Human regnases are evolutionarily diversified antiviral restriction factors targeting viral RNA

The regnase family of endoribonucleases regulates immune gene expression through degradation of cellular mRNAs. Regnase-1 is known to also target viral RNA, but whether antiviral activity is a conserved property of all human regnases remains unknown. Here, we systematically compare the antiviral properties of all four human regnases. Regnases-1-4 expression inhibited HIV-1, HIV-2, MLV, RSV and hCoVs OC43 and SARS-CoV-2, but not the DNA virus HSV-1. Endogenous knockout and knockdown experiments demonstrated that physiological regnase expression restricts HIV-1 replication in a cell-type-dependent manner. Regnase-1 and regnase-4 were induced by interferons in macrophages, and all four regnases displayed signatures of positive selection during mammalian evolution, consistent with their potential roles as antiviral restriction factors. Mechanistically, antiviral activity of regnases required intact catalytic core and CCCH zinc finger domains, while nuclear shuttling and dimerisation site conservation were not shared features of all family members. Domain-swap and reporter analyses further showed that differences in antiviral potency between regnases primarily reflect differential RNA target recognition rather than catalytic activity. Regnase-1 exhibited broad RNA targeting, whereas regnases-2-4 displayed more selective targeting profiles. Collectively, our findings establish the human regnase family as evolutionarily diversified antiviral RNA restriction factors with distinct substrate specificities.