Valerie Odon, Iliana Georgana, Joe Holley, Jordi Morata, Carlos Maluquer de Motes

Ankyrin repeat (ANK) domains are one of the most abundant motifs in eukaryotic proteins. ANK proteins are rare amongst viruses with the exception of poxviruses, which presumably acquired them from the host via horizontal gene transfer. The architecture of poxvirus ANK proteins is however different from their cellular counterparts and this precludes a direct acquisition event. Here we combine bioinformatics analysis and quantitative proteomics to discovera new class of viral ANK proteins with a domain organisation that relates to cellular ANK proteins. These non-canonical viral ANK proteins,termed ANK/BC, interact with host Cullin-2 via a C-terminal BCbox resembling that of cellular Cullin-2 substrate adaptors such as the von Hippel-Lindau protein. Mutagenesis of the BC box-like sequence abrogatesbinding to Cullin-2, whereas fusion of this motif to an ANK-only protein confersCullin-2 association. We demonstrate that these viral ANK/BC proteins are potent immunomodulatory proteins suppressing the activation of the pro-inflammatory transcription factors NF-κB and IRF-3 and the production of cytokines and chemokines including interferon, and that association with Cullin-2 is required for optimal inhibitory activity. ANK/BC proteins exist in several orthopoxvirusesand cluster into 2 closely relatedorthologue groups in a phylogenetic lineage that is separate from canonical ANK/F-box proteins. Given the existence of cellular proteins with similar architecture,viral ANK/BC proteins may be closely related to the original ANK gene acquired by an ancestral orthopoxvirus.These findingsuncover a novel viral strategy to antagonise innate immunity and shed light on the origin of the poxviral ANK protein family.IMPORTANCEViruses encode multiple proteins aimed at modulating cellular homeostasis and antagonising the host anti-viral response. Most of these genes were originally acquired from the host and subsequently adapted to the virus advantage. ANK proteins are common in eukaryotes, but unusual amongst viruses with the exception of poxviruses where they represent one of the largest protein families. We discover here the existence of a new class of viral ANK proteins, termed ANK/BC, that provide new insights on the origin of poxvirus ANK proteins. ANK/BC proteins target the host E3 ubiquitin ligase Cullin-2 via a C-terminal BC box domain and are potent suppressors of the production of inflammatory cytokines including interferon. The existence of cellular ANK proteins with similar architecture suggests the acquisition of a host ANK/BC gene by an ancestral orthopoxvirus and its subsequent duplication and adaptation to widen the repertoire of immune evasion strategies.