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 (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-κB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 Å and 2.7 Å resolution, respectively. Strikingly, both these proteins adopt a Bcl-2–like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2–like proteins. Unlike cellular and viral Bcl-2–like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2–like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2–like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2–like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2–like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways.

The IκB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-κB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-κB activation in response to TNFα, IL-1β, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vΔB14) there was a higher level of phosphorylated IκBα but a similar level of IκBα compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKβ but not IKKα, suggesting the interaction occurs via IKKβ. B14 inhibited NF-κB activation induced by overexpression of IKKα, IKKβ, and a constitutively active mutant of IKKα, S176/180E, but did not inhibit a comparable mutant of IKKβ, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKβ is targeted by B14, and this was confirmed using Ab specific for phospho-IKKβ.

Over the course of evolution, viruses have developed the ability to modulate a variety of host cell signalling pathways. Inhibition of apoptosis, in particular, has become recognized as an important contributory factor in virus survival. Apoptotic inhibition contributes to the establishment of latent and chronic infections and has been implicated in viral oncogenesis. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is utilized by many cell types for inhibition of apoptosis and cellular survival. Virus modulation of this pathway provides an alternative to the expression of viral oncogenes or the direct inhibition of pro-apoptotic proteins. It has become evident that many viruses require up-regulation of this pathway to sustain long-term infections and it is modulated, in some cases, by specific viral products to create an environment favourable for cellular transformation. In other cases, PI3K-Akt signalling simply helps to create an environment favourable for virus replication and virion assembly. This review details the modulation and function of PI3K-Akt signalling for virus survival.