On the 24 th November 2021 the sequence of a new SARS CoV-2 viral isolate Omicron-B.1.1.529 was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.

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.

Vaccinia virus (VACV) protein N1 is an intracellular virulence factor and belongs to a family of VACV B-cell lymphoma (Bcl)-2-like proteins whose members inhibit apoptosis or activation of pro-inflammatory transcription factors, such as interferon (IFN) regulatory factor-3 (IRF-3) and nuclear factor-κB (NF-κB). Unusually, N1 inhibits both apoptosis and NF-κB activation. To understand how N1 exerts these different functions, we have mutated residues in the Bcl-2-like surface groove and at the interface used to form N1 homodimers. Mutagenesis of the surface groove abolished only the N1 anti-apoptotic activity and protein crystallography showed these mutants differed from wild-type N1 only at the site of mutation. Conversely, mutagenesis of the dimer interface converted N1 to a monomer and affected only inhibition of NF-κB activation. Collectively, these data show that N1 inhibits pro-inflammatory and pro-apoptotic signalling using independent surfaces of the protein. To determine the relative contribution of each activity to virus virulence, mutant N1 alleles were introduced into a VACV strain lacking N1 and the virulence of these viruses was analysed after intradermal and intranasal inoculation in mice. In both models, VACV containing a mutant N1 unable to inhibit apoptosis had similar virulence to wild-type virus, whereas VACV containing a mutant N1 impaired for NF-κB inhibition induced an attenuated infection similar to that of the N1-deleted virus. This indicates that anti-apoptotic activity of N1 does not drive virulence in these in vivo models, and highlights the importance of pro-inflammatory signalling in the immune response against viral infections.

Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized virus-like particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single-particle reconstruction reveals a structure almost indistinguishable from wild-type PV3.

SummaryThe morphogenesis of poxviruses such as vaccinia virus (VACV) sees the virion shape mature from spherical to brick-shaped. Trimeric capsomers of the VACV D13 protein form a transitory, stabilizing lattice on the surface of the initial spherical immature virus particle. The crystal structure of D13 reveals that this major scaffolding protein comprises a double β barrel “jelly-roll” subunit arranged as pseudo-hexagonal trimers. These structural features are characteristic of the major capsid proteins of a lineage of large icosahedral double-stranded DNA viruses including human adenovirus and the bacteriophages PRD1 and PM2. Structure-based phylogenetic analysis confirms that VACV belongs to this lineage, suggesting that (analogously to higher organism embryogenesis) early poxvirus morphogenesis reflects their evolution from a lineage of viruses sharing a common icosahedral ancestor.

The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 Å resolution. The protein has a globular β sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI–chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (μM–nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine–chemokine receptor interactions.

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced. Omicron contains a total of 30 substitutions plus deletions and an insertion in Spike, far more than any previously reported variant. The mutations include those previously identified by In-vitro evolution to contribute to high-affinity binding to ACE2, including mutations Q498R and N501Y critical in forming additional interactions in the interface. Together with increased charge complementarity between the RBD and ACE2, these substantially increase affinity and potentially virus transmissibility through increased syncytia formation. Further mutations promote immune evasion. We have studied the binding of a large panel of potent monoclonal antibodies generated from early pandemic or Beta infected cases. Mutations in Omicron will likely compromise the binding of many of these and additionally, the binding of antibodies under commercial development, however residual binding should provide protection from severe disease.