Zalpha domains are a subfamily of the winged helix-turn-helix domains sharing the unique ability to recognize CpG repeats in the left-handed Z-DNA conformation. In vertebrates, domains of this family are found exclusively in proteins that detect foreign nucleic acids and activate components of the antiviral interferon response. Moreover, poxviruses encode the Zalpha domaincontaining protein E3L, a well-studied and potent inhibitor of interferon response. Here we describe a herpesvirus Zalpha-domain-containing protein (ORF112) from cyprinid herpesvirus 3. We demonstrate that ORF112 also binds CpG repeats in the left-handed conformation, and moreover, its structure at 1.75 Å reveals the Zalpha fold found in ADAR1, DAI, PKZ, and E3L. Unlike other Zalpha domains, however, ORF112 forms a dimer through a unique domain-swapping mechanism. Thus, ORF112 may be considered a new member of the Z-domain family having DNA binding properties similar to those of the poxvirus E3L inhibitor of interferon response.
The African Swine Fever Virus (ASFV) is an economically important, large DNA virus which causes a highly contagious and frequently fatal disease in domestic pigs. Due to the acute nature of the infection and the complexity of the protective porcine anti-ASFV response, there is no accepted vaccine in use. As resistance to ASFV is known to correlate with a robust IFN response, the virus is predicted to have evolved strategies to inhibit innate immunity by modulating the IFN response. The deletion of virus host evasion gene(s) inhibiting IFN is a logical solution to develop an attenuated virus vaccine. One such candidate, the ASFV ORF I329L gene, is highly conserved in pathogenic and non-pathogenic virus isolates and in this study we confirm and extend the conclusion that it has evolved for the inhibition of innate immunity initiated through Toll-like receptors (TLRs). Specifically, the ASFV I329L extracellular (ECD) and intracellular (ICD) domains inhibit TLR signalling by two entirely different mechanisms. Bioinformatics modelling suggests that the ECD inhibits several TLR signalling pathways through a short sequence homologous to the conserved TLR dimerization domain, here termed the putative dimerization domain (PDD). Remarkably, both full length and PDD constructs of I329L were demonstrated to inhibit activation, not only of TLR3, but also TLR4, TLR5, TLR8 and TLR9. Additionally, the demonstration of a weak association of I329L with TLR3 is consistent with the formation of a non-signalling I329L-TLR3 heterodimer, perhaps mediated through the PDD of I329L. Finally, the ICD associates with TRIF, thereby impacting on both TLR3 and TLR4 signalling. Thus, I329L offers potential as a general inhibitor of TLR responses and is a rational candidate for construction and testing of an I329L deletion mutant vaccine.
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