No 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 (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.

RNA polymerase transcription initiation sites are largely unknown in Caenorhabditis elegans. The initial 59 end of most protein-coding transcripts is removed by trans-splicing, and noncoding initiation sites have not been investigated. We characterized the landscape of RNA Pol II transcription initiation, identifying 73,500 distinct clusters of initiation. Bidirectional transcription is frequent, with a peak of transcriptional pairing at 120 bp. We assign transcription initiation sites to 7691 protein-coding genes and find that they display features typical of eukaryotic promoters. Strikingly, the majority of initiation events occur in regions with enhancer-like chromatin signatures. Based on the overlap of transcription initiation clusters with mapped transcription factor binding sites, we define 2361 transcribed intergenic enhancers. Remarkably, productive transcription elongation across these enhancers is predominantly in the same orientation as that of the nearest downstream gene. Directed elongation from an upstream enhancer toward a downstream gene could potentially deliver RNA polymerase II to a proximal promoter, or alternatively might function directly as a distal promoter. Our results provide a new resource to investigate transcription regulation in metazoans.

Piwi-interacting RNAs (piRNA) are small regulatory RNAs with essential roles in maintaining genome integrity in animals and protists. Most Caenorhabditis elegans piRNAs are transcribed from two genomic clusters that likely contain thousands of individual transcription units; however, their biogenesis is not understood. Here we identify and characterize prde-1 (piRNA silencing-defective) as the first essential C. elegans piRNA biogenesis gene. Analysis of prde-1 provides the first direct evidence that piRNA precursors are 28- to 29-nucleotide (nt) RNAs initiating 2 nt upstream of mature piRNAs. PRDE-1 is a nuclear germline-expressed protein that localizes to chromosome IV. PRDE-1 is required specifically for the production of piRNA precursors from genomic loci containing an 8-nt upstream motif, the Ruby motif. The expression of a second class of motif-independent piRNAs is unaffected in prde-1 mutants. We exploited this finding to determine the targets of the motif-independent class of piRNAs. Together, our data provide new insights into both the biogenesis and function of piRNAs in gene regulation.

A characterization of the B14R gene from Vaccinia virus (VACV) strain Western Reserve (WR) is presented. Computational analyses of the B14R gene indicated high conservation in orthopoxviruses but no orthologues outside the Poxviridae. To characterize the B14 protein, the B14R gene was expressed in Escherichia coli and recombinant protein was purified and used to generate a rabbit polyclonal antiserum. This antiserum recognized a 15 kDa cytoplasmic protein in mammalian cells that were transfected with the B14R gene or infected with VACV WR, but not from cells infected with a VACV mutant (vDB14) from which the B14R gene was deleted. Compared to wild-type and revertant virus controls, vDB14 had normal growth kinetics in cell culture. The virulence of vDB14 was assessed in two in vivo models. Mice infected intranasally with vDB14 had similar weight loss compared to the controls, but in C57BL/6 mice infected intradermally vDB14 induced a smaller lesion size compared with controls. Moreover, intradermal infection with vDB14 caused an increased infiltration of cells into the infected lesion despite the smaller lesion size. Therefore, B14 is an intracellular protein that is non-essential for virus replication in cell culture but contributes to virus virulence in vivo and affects the host response to infection.

Viruses are obligate intracellular parasites and are some of the most rapidly evolving and diverse pathogens encountered by the host immune system. Large complicated viruses, such as poxviruses, have evolved a plethora of proteins to disrupt host immune signalling in their battle against immune surveillance. Recent X-ray crystallographic analysis of these viral immunomodulators has helped form an emerging picture of the molecular details of virus-host interactions. In this review we consider some of these immune evasion strategies as they apply to poxviruses, from a structural perspective, with specific examples from the European SPINE2-Complexes initiative. Structures of poxvirus immunomodulators reveal the capacity of viruses to mimic and compete against the host immune system, using a diverse range of structural folds that are unique or acquired from their hosts with both enhanced and unexpectedly divergent functions.