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Comparative Study
. 2007 Jun;88(Pt 6):1656-1666.
doi: 10.1099/vir.0.82772-0.

Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein

Samantha Cooray  1 , Mohammad W Bahar  2 , Nicola G A Abrescia  2 , Colin E McVey  1 , Nathan W Bartlett  1 , Ron A-J Chen  1 , David I Stuart  2 , Jonathan M Grimes  2 , Geoffrey L Smith  1
Affiliations
Comparative Study

Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein

Samantha Cooray et al. J Gen Virol. 2007 Jun.

Abstract

Vaccinia virus (VACV) encodes many immunomodulatory proteins, including inhibitors of apoptosis and modulators of innate immune signalling. VACV protein N1 is an intracellular homodimer that contributes to virus virulence and was reported to inhibit nuclear factor (NF)-kappaB signalling. However, analysis of NF-kappaB signalling in cells infected with recombinant viruses with or without the N1L gene showed no difference in NF-kappaB-dependent gene expression. Given that N1 promotes virus virulence, other possible functions of N1 were investigated and this revealed that N1 is an inhibitor of apoptosis in cells transfected with the N1L gene and in the context of VACV infection. In support of this finding virally expressed N1 co-precipitated with endogenous pro-apoptotic Bcl-2 proteins Bid, Bad and Bax as well as with Bad and Bax expressed by transfection. In addition, the crystal structure of N1 was solved to 2.9 A resolution (0.29 nm). Remarkably, although N1 shows no sequence similarity to cellular proteins, its three-dimensional structure closely resembles Bcl-x(L) and other members of the Bcl-2 protein family. The structure also reveals that N1 has a constitutively open surface groove similar to the grooves of other anti-apoptotic Bcl-2 proteins, which bind the BH3 motifs of pro-apoptotic Bcl-2 family members. Molecular modelling of BH3 peptides into the N1 surface groove, together with analysis of their physico-chemical properties, suggests a mechanism for the specificity of peptide recognition. This study illustrates the importance of the evolutionary conservation of structure, rather than sequence, in protein function and reveals a novel anti-apoptotic protein from orthopoxviruses.

