3 peptide binding groove and does not bind the proapoptotic proteins Bax and Bak. Although A49 self-associates via a hydrophobic 4-6 face in crystals, it does not self-associate in solution or in cells, suggesting that this 4-6 face may mediate binding to yet undetermined cellular partners. Conservation of the Bcl-2 fold by poxvirus proteins with highly divergent sequences is consistent with duplication and divergence of an ancestral gene encoding a Bcl-2 family protein.

      Acknowledgments

      We thank Diamond Light Source for access to beamline I04-1 (mx8547) and the European Synchrotron Radiation Facility for access to beamline ID14-2, which contributed to the results presented here. We thank David Stuart and Jonathan Grimes for advice and helpful comments, Janne Ravantti for access to the HSF analysis software, and Janet Deane for assistance with SEC-MALS and helpful discussions.

      REFERENCES

        • Moss B.
        Fields B.N. Knipe D.M. Howley P.M. Fields' Virology. 5th Ed. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia2007: 2905-2946
        • Gubser C.
        • Hué S.
        • Kellam P.
        • Smith G.L.
        Poxvirus genomes: a phylogenetic analysis.
        J. Gen. Virol. 2004; 85: 105-117
        • Ferguson B.J.
        • Mansur D.S.
        • Peters N.E.
        • Ren H.
        • Smith G.L.
        DNA-PK is a DNA sensor for IRF-3-dependent innate immunity.
        eLife. 2012; 1: e00047
        • Walsh S.R.
        • Dolin R.
        Vaccinia viruses: vaccines against smallpox and vectors against infectious diseases and tumors.
        Expert Rev. Vaccines. 2011; 10: 1221-1240
        • Mansur D.S.
        • Maluquer de Motes C.
        • Unterholzner L.
        • Sumner R.P.
        • Ferguson B.J.
        • Ren H.
        • Strnadova P.
        • Bowie A.G.
        • Smith G.L.
        Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.
        PLoS Pathog. 2013; 9: e1003183
        • Oeckinghaus A.
        • Ghosh S.
        The NF-κB family of transcription factors and its regulation.
        Cold Spring Harb. Perspect. Biol. 2009; 1: a000034
        • Balachandran S.
        • Beg A.A.
        Defining emerging roles for NF-kappaB in antivirus responses: revisiting the interferon-β enhanceosome paradigm.
        PLoS Pathog. 2011; 7: e1002165
        • Wu G.
        • Xu G.
        • Schulman B.A.
        • Jeffrey P.D.
        • Harper J.W.
        • Pavletich N.P.
        Structure of a β-TrCP1-Skp1-β-catenin complex: destruction motif binding and lysine specificity of the SCFβ-TrCP1 ubiquitin ligase.
        Mol. Cell. 2003; 11: 1445-1456
        • Smith G.L.
        • Benfield C.T.
        • Maluquer de Motes C.
        • Mazzon M.
        • Ember S.W.
        • Ferguson B.J.
        • Sumner R.P.
        Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity.
        J. Gen. Virol. 2013; 94: 2367-2392
        • van Buuren N.
        • Burles K.
        • Schriewer J.
        • Mehta N.
        • Parker S.
        • Buller R.M.
        • Barry M.
        EVM005: an ectromelia-encoded protein with dual roles in NF-κB inhibition and virulence.
        PLoS Pathog. 2014; 10: e1004326
        • Aoyagi M.
        • Zhai D.
        • Jin C.
        • Aleshin A.E.
        • Stec B.
        • Reed J.C.
        • Liddington R.C.
        Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein.
        Protein Sci. 2007; 16: 118-124
        • Cooray S.
        • Bahar M.W.
        • Abrescia N.G.
        • McVey C.E.
        • Bartlett N.W.
        • Chen R.A.
        • Stuart D.I.
        • Grimes J.M.
        • Smith G.L.
        Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein.
        J. Gen. Virol. 2007; 88: 1656-1666
        • Graham S.C.
        • Bahar M.W.
        • Cooray S.
        • Chen R.A.
        • Whalen D.M.
        • Abrescia N.G.
        • Alderton D.
        • Owens R.J.
        • Stuart D.I.
        • Smith G.L.
        • Grimes J.M.
        Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-κB rather than apoptosis.
        PLoS Pathog. 2008; 4: e1000128
        • Kvansakul M.
        • van Delft M.F.
