The Structure of the Cyprinid herpesvirus 3 ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response

Abstract

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Z␣ domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Z␣ domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Z␣ in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Z␣ domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.

The Role of the Z-DNA Binding Domain in Innate Immunity and Stress Granules

“…In line with other ZDBD-containing proteins discussed so far, ORF112 also localizes to SGs during oxidative stress ( 24 ) ( Figure 2 ).…”

“…Indeed, the Zα ORF112 protein binds Z-DNA/Z-RNA in left-handed conformation resembles that of ADAR1, ZBP1, PKZ and E3L despite of low overall sequence identity and different binding kinetics ( 128 ). Significant structure resemblance between Zα ORF112 with Zα PKZ suggests common ancestry or convergent evolution as a competitive inhibitor for PKZ sensing ( 24 ) ( Figure 2 ).…”

“…Since the initial discovery of Zα domain in ADAR1, Z-DNA/Z-RNA binding has been thought to be involved in mediating innate immunity, as Zα domain is only present in the interferon-inducible ADAR1 p150 isoform, instead of the constitutively expressed ADAR1 p110 isoform ( 19 , 20 ). In addition to mammalian ADAR1, Zα domains were subsequently discovered to be encoded in proteins across different classes of metazoans, including immunity-related Z-DNA Binding Protein 1 (ZBP1) in mammals ( 21 ) and Protein Kinase Containing Z-DNA binding domains (PKZ) in fishes ( 22 ); and viruses affecting them, such as E3L in poxviruses ( 23 ), and ORF112 in fish herpesviruses ( 24 ). RBP7910 is recently discovered in kinetoplastids as another ZDBD-containing protein ( 25 ).…”

Z-DNA and Z-RNA in human disease

“…The winged-helix-turn-helix Zα motif was found in 182 other proteins (SMART Domain SM00550) representing orthologs of ADAR1, ZBP1, PKZ, E3L and ORF112 from different species. Structural studies have confirmed that many of these bind Z-DNA, including a number of viral proteins, that, like ADAR, play a role in the innate immune response and are essential for viral infectivity 12,21–26 . The related Zβ domain of human ZBP1 also binds to Z-DNA but uses a different set of contact residues 22 , suggesting that there are even more divergent members of the winged-helix-turn-helix Z-DNA binding family.…”

“…This domain has a helix-turn-helix motif similar to that found in B-DNA binding proteins and a binding site of 6 basepairs 6,11 . Cocrystallization of the Cyprinid herpes virus 3 ORF11 Zα with d(T(CG) 9 ) DNA identified a guanosine base-specific contact outside the core-binding site that may help stabilize the B−Z transformation 12 . Further structural studies, enabled by Zα, detailed B−Z and Z−Z junctions at atomic resolution.…”