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Figures

Fig. 1.
Fig. 1.
N1 does not inhibit IL-1-induced NF-κB-dependent gene expression in VACV-infected cells. HeLa cells were transfected with the NF-κB luciferase reporter, reseeded and mock-infected or infected with VACV, vΔN1 or vN1-rev at 2 p.f.u. per cell for 2 h. Cells were then treated with 100 ng IL-1β ml−1 for 2 h and lysates were assayed for luciferase activity. Data are expressed as the mean fold induction compared to the mean of the normalized luciferase activity in the mock infection. Data are means±sd.
Fig. 2.
Fig. 2.
N1 inhibits ST-induced apoptosis in transfected HeLa cells. HeLa cells were transfected with vectors expressing N1, C40S, Bcl-xL or empty control vector (pCI) together with a cell surface CD20 expression vector. After 24 h, transfected cells were selected on MACS columns using anti-CD20-coated magnetic beads as described in Methods and replated into culture medium. After a further 24 h, cells were treated with 1 μM ST (black bars) for 4 h or left untreated (white bars) and assayed for caspase activity (a) and mitochondrial dysfunction (b) as described in Methods. Bcl-xL and Bcl-2 (not shown) were used as positive controls. Asterisks indicate significant difference compared with pCI+ST samples. Data are means±sd (*P<0.05, **P<0.005; Student's t-test, n=3).
Fig. 3.
Fig. 3.
N1 inhibits ST-induced apoptosis in VACV-infected cells. HeLa cells were mock-infected or infected with vN1, vΔN1 or vN1-rev at 2 p.f.u. per cell for 2 h followed by treatment with (black bars) or without (white bars) 1 μM ST for 2 h (panel a) or the indicated times (panel b), and were assayed for caspase activity (a), and caspase-3 (p17 fragment) and PARP (p89 fragment) cleavage (b). (c) HeLa cells were mock-infected or infected with vN1 or vΔN1 at 10 p.f.u. per cell for 6 h and mitochondrial (M), organelle (O) and cytosolic (C) fractions were isolated by subcellular fractionation. Fractions were analysed by immunoblotting for N1, cytochrome c, α-tubulin and Bcl-xL. Data are means±sd (**/##P<0.005, ***P<0.0005; Student's t-test, n=3). Blots are representative of three individual experiments.
Fig. 4.
Fig. 4.
N1 interacts with Bad, Bax and Bid. Endogenous Bax, Bid and Bad (a) or overexpressed HA-tagged Bax, Bad or eiF4E control protein (b) were immunoprecipitated from lysates of HeLa cells infected with wild-type VACV (vN1) or N1 deletion virus (vΔN1). The input lysates and immunoprecipitates (IP) were fractionated by SDS-PAGE and immunoblotted for α-tubulin, N1, HA or Bax, Bid and Bad (Methods).
Fig. 5.
Fig. 5.
The structure of N1. (a) The dimer of N1 is viewed looking along the molecular twofold axis. BH1, BH2, BH3 and BH4 motifs are coloured green, magenta, yellow and blue, respectively and N and C termini are labelled. The right hand view is the same as in (b) and looks onto the surface groove and the mutated C40S residue is shown in red. For clarity, the helices of one monomer have been labelled. In the crystal structure of N1, the asymmetric unit has three dimers, each held together by anti-parallel interactions between helix α1 and α6 of each subunit, with a loss of 950 Å2 of solvent accessible surface. The interface does not involve the surface groove, which consequently is exposed and available to bind BH3 motifs. (b and c) Comparison of the structure of VACV N1 with Bcl-xL. Cartoons of N1 (b) and Bcl-xL (c), with structurally equivalent BH1, BH2, BH3 and BH4 motifs coloured as in panel (a). The position adopted by helix 3 of Bcl-xL when complexed with the Bim BH3 helix is drawn as a red semi-transparent loop and red arrows show movement of this helix upon peptide binding. (d) Sequence alignment of N1 and Bcl-xL based on structural alignment from SHP (Stuart et al., 1979). The core of the molecules, used in the structure based alignment, are highlighted in pink. Strictly conserved residues are in red blocks and similar residues in blue boxes. Secondary structural elements are coloured by BH motif definition, as above. To avoid breaking up the VACV N1 sequence, residues for Bcl-xL that are not matched by N1 are omitted, and the position and number of residues removed are indicated under the Bcl-xL sequence. The position of a disordered loop in Bcl-xL is marked by the green triangle.
Fig. 6.
Fig. 6.
Comparison of N1 and Bcl-xL surface grooves and molecular modelling of BH3 peptides. Molecular surfaces of N1 (a) and Bcl-xL (b), with the grooves highlighted. Lys, Arg and His are coloured in blue, Asp and Glu are coloured in red and Leu, Ile, Val, Met, Tyr, Phe and Trp are coloured in yellow. All other residue types are coloured in grey. (c) Molecular surface of N1 with the Bim BH3 helix (PDB code 1PQ1) modelled into the surface groove as described in the text. The surface of N1 is coloured as described in (a). The peptide is coloured from cyan to magenta from the N to the C terminus. The middle panel shows the Bim peptide from residue 85 to 105 with contacting residues drawn in atomic detail. Residues of the peptide contacting N1 are labelled in black and residues of N1 contacting the peptide are labelled in white. The lower panel shows the Bim BH3 peptide helix skeleton coloured from cyan to magenta, with the contacting residues of Bcl-xL and their equivalents of N1 boxed (N1 residues are in parentheses), and residues coloured according to their properties, as in (a). (d) Helical wheel representation of the Bim BH3 peptide (residues 85–105), drawn rotating the peptide in (c) by 90 ° around an axis vertical to the page, showing the predominantly amphipathic nature of the BH3 helix.
Fig. 7.
Fig. 7.
The hydrophobic properties of the BH3 motifs (boxed in green) and flanking regions (boxed in red) of pro-apoptotic Bcl-2 proteins Bad, Bid, Bax, Bim, Hrk and Bak are shown as GRAVY (GRand AVerage of hydropathY) scores (Kyte & Doolittle, 1982). The numbers above the alignment denote the residues in the BH3 motif, and the conserved hydrophobic residues are coloured yellow.
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