        • Lee E.F.
        • Gulbis J.M.
        • Fairlie W.D.
        • Huang D.C.
        • Colman P.M.
        A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak.
        Mol. Cell. 2007; 25: 933-942
        • Kvansakul M.
        • Yang H.
        • Fairlie W.D.
        • Czabotar P.E.
        • Fischer S.F.
        • Perugini M.A.
        • Huang D.C.
        • Colman P.M.
        Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands.
        Cell Death Differ. 2008; 15: 1564-1571
        • González J.M.
        • Esteban M.
        A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history.
        Virol. J. 2010; 7: 59
        • Czabotar P.E.
        • Lessene G.
        • Strasser A.
        • Adams J.M.
        Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy.
        Nat. Rev. Mol. Cell Biol. 2014; 15: 49-63
        • Maluquer de Motes C.
        • Cooray S.
        • Ren H.
        • Almeida G.M.
        • McGourty K.
        • Bahar M.W.
        • Stuart D.I.
        • Grimes J.M.
        • Graham S.C.
        • Smith G.L.
        Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence.
        PLoS Pathog. 2011; 7: e1002430
        • Berrow N.S.
        • Alderton D.
        • Sainsbury S.
        • Nettleship J.
        • Assenberg R.
        • Rahman N.
        • Stuart D.I.
        • Owens R.J.
        A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.
        Nucleic Acids Res. 2007; 35: e45
        • Teo H.
        • Perisic O.
        • González B.
        • Williams R.L.
        ESCRT-II, an endosome-associated complex required for protein sorting: crystal structure and interactions with ESCRT-III and membranes.
        Dev. Cell. 2004; 7: 559-569
        • Bartlett N.
        • Symons J.A.
        • Tscharke D.C.
        • Smith G.L.
        The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence.
        J. Gen. Virol. 2002; 83: 1965-1976
        • Chen R.A.
        • Jacobs N.
        • Smith G.L.
        Vaccinia virus strain Western Reserve protein B14 is an intracellular virulence factor.
        J. Gen. Virol. 2006; 87: 1451-1458
        • Walter T.S.
        • Diprose J.M.
        • Mayo C.J.
        • Siebold C.
        • Pickford M.G.
        • Carter L.
        • Sutton G.C.
        • Berrow N.S.
        • Brown J.
        • Berry I.M.
        • Stewart-Jones G.B.
        • Grimes J.M.
        • Stammers D.K.
        • Esnouf R.M.
        • Jones E.Y.
        • Owens R.J.
        • Stuart D.I.
        • Harlos K.
        A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization.
        Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 651-657
        • Holyoak T.
        • Fenn T.D.
        • Wilson M.A.
        • Moulin A.G.
        • Ringe D.
        • Petsko G.A.
        Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals.
        Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 2356-2358
        • Kabsch W.
        XDS.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 125-132
        • Winter G.
        xia2: an expert system for macromolecular crystallography data reduction.
        J. Appl. Cryst. 2010; 43: 186-190
        • Vonrhein C.
        • Blanc E.
        • Roversi P.
        • Bricogne G.
        Automated structure solution with autoSHARP.
        Methods Mol. Biol. 2007; 364: 215-230
        • Perrakis A.
        • Morris R.
        • Lamzin V.S.
        Automated protein model building combined with iterative structure refinement.
        Nat. Struct. Biol. 1999; 6: 458-463
        • Emsley P.
        • Lohkamp B.
        • Scott W.G.
        • Cowtan K.
        Features and development of Coot.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501
        • Murshudov G.N.
        • Skubák P.
        • Lebedev A.A.
        • Pannu N.S.
        • Steiner R.A.
        • Nicholls R.A.
        • Winn M.D.
        • Long F.
        • Vagin A.A.
        REFMAC5 for the refinement of macromolecular crystal structures.
        Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 355-367
        • Vagin A.
        • Teplyakov A.
        MOLREP: an automated program for molecular replacement.
        J. Appl. Cryst. 1997; 30: 1022-1025
        • McCoy A.J.
        • Grosse-Kunstleve R.W.
        • Adams P.D.
        • Winn M.D.
        • Storoni L.C.
        • Read R.J.
        Phaser crystallographic software.
        J. Appl. Cryst. 2007; 40: 658-674
        • Davis I.W.
        • Leaver-Fay A.
        • Chen V.B.
        • Block J.N.
