A fish herpesvirus highlights functional diversities among Zα domains related to phase separation induction and A-to-Z conversion

Abstract

Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.

Virus-induced interference as a means for accelerating fitness-based selection of cyprinid herpesvirus 3 single-nucleotide variantsin vitroandin vivo

“…For a good example, see Fig. 4 of Diallo et al (2022). Confocal microscopy analysis revealed that CyHV-3 syncytia contained only nuclei expressing marginalized chromatin and so resulting from the fusion of infected cells at a late stage of infection (data not shown).…”

Z-RNA biology: a central role in the innate immune response?

“…Point mutations within Z domains also disrupt Z localization to biological condensates (Ng et al 2013;Gabriel et al 2021). For example, mutating Z-RNA-stabilizing amino acids within ORF112 leads to a disruption of the liquid-liquid phase separation necessary for the formation of condensates (Diallo et al 2022).…”

“…3 B; Koehler et al 2017 ; Balachandran and Mocarski 2021 ). In fact, both E3L and ORF112 have been reported to outcompete binding of a Z-RNA antibody to its epitope ( Diallo et al 2022 ). E3L and E3L-like proteins are particularly important for preventing ZBP1 activation during early viral infection when viral RNAs are being heavily transcribed ( Koehler et al 2021 ).…”

“…Point mutations within Zα domains also disrupt Zα localization to biological condensates ( Ng et al 2013 ; Gabriel et al 2021 ). For example, mutating Z-RNA-stabilizing amino acids within ORF112 leads to a disruption of the liquid–liquid phase separation necessary for the formation of condensates ( Diallo et al 2022 ). Mutating Lys 169 or Tyr 177 to Ala in the Zα domain of ADAR1 or the equivalent residues in E3L (Lys 40 and Tyr 48) leads to impaired localization of ADAR1 and E3L to condensates ( Ng et al 2013 ).…”

“…A rigorous biochemical and structural characterization of these proteins would be worthwhile, to expose further clues about Z-RNA recognition. A wider pool of Zα domains may offer additional model systems for exploring Z-RNA biology, as exemplified most recently with a study of ORF112 from a fish herpesvirus ( Diallo et al 2022 ).…”