A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history

Abstract

Background: Poxviruses evade the immune system of the host through the action of viral encoded inhibitors that block various signalling pathways. The exact number of viral inhibitors is not yet known. Several members of the vaccinia virus A46 and N1 families, with a Bcl-2-like structure, are involved in the regulation of the host innate immune response where they act non-redundantly at different levels of the Toll-like receptor signalling pathway. N1 also maintains an anti-apoptotic effect by acting similarly to cellular Bcl-2 proteins. Whether there are related families that could have similar functions is the main subject of this investigation.

Results: We describe the sequence similarity existing among poxvirus A46, N1, N2 and C1 protein families, which share a common domain of approximately 110-140 amino acids at their C-termini that spans the entire N1 sequence. Secondary structure and fold recognition predictions suggest that this domain presents an all-alpha-helical fold compatible with the Bcl-2-like structures of vaccinia virus proteins N1, A52, B15 and K7. We propose that these protein families should be merged into a single one. We describe the phylogenetic distribution of this family and reconstruct its evolutionary history, which indicates an extensive gene gain in ancestral viruses and a further stabilization of its gene content.

Conclusions: Based on the sequence/structure similarity, we propose that other members with unknown function, like vaccinia virus N2, C1, C6 and C16/B22, might have a similar role in the suppression of host immune response as A46, A52, B15 and K7, by antagonizing at different levels with the TLR signalling pathways.

Figure 1

Sequence conservation in A46 and related families. (A) Multiple sequence alignment with the common sequence domain found in protein families A46, N1, N2 and C1. The alignment is non-redundant at 90% sequence identity. Sequences are identified by species/strain and gene locus number: SWPV-NEB, swinepox virus strain Nebraska 17077-99; SPPV-TU, sheeppox virus strain TU-V02127; DPV-W848_83, deerpox virus strain W-848-83; MYXV-LAU, myxoma virus strain Lausanne; RFV-KAS, rabbit fibroma virus strain Kasza; VACV-WR, vaccinia virus strain Western Reserve; YLDV-Davis, yaba-like disease virus strain Davis; RPXV-UTR, rabbitpox virus strain Utrecht; LSDV-NW_LW, lumpy skin disease virus strain Neethling Warmbaths LW; ECTV-NAV, ectromelia virus strain Naval. Shading indicates degree of sequence similarity. Conserved motifs are indicated with horizontal bars on the top of the alignment. Predicted secondary structure is indicated below each block of sequences (orange: alpha-helix; blue: beta-sheet), except for A46 and N1, for which secondary structures of A52 (PDB:2VVW) and N1 (PDB:2I39), respectively, are shown. Green arrowheads indicate N1 protein residues putatively involved in BH3 peptide binding [11]. (B) Structural distribution of conserved motifs. Conserved residues in the multiple sequence alignment were mapped on the N1 structure (PDB:2I39). Secondary structure elements are depicted in yellow, except conserved residues, in orange. Side chains are coloured in red. Surface is shown in light grey. Structures were rendered with UCSF Chimera [60].

Figure 2

Groups of orthologous genes in A46 and related families. (A) Phylogenetic relationships among the orthologue groups obtained from A46, N1, N2 and C1 families. A Bayesian phylogenetic tree was constructed from a multiple sequence alignment of proteins encoded by genes in the ten orthologue groups. For simplicity only a representative species of every poxvirus genus, as depicted in (B), was selected. Posterior probabilities of every node are shown. (B) Virus genomes representing genera Orthopoxvirus (VACV-COP), Leporipoxvirus (MYXV-LAU), Capripoxvirus (LSDV-NW_LW), Suipoxvirus (SWPV-NEB), Yatapoxvirus (YLDV-Davis) and Cervidpoxvirus (DPV-W848_83) are depicted, indicating the relative genome positions of genes included in the orthologue groups. Species/strain names as in Figure 1A; VACV-COP, vaccinia virus strain Copenhagen. Numbers above every line represent the gene positions in the genome. Symbols below every line represent gene names. Genes drawn in the same colour belong to the same orthologue group.

Figure 3

Reconstruction of ancestral gene repertoires in the evolutionary history of A46 and related families. The number in every node represents the inferred or real number of groups of orthologues present in each genome. This number was inferred for ancestral species by the maximum likelihood method implemented in the Count program [30]. The background colour of the number indicates the kind of variation in the gene content since the preceding node: green for nodes with a net gene gain, red for nodes with a net gene loss, and grey if the gene content remained unchanged. The tree contains a representative strain for every species of the subfamily Chordopoxvirinae with a completely sequenced genome and is based on a maximum likelihood phylogenetic tree (Additional File 3). Species/strain names as in Figures 1 and 2; TATV-DAH68, Taterapox virus strain Dahomey 1968; CMLV-CMS, Camelpox virus strain CMS; VARV-IND3_1967, Variola virus strain India 3 Major 1967; CPXV-GRI, Cowpox virus strain GRI-90; MPXV-SLE, Monkeypox virus strain Sierra Leone; YMTV-Amano, Yaba monkey tumor virus strain Amano; RFV-Kas, Rabbit fibroma virus strain Kasza; SPPV-A, Sheeppox virus strain A; GTPV-G20LKV, Goatpox virus strain G20-LKV; BPSV-AR02, Bovine papular stomatitis virus strain BV-AR02; ORFV-NZ2, Orf virus strain NZ2; MOCV-st1, Molluscum contagiosum virus strain subtype 1; CNPV-VR111, Canarypox virus strain ATCC VR111; FWPV-Iowa, Fowlpox virus strain Iowa; CRV-ZWE, Crocodilepox virus strain Zimbabwe.

Figure 4

Inhibition of host signalling pathways by VACV members of A46 and related families. TLRs are distributed in the plasma membrane and endosomes. When a pathogen is recognized by a TLR adaptor proteins are recruited which transmit the signal further downstream until specific transcription factors are activated and enhance the expression of genes encoding type I IFNs and pro-inflammatory cytokines. VACV proteins belonging to A46 and N1 families interfere with the TLR signalling pathway at different levels. A46 targets all known adaptor proteins: MyD88, MAL (TIRAP), TRIF and TRAM. A52 targets IRAK2 and TRAF6, intermediary between adaptors and transcription factors. K7 inhibits IRAK2, TRAF6 and also DDX3, which is part of the complex that activates transcription factor IRF3. B15 targets the IKK complex by avoiding IKKbeta phosphorylation, what eventually causes the inhibition of NF-kappaB. N1 associates with several components of the IKK complex and with TBK1, inhibiting NF-kappaB and IRF3 activation, respectively.