Department of Hepatology, Center for Pathogen Biology and Infectious Diseases, Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin 130000, China.
Department of Nephrology, The First Hospital of Jilin University, Changchun, Jilin 130000, China.
Chin Med J (Engl). 2023 Apr 5;136(7):799-806. doi: 10.1097/CM9.0000000000002617.
The hepatitis B virus (HBV) vaccine has been efficiently used for decades. However, hepatocellular carcinoma caused by HBV is still prevalent globally. We previously reported that interferon (IFN)-induced tripartite motif-containing 25 (TRIM25) inhibited HBV replication by increasing the IFN expression, and this study aimed to further clarify the anti-HBV mechanism of TRIM25.
The TRIM25-mediated degradation of hepatitis B virus X (HBx) protein was determined by detecting the expression of HBx in TRIM25-overexpressed or knocked-out HepG2 or HepG2-NTCP cells via Western blotting. Co-immunoprecipitation was performed to confirm the interaction between TRIM25 and HBx, and colocalization of TRIM25 and HBx was identified via immunofluorescence; HBV e-antigen and HBV surface antigen were qualified by using an enzyme-linked immunosorbent assay (ELISA) kit from Kehua Biotech. TRIM25 mRNA, pregenomic RNA (pgRNA), and HBV DNA were detected by quantitative real-time polymerase chain reaction. The retinoic acid-inducible gene I (RIG-I) and pgRNA interaction was verified by RNA-binding protein immunoprecipitation assay.
We found that TRIM25 promoted HBx degradation, and confirmed that TRIM25 could enhance the K90-site ubiquitination of HBx as well as promote HBx degradation by the proteasome pathway. Interestingly, apart from the Really Interesting New Gene (RING) domain, the SPRY domain of TRIM25 was also indispensable for HBx degradation. In addition, we found that the expression of TRIM25 increased the recognition of HBV pgRNA by interacting with RIG-I, which further increased the IFN production, and SPRY, but not the RING domain is critical in this process.
The study found that TRIM25 interacted with HBx and promoted HBx-K90-site ubiquitination, which led to HBx degradation. On the other hand, TRIM25 may function as an adaptor, which enhanced the recognition of pgRNA by RIG-I, thereby further promoting IFN production. Our study can contribute to a better understanding of host-virus interaction.
乙型肝炎病毒 (HBV) 疫苗已被有效使用了几十年。然而，由 HBV 引起的肝细胞癌在全球仍然普遍存在。我们之前曾报道，干扰素 (IFN)-诱导的三结构域包含蛋白 25 (TRIM25) 通过增加 IFN 的表达来抑制 HBV 复制，本研究旨在进一步阐明 TRIM25 的抗 HBV 机制。
通过 Western blot 检测 TRIM25 过表达或敲除 HepG2 或 HepG2-NTCP 细胞中 HBx 蛋白的表达，确定 TRIM25 介导的乙型肝炎病毒 X (HBx) 蛋白降解。通过共免疫沉淀证实 TRIM25 与 HBx 的相互作用，并通过免疫荧光鉴定 TRIM25 和 HBx 的共定位；使用科华生物的酶联免疫吸附测定 (ELISA) 试剂盒定性 HBV e 抗原和 HBV 表面抗原。通过实时定量聚合酶链反应检测 TRIM25 mRNA、前基因组 RNA (pgRNA) 和 HBV DNA。通过 RNA 结合蛋白免疫沉淀测定验证 RIG-I 和 pgRNA 的相互作用。
我们发现 TRIM25 促进 HBx 降解，并证实 TRIM25 可以增强 HBx 的 K90 位点泛素化，并通过蛋白酶体途径促进 HBx 降解。有趣的是，除了 Really Interesting New Gene (RING) 结构域外，TRIM25 的 SPRY 结构域对于 HBx 降解也是必不可少的。此外，我们发现 TRIM25 通过与 RIG-I 相互作用增加了对 HBV pgRNA 的识别，从而进一步增加了 IFN 的产生，而 SPRY，但不是 RING 结构域在这个过程中是关键的。
该研究发现 TRIM25 与 HBx 相互作用并促进 HBx-K90 位点泛素化，导致 HBx 降解。另一方面，TRIM25 可能作为一种衔接蛋白，增强了 RIG-I 对 pgRNA 的识别，从而进一步促进 IFN 的产生。我们的研究有助于更好地理解宿主-病毒相互作用。
