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

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)-κB signalling. However, analysis of NF-κB signalling in cells infected with recombinant viruses with or without the N1L gene showed no difference in NF-κB-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 Å resolution (0.29 nm). Remarkably, although N1 shows no sequence similarity to cellular proteins, its three-dimensional structure closely resembles Bcl-xL 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.

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

“…23,24 N1L also exists as a dimer, 14 but no domain swapping occurs in that case. 12,13 Other structural studies of Bcl-2 family proteins suggest that the fold is largely unaffected by the presence or absence of the C-terminal membrane anchor. If the C-terminal segments, absent in our construct, are taken into consideration, the dimer structure we describe is compatible with membrane anchoring (see, for example, Figure 1b, and note the dyadrelated a7 segments projecting towards the viewer).…”

“…14 N1L reportedly binds BH3 peptides of Bim, Bak and Bid, 12 and interacts with Bad, Bax and Bid in cells transfected with the N1L gene. 13 In contrast, virus lacking F1L (VVDF1L) results in apoptosis of infected cells in culture, 10,15 suggesting that Figure 5 Novel consensus sequence motif redefining the BH4 domain. The novel BH4 motif is formed by f 1 f 2 X X f 3 f 4 , where X is any amino acid, f is a hydrophobic residue and f 3 is an aromatic residue.…”

“…11 Moreover, the structure of M11L in complex with the BH3 peptide from Bak revealed that the canonical BH3-binding groove is utilized in this interaction, and tests of M11L mutants showed that its anti-apoptotic action relies on binding to Bak and/or Bax, less so to BH3-only proteins. 11 The vaccinia virus N1L protein also displays a Bcl-2-like fold, 12,13 even though, like myxoma virus M11L, its sequence does not resemble that of Bcl-2. Vaccinia virus lacking N1L replicate normally in cell culture, but are attenuated in animal models.…”

“…14 The function of N1L is not fully understood, but its three-dimensional fold and ability to bind BH3 peptides suggest a role in modulating apoptosis. 12,13 Vaccinia virus F1L inhibits the mitochondrial pathway of apoptosis and, when overexpressed in mammalian cells, it localizes via its C-terminal tail to the mitochondria. 9 F1L interacts with the isolated BH3 domain of Bim and an F1L-deficient virus potently causes Bak/Bax-mediated apoptosis.…”

The N Terminus of the Vaccinia Virus Protein F1L Is an Intrinsically Unstructured Region That Is Not Involved in Apoptosis Regulation

“…Structural studies of M11L (37,38) revealed that although it lacks sequence similar-ity to the Bcl-2 family of proteins it adopts a Bcl-2-like fold and engages BH3 ligands utilizing the canonical BH3 domain binding groove (38). Vaccinia virus N1L was shown to also adopt a Bcl-2-like fold (39,40), which enabled it to assume dual functionality by mediating intrinsic apoptosis via the canonical Bcl-2 binding groove as well as NF-B signaling via an additional non-canonical site (41). Similarly, deerpox virus DPV022 was shown to be a Bcl-2-like protein, albeit with a dimeric topology due to a domain swap (42).…”