Identification and Characterization of Multiple TRIM Proteins That Inhibit Hepatitis B Virus Transcription

Tripartite motif (TRIM) proteