Association of Zinc Finger Antiviral Protein Binding to Viral Genomic RNA with Attenuation of Replication of Echovirus 7

Previous studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNase L in the attenuation of RNA viruses with elevated CpG and UpA dinucleotides. Mechanisms and interrelationships 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 on replication or specific binding to ZAP by IP. However, the bases 3′ and 5′ of 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 noncompetitively 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 colocalized 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/RNase L, KHNYN, and other RNA degradation pathways. IMPORTANCE We recently discovered that the OAS3/RNase L 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 reconceptualization of the pathways associated with this aspect of antiviral defense.