        • Kapral G.J.
        • Wang X.
        • Murray L.W.
        • Arendall 3rd, W.B.
        • Snoeyink J.
        • Richardson J.S.
        • Richardson D.C.
        MolProbity: all-atom contacts and structure validation for proteins and nucleic acids.
        Nucleic Acids Res. 2007; 35: W375-W383
        • Hooft R.W.W.
        • Vriend G.
        • Sander C.
        • Abola E.E.
        Errors in protein structures.
        Nature. 1996; 381 (272): 272
        • Finn R.D.
        • Bateman A.
        • Clements J.
        • Coggill P.
        • Eberhardt R.Y.
        • Eddy S.R.
        • Heger A.
        • Hetherington K.
        • Holm L.
        • Mistry J.
        • Sonnhammer E.L.
        • Tate J.
        • Punta M.
        Pfam: the protein families database.
        Nucleic Acids Res. 2014; 42: D222-D230
        • Kleywegt G.J.
        • Jones T.A.
        Detecting folding motifs and similarities in protein structures.
        Methods Enzymol. 1997; 277: 525-545
        • Kelley L.A.
        • Sutcliffe M.J.
        OLDERADO: on-line database of ensemble representatives and domains.
        Protein Sci. 1997; 6: 2628-2630
        • Ravantti J.
        • Bamford D.
        • Stuart D.I.
        Automatic comparison and classification of protein structures.
        J. Struct. Biol. 2013; 183: 47-56
        • Huson D.H.
        • Scornavacca C.
        Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks.
        Syst. Biol. 2012; 61: 1061-1067
        • Bond C.S.
        • Schüttelkopf A.W.
        ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments.
        Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 510-512
        • Gloeckner C.J.
        • Boldt K.
        • Schumacher A.
        • Roepman R.
        • Ueffing M.
        A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes.
        Proteomics. 2007; 7: 4228-4234
        • Ember S.W.
        • Ren H.
        • Ferguson B.J.
        • Smith G.L.
        Vaccinia virus protein C4 inhibits NF-κB activation and promotes virus virulence.
        J. Gen. Virol. 2012; 93: 2098-2108
        • Falkner F.G.
        • Moss B.
        Transient dominant selection of recombinant vaccinia viruses.
        J. Virol. 1990; 64: 3108-3111
        • Parkinson J.E.
        • Smith G.L.
        Vaccinia virus gene A36R encodes a Mr 43–50 K protein on the surface of extracellular enveloped virus.
        Virology. 1994; 204: 376-390
        • Lama D.
        • Sankararamakrishnan R.
        Identification of core structural residues in the sequentially diverse and structurally homologous Bcl-2 family of proteins.
        Biochemistry. 2010; 49: 2574-2584
        • Krissinel E.
        • Henrick K.
        Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions.
        Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2256-2268
        • Kvansakul M.
        • Hinds M.G.
        Structural biology of the Bcl-2 family and its mimicry by viral proteins.
        Cell Death Dis. 2013; 4: e909
        • Wang G.
        • Barrett J.W.
        • Nazarian S.H.
        • Everett H.
        • Gao X.
        • Bleackley C.
        • Colwill K.
        • Moran M.F.
        • McFadden G.
        Myxoma virus M11L prevents apoptosis through constitutive interaction with Bak.
        J. Virol. 2004; 78: 7097-7111
        • Day C.L.
        • Smits C.
        • Fan F.C.
        • Lee E.F.
        • Fairlie W.D.
        • Hinds M.G.
        Structure of the BH3 domains from the p53-inducible BH3-only proteins Noxa and Puma in complex with Mcl-1.
        J. Mol. Biol. 2008; 380: 958-971
        • Sattler M.
        • Liang H.
        • Nettesheim D.
        • Meadows R.P.
        • Harlan J.E.
        • Eberstadt M.
        • Yoon H.S.
        • Shuker S.B.
        • Chang B.S.
        • Minn A.J.
        • Thompson C.B.
        • Fesik S.W.
        Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis.
        Science. 1997; 275: 983-986
        • Fedosyuk S.
        • Grishkovskaya I.
        • de Almeida Ribeiro Jr., E.
        • Skern T.
        Characterization and structure of the vaccinia virus NF-κB antagonist A46.
        J. Biol. Chem. 2014; 289: 3749-3762
        • Oda S.
        • Schröder M.
        • Khan A.R.
