Department of Infectious Disease, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Department of Pediatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Mediators Inflamm. 2023 Jun 23;2023:2546278. doi: 10.1155/2023/2546278. eCollection 2023.
Using a CRISPR/Cas9 gene-editing system, single allele knockout HepG2.2.15 cells were constructed. Subsequently, the HBV biomarkers in HepG2.2.15 cells and wild-type (WT) cells with or without IFN- treatment 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 in HepG2.2.15 cells was performed by EFTUD2 overexpression.
IFN-induced anti-HBV activity was found to be restricted in HepG2.2.15 cells. The mRNA sequence showed that EFTUD2 could regulate classical IFN and virus response genes. Mechanistically, single 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 from single allele knockout.
, 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, including , , and . 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.
使用 CRISPR/Cas9 基因编辑系统构建单等位基因敲除 HepG2.2.15 细胞。随后，检测 IFN-处理前后 HepG2.2.15 细胞和野生型（WT）细胞中的 HBV 生物标志物。然后，使用 mRNA 序列鉴定 EFTUD2 调节的基因。通过 qRT-PCR 和 Western blot 检测选定基因的 mRNA 变体及其蛋白。为了确认 EFTUD2 对 HBV 复制和 IFN 刺激基因（ISG）表达的影响，在 HepG2.2.15 细胞中通过 EFTUD2 过表达进行了挽救实验。
发现 IFN 诱导的抗 HBV 活性在 HepG2.2.15 细胞中受到限制。mRNA 序列显示 EFTUD2 可以调节经典 IFN 和病毒反应基因。在机制上，单等位基因敲除通过介导基因剪接降低了 ISG 编码蛋白的表达，包括 Mx1、OAS1 和 PKR（EIF2AK2）。然而，EFTUD2 不影响 Jak-STAT 途径基因的表达。此外，EFTUD2 过表达可以恢复单等位基因敲除导致的 IFN 抗 HBV 活性减弱和 ISG 减少。
EFTUD2 是剪接体因子，不是 IFN 诱导的，但却是 IFN 效应基因。EFTUD2 通过调节某些 ISGs 的基因剪接介导 IFN 抗 HBV 作用，包括 EIF2AK2、DDX58 和 IFIT1。EFTUD2 不影响 IFN 受体或经典信号转导成分。因此，可以得出结论，EFTUD2 通过一种新的非经典机制调节 ISGs。
