ZAP Inhibits Double-Stranded RNA Virus Infection by Degrading Negative-Strand RNA and Blocking Elongation Phase of Viral Protein Synthesis

Zinc-finger antiviral protein (ZAP) is a crucial host restriction factor that recognizes CpG dinucleotides in single-strand RNAs, yet its role in double-stranded RNA (dsRNA) virus replication remains uncharacterized. Here we demonstrate that ZAP broadly inhibits dsRNA viruses, including bluetongue virus (BTV) serotypes and epizootic hemorrhagic disease virus (EHDV), but not rotavirus (RV). Using BTV as a model, we reveal that ZAP inhibits replication via two mechanisms: 1) ZAP interacts with eukaryotic translation elongation factor 1A to block elongation during viral protein synthesis and 2) it binds preferentially to negative-sense RNA strands to stimulate their degradation. Additionally, BTV-NS1, encoded by segment 5, antagonizes ZAP by impairing its RNA-binding ability. Notably, synonymously CpG enrichment in BTV segment 5 significantly attenuated viral replication both in vitro and in vivo. Together, these findings uncover a dynamic interplay between ZAP and dsRNA viruses and suggest CpG-elevated BTV as a potential live attenuated vaccine candidate.

锌指抗病毒蛋白（Zinc-finger Antiviral Protein, ZAP）是一类关键的宿主限制因子，可识别单链RNA中的CpG二核苷酸（CpG dinucleotides），但其在双链RNA（double-stranded RNA, dsRNA）病毒复制过程中的功能仍未被系统阐明。本研究证实，ZAP可广谱抑制蓝舌病毒（bluetongue virus, BTV）各血清型、流行性出血病病毒（epizootic hemorrhagic disease virus, EHDV）等双链RNA病毒的复制，但对轮状病毒（rotavirus, RV）无效。以蓝舌病毒为模型，本研究揭示了ZAP通过双重机制抑制病毒复制：其一，ZAP与真核翻译延伸因子1A（eukaryotic translation elongation factor 1A）相互作用，阻断病毒蛋白合成过程中的翻译延伸阶段；其二，ZAP优先结合负链RNA链（negative-sense RNA strands），促进其降解。此外，由BTV第5基因组节段编码的BTV-NS1蛋白可通过削弱ZAP的RNA结合能力，拮抗其抗病毒活性。值得注意的是，通过同义突变提升BTV第5基因组节段的CpG富集水平，可显著减弱病毒在体外及体内的复制能力。综上，本研究揭示了ZAP与双链RNA病毒之间的动态互作关系，并提示CpG富集型BTV有望成为潜在的减毒活疫苗候选株。