        Structural basis for targeting of human RNA helicase DDX3 by poxvirus protein K7.
        Structure. 2009; 17: 1528-1537
        • Krissinel E.
        • Henrick K.
        Inference of macromolecular assemblies from crystalline state.
        J. Mol. Biol. 2007; 372: 774-797
        • Benfield C.T.
        • Mansur D.S.
        • McCoy L.E.
        • Ferguson B.J.
        • Bahar M.W.
        • Oldring A.P.
        • Grimes J.M.
        • Stuart D.I.
        • Graham S.C.
        • Smith G.L.
        Mapping the IκB kinase β (IKKβ)-binding interface of the B14 protein, a vaccinia virus inhibitor of IKKβ-mediated activation of nuclear factor κB.
        J. Biol. Chem. 2011; 286: 20727-20735
        • Finn R.D.
        • Clements J.
        • Eddy S.R.
        HMMER web server: interactive sequence similarity searching.
        Nucleic Acids Res. 2011; 39: W29-W37
        • Söding J.
        • Remmert M.
        • Biegert A.
        • Lupas A.N.
        HHsenser: exhaustive transitive profile search using HMM-HMM comparison.
        Nucleic Acids Res. 2006; 34: W374-W378
        • Rossmann M.G.
        • Argos P.
        Exploring structural homology of proteins.
        J. Mol. Biol. 1976; 105: 75-95
        • Veyer D.L.
        • Maluquer de Motes C.
        • Sumner R.P.
        • Ludwig L.
        • Johnson B.F.
        • Smith G.L.
        Analysis of the anti-apoptotic activity of four vaccinia virus proteins demonstrates that B13 is the most potent inhibitor in isolation and during viral infection.
        J. Gen. Virol. 2014; 95: 2757-2768
        • Chen R.A.
        • Ryzhakov G.
        • Cooray S.
        • Randow F.
        • Smith G.L.
        Inhibition of IκB kinase by vaccinia virus virulence factor B14.
        PLoS Pathog. 2008; 4: e22
        • Harte M.T.
        • Haga I.R.
        • Maloney G.
        • Gray P.
        • Reading P.C.
        • Bartlett N.W.
        • Smith G.L.
        • Bowie A.
        • O'Neill L.A.
        The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense.
        J. Exp. Med. 2003; 197: 343-351
        • Stack J.
        • Haga I.R.
        • Schröder M.
        • Bartlett N.W.
        • Maloney G.
        • Reading P.C.
        • Fitzgerald K.A.
        • Smith G.L.
        • Bowie A.G.
        Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence.
        J. Exp. Med. 2005; 201: 1007-1018
        • Schröder M.
        • Baran M.
        • Bowie A.G.
        Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKϵ-mediated IRF activation.
        EMBO J. 2008; 27: 2147-2157
        • DiPerna G.
        • Stack J.
        • Bowie A.G.
        • Boyd A.
        • Kotwal G.
        • Zhang Z.
        • Arvikar S.
        • Latz E.
        • Fitzgerald K.A.
        • Marshall W.L.
        Poxvirus protein N1L targets the I-κB kinase complex, inhibits signaling to NF-κB by the tumor necrosis factor superfamily of receptors, and inhibits NF-κB and IRF3 signaling by Toll-like receptors.
        J. Biol. Chem. 2004; 279: 36570-36578
        • Li H.
        • Zhu H.
        • Xu C.J.
        • Yuan J.
        Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.
        Cell. 1998; 94: 491-501
        • Luo X.
        • Budihardjo I.
        • Zou H.
        • Slaughter C.
        • Wang X.
        Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors.
        Cell. 1998; 94: 481-490
        • Wang Y.
        • Tjandra N.
        Structural insights of tBid, the caspase-8-activated Bid, and its BH3 domain.
        J. Biol. Chem. 2013; 288: 35840-35851
        • Bergsten J.
        A review of long-branch attraction.
        Cladistics. 2005; 21: 163-193
        • Bour S.
        • Perrin C.
        • Akari H.
        • Strebel K.
        The human immunodeficiency virus type 1 Vpu protein inhibits NF-κB activation by interfering with β TrCP-mediated degradation of IκB.
        J. Biol. Chem. 2001; 276: 15920-15928
        • Douglas J.L.
        • Viswanathan K.
        • McCarroll M.N.
        • Gustin J.K.
        • Früh K.
