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. 2015 Mar 6;290(10):5991-6002.
doi: 10.1074/jbc.M114.624650. Epub 2015 Jan 20.

Vaccinia virus protein A49 is an unexpected member of the B-cell Lymphoma (Bcl)-2 protein family

Sarah Neidel  1 , Carlos Maluquer de Motes  1 , Daniel S Mansur  2 , Pavla Strnadova  1 , Geoffrey L Smith  1 , Stephen C Graham  3
Affiliations

Vaccinia virus protein A49 is an unexpected member of the B-cell Lymphoma (Bcl)-2 protein family

Sarah Neidel et al. J Biol Chem. .

Abstract

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.

Keywords: B-cell Lymphoma 2 (Bcl-2) Family; Innate Immunity; NF-κB (NF-KB); Poxvirus; Protein Structure; Structure-based Phylogenetics; Vaccinia Virus; Viral Protein.

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Figures

FIGURE 1.
FIGURE 1.
A49 adopts a Bcl-2-like fold. A, the structure of A49 Δ12 is shown in two orthogonal views as a ribbon colored from blue (N terminus) to red (C terminus). The inset shows the alternative conformations of residues 13–17 observed, highlighting the mobility of this region. In crystals of A49 grown from full-length protein, no density was observed for these residues. B, sequence of VACV WR A49. The secondary structure is shown above the sequence, and residues thought to interact with the β-propeller domain of β-TrCP are in boldface type.
FIGURE 2.
FIGURE 2.
A49 lacks a surface groove and does not bind BH3 peptides. A, the structures of MYXV M11 (blue ribbons, PDB 2JBY (14)) and VACV A49 (yellow ribbons) are shown, superposed, in two orthogonal views. Selected helices are labeled. B, the structure of the human Bak BH3 peptide (green ribbon and side chains) bound to M11 (blue molecular surface) is shown (PDB 2JBY (14)). C, model of A49 in complex with the human Bak BH3 peptide generated by superposing A49 onto the M11-Bak complex (14). Asterisks denote significant clashes. D, unlike M11, A49 does not co-immunoprecipitate with human Bak or Bax. HEK293T cells were transfected with Myc-β-TrCP (lanes 5 and 8), HA-Bak (lanes 1, 2, and 6), or HA-Bax (lanes 3, 4, and 7) and nTAP-A49 (lanes 1–5) or FLAG-M11 (lanes 6–8). After 24 h, cells were infected with vΔA49 at 5 pfu/cell (lanes 2 and 4) or mock-infected (lanes 1, 3, and 5–8). Cells were lysed 6 h after infection, and lysates were immunoprecipitated (IP) with an anti-FLAG matrix before immunoblotting using the antibodies specified. Tubulin served as a loading control, and D8, a VACV envelope protein, served as a positive marker of infection.
FIGURE 3.
FIGURE 3.
A49 does not oligomerize in solution or in cells. A, two molecules of A49 Δ12, which interact via helices α4 and α6 (the “4-6 face”) are shown in two orthogonal views, colored as in Fig. 1A. B, the 4-6 face of A49 Δ12, with side chains of residues that form the homotypic contact surface shown as sticks. C, molecular surface representation of the A49 Δ12 4-6 face colored by amino acid hydrophobicity from white (polar) to green (hydrophobic). D, SEC-MALS of full-length A49 (top), A49 Δ12 (middle), and N1 (bottom), each analyzed at three concentrations. Weight-averaged molar masses (colored solid lines) are shown across the elution profiles (normalized differential refractive index, colored dashed lines). The expected molar masses for monomers and dimers of each protein are shown (gray dotted lines). E, A49 does not self-associate in cells. HEK293T cells were transfected with TAP-tagged (lane 1) or Myc-tagged (lane 2) β-TrCP and nHis-A49 (lanes 1 and 3) or FLAG-B14 (lane 4). After 24 h, cells were mock-infected (lane 1) or infected with vA49-cTAP (lanes 2 and 3) or vB14-HA (lane 4) at 2 pfu/cell or co-infected with both viruses at 1.5 pfu for each virus per cell (lane 5). Cells were lysed 16 h after infection, and lysates were immunoprecipitated (IP) with an anti-FLAG matrix before immunoblotting using the antibodies specified. Tubulin served as a loading control, and D8 served as a positive marker of infection.
FIGURE 4.
FIGURE 4.
Poxvirus Bcl-2 proteins are structurally closer to each other than to other cellular or viral Bcl-2 proteins. A, clustering of cellular and viral Bcl-2 proteins to determine the minimal structural “core” of the Bcl-2 fold. PDB identifiers of structures used are shown as leaves on the tree, colored by source organism (black, cellular; magenta, herpesvirus; blue, poxvirus). Cα atom root mean square deviation (Å) and number of equivalent Cα atoms, which corresponds to the size of the common core, are shown at each branch point. For higher branches in the tree, superpositions were performed between these common cores to iteratively determine the minimal core of the Bcl-2 fold. B, structure-based phylogenetic tree showing the relationship between cellular, herpesvirus, and poxvirus Bcl-2 proteins. PDB codes for each structure used are given with their species of origin in parentheses (human (H), mouse (M), rat (R), Caenorhabditis elegans (C), Schistosoma japonicum (SJ), vaccinia virus (VACV), myxoma virus (MYXV), Kaposi sarcoma herpesvirus (KSHV), murine γ-herpesvirus 68 (MHV-68), and Epstein-Barr virus (EBV)). Structures determined by x-ray crystallography are labeled in black, NMR models are shown in gray, and structures determined with BH3 peptides or C-terminal tails bound in the surface groove are italicized. Ribbon diagrams of representative structures for each protein are shown, color-ramped from blue (N terminus) to red (C terminus). The inset shows the 56 Cα atoms that comprise the minimal core of the Bcl-2 fold, colored as above. Coordinate files containing the core Cα atoms of the Bcl-2 fold plus superpositions of the 35 representative Bcl-2 family structures upon this core are supplied as supplemental material.
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