Abstract
The human zinc finger antiviral protein (ZAP) recognizes RNA by binding to CpG dinucleotides. Mammalian transcriptomes are CpG-poor, and ZAP may have evolved to exploit this feature to specifically target non-self viral RNA. Phylogenetic analyses reveal that ZAP and its paralogue PARP12 share an ancestral gene that arose prior to extensive eukaryote divergence, and the ZAP lineage diverged from the PARP12 lineage in tetrapods. Notably, the CpG content of modern eukaryote genomes varies widely, and ZAP-like genes arose subsequent to the emergence of CpG-suppression in vertebrates. Human PARP12 exhibited no antiviral activity against wild type and CpG-enriched HIV-1, but ZAP proteins from several tetrapods had antiviral activity when expressed in human cells. In some cases, ZAP antiviral activity required a TRIM25 protein from the same or related species, suggesting functional co-evolution of these genes. Indeed, a hypervariable sequence in the N-terminal domain of ZAP contributed to species-specific TRIM25 dependence in antiviral activity assays. Crosslinking immunoprecipitation coupled with RNA sequencing revealed that ZAP proteins from human, mouse, bat and alligator exhibit a high degree of CpG-specificity, while some avian ZAP proteins appear more promiscuous. Together, these data suggest that the CpG- rich RNA directed antiviral activity of ZAP-related proteins arose in tetrapods, subsequent to the onset of CpG suppression in certain eukaryote lineages, with subsequent species-specific adaptation of cofactor requirements and RNA target specificity.