Key result
Specific CpG and UpA contexts alter echovirus 7 replication and ZAP/OAS3 binding within stress granules.
Why the study?
Previous studies implicated ZAP and OAS3/RNASeL in attenuating RNA viruses with elevated CpG and UpA dinucleotides, but the mechanisms and inter-relationships between these pathways were unclear.
The study demonstrates that specific dinucleotide contexts influence ZAP and OAS3 binding, which colocalize with stress granules to mediate antiviral defense against RNA viruses.
Preclinical only; leaves open whether dinucleotide motifs can be targeted for enteroviral antivirals.
Previous studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNASeL in the attenuation of RNA viruses with elevated CpG and UpA dinucleotides. Mechanisms and inter-relationships between these two pathways were investigated using an echovirus 7 (E7) replicon with compositionally modified sequences inserted into the 3’untranslated region. ZAP and OAS3 immunoprecipitation (IP) assays provided complementary data on dinucleotide composition effects on binding. Elevated frequencies of alternative pyrimidine/purine (CpA and UpG) and reversed (GpC and ApU) dinucleotides showed no attenuating effect nor specific binding to ZAP by IP. However, the bases 3’ and 5’ to CpG motifs influenced replication and ZAP binding; UCGU enhanced CpG-mediated attenuation and ZAP-binding while A residues shielded CpGs from ZAP recognition. Attenuating effects of elevated frequencies of UpA on replication occurred independently of CpG dinucleotides and bound non-competitively with CpG-enriched RNA consistent with a separate recognition site from CpG. Remarkably, immunoprecipitation with OAS3 antibody reproduced the specific binding to CpG- and UpA-enriched RNA sequences. However, OAS3 and ZAP were coimmunoprecipitated in both ZAP and OAS3 IP, and colocalised with E7 and stress granules (SGs) by confocal microscopy analysis of infected cells. ZAP’s association with larger cellular complexes may mediate the recruitment of OAS3/RNAseL, KHNYN and other RNA degradation pathways. Importance We have recently discovered that the OAS3/RNAseL antiviral pathway is essential for restriction of CpG- and UpA-enriched viruses, in addition to the requirement for zinc finger antiviral protein (ZAP). The current study provides evidence for the specific dinucleotide and wider recognition contexts associated with virus recognition and attenuation. It further documents the association of ZAP and OAS3 and association with stress granules and a wider protein interactome that may mediate antiviral effects in different cellular compartments. The study provides a striking re-conceptualisation of the pathways associated with this aspect of antiviral defence.
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Goonawardane et al. (2020) studied Echovirus 7 infection. Compositionally modified sequences in echovirus 7 replicon was evaluated on Viral replication attenuation and ZAP/OAS3 binding. Compositionally modified echovirus 7 replicons showed that specific CpG and UpA dinucleotide contexts influence viral replication and ZAP/OAS3 binding, which co-localize with stress granules.
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