        • Moses A.V.
        Vpu directs the degradation of the human immunodeficiency virus restriction factor BST-2/Tetherin via a βTrCP-dependent mechanism.
        J. Virol. 2009; 83: 7931-7947
        • Margottin F.
        • Bour S.P.
        • Durand H.
        • Selig L.
        • Benichou S.
        • Richard V.
        • Thomas D.
        • Strebel K.
        • Benarous R.
        A novel human WD protein, h-β TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif.
        Mol. Cell. 1998; 1: 565-574
        • Stack J.
        • Hurst T.P.
        • Flannery S.M.
        • Brennan K.
        • Rupp S.
        • Oda S.
        • Khan A.R.
        • Bowie A.G.
        Poxviral protein A52 stimulates p38 mitogen-activated protein kinase (MAPK) activation by causing tumor necrosis factor receptor-associated factor 6 (TRAF6) self-association leading to transforming growth factor β-activated kinase 1 (TAK1) recruitment.
        J. Biol. Chem. 2013; 288: 33642-33653
        • Smith G.L.
        • Chan Y.S.
        • Howard S.T.
        Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat.
        J. Gen. Virol. 1991; 72: 1349-1376
        • Kotwal G.J.
        • Moss B.
        Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant.
        Virology. 1988; 167: 524-537
        • Pickup D.J.
        • Ink B.S.
        • Parsons B.L.
        • Hu W.
        • Joklik W.K.
        Spontaneous deletions and duplications of sequences in the genome of cowpox virus.
        Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 6817-6821
        • Elde N.C.
        • Child S.J.
        • Eickbush M.T.
        • Kitzman J.O.
        • Rogers K.S.
        • Shendure J.
        • Geballe A.P.
        • Malik H.S.
        Poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses.
        Cell. 2012; 150: 831-841
        • French A.R.
        • Pingel J.T.
        • Wagner M.
        • Bubic I.
        • Yang L.
        • Kim S.
        • Koszinowski U.
        • Jonjic S.
        • Yokoyama W.M.
        Escape of mutant double-stranded DNA virus from innate immune control.
        Immunity. 2004; 20: 747-756
        • Unterholzner L.
        • Sumner R.P.
        • Baran M.
        • Ren H.
        • Mansur D.S.
        • Bourke N.M.
        • Randow F.
        • Smith G.L.
        • Bowie A.G.
        Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.
        PLoS Pathog. 2011; 7: e1002247
        • Ferguson B.J.
        • Benfield C.T.
        • Ren H.
        • Lee V.H.
        • Frazer G.L.
        • Strnadova P.
        • Sumner R.P.
        • Smith G.L.
        Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence.
        J. Gen. Virol. 2013; 94: 2070-2081
        • Kalverda A.P.
        • Thompson G.S.
        • Vogel A.
        • Schröder M.
        • Bowie A.G.
        • Khan A.R.
        • Homans S.W.
        Poxvirus K7 protein adopts a Bcl-2 fold: biochemical mapping of its interactions with human DEAD box RNA helicase DDX3.
        J. Mol. Biol. 2009; 385: 843-853
        • Gerlic M.
        • Faustin B.
        • Postigo A.
        • Yu E.C.
        • Proell M.
        • Gombosuren N.
        • Krajewska M.
        • Flynn R.
        • Croft M.
        • Way M.
        • Satterthwait A.
        • Liddington R.C.
        • Salek-Ardakani S.
        • Matsuzawa S.
        • Reed J.C.
        Vaccinia virus F1L protein promotes virulence by inhibiting inflammasome activation.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 7808-7813
        • Postigo A.
        • Cross J.R.
        • Downward J.
        • Way M.
        Interaction of F1L with the BH3 domain of Bak is responsible for inhibiting vaccinia-induced apoptosis.
        Cell Death Differ. 2006; 13: 1651-1662
        • Wasilenko S.T.
        • Stewart T.L.
        • Meyers A.F.
        • Barry M.
        Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 14345-14350
        • Bowie A.
        • Kiss-Toth E.
        • Symons J.A.
        • Smith G.L.
        • Dower S.K.
        • O'Neill L.A.
        A46R and A52R from vaccinia virus are antagonists of host IL-1 and Toll-like receptor signaling.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10162-10167
        • Karplus P.A.
        • Diederichs K.
        Linking crystallographic model and data quality.
        Science. 2012; 336: 1030-1033