MethodsUsing a CRISPR/Cas9 gene-editing system,EFTUD2single allele knockout HepG2.2.15 cells were constructed. Subsequently, the HBV biomarkers inEFTUD2+/–HepG2.2.15 cells and wild-type (WT) cells with or without IFN-αtreatment were detected. And the EFTUD2-regulated genes were then identified using mRNA sequence. Selected gene mRNA variants and their proteins were examined by qRT-PCR and Western blotting. To confirm the effects of EFTUD2 on HBV replication and IFN-stimulated gene (ISG) expression, a rescue experiment inEFTUD2+/–HepG2.2.15 cells was performed by EFTUD2 overexpression.ResultsIFN-induced anti-HBV activity was found to be restricted inEFTUD2+/–HepG2.2.15 cells. The mRNA sequence showed that EFTUD2 could regulate classical IFN and virus response genes. Mechanistically,EFTUD2single allele knockout decreased the expression of ISG-encoded proteins, comprising Mx1, OAS1, and PKR (EIF2AK2), through mediated gene splicing. However, EFTUD2 did not affect the expression of Jak-STAT pathway genes. Furthermore, EFTUD2 overexpression could restore the attenuation of IFN anti-HBV activity and the reduction of ISG resulting fromEFTUD2single allele knockout.ConclusionEFTUD2, the spliceosome factor, is not IFN-inducible but is an IFN effector gene. EFTUD2 mediates IFN anti-HBV effect through regulation of gene splicing for certain ISGs, includingMx1,OAS1, andPKR. EFTUD2 does not affect IFN receptors or canonical signal transduction components. Therefore, it can be concluded that EFTUD2 regulates ISGs using a novel, nonclassical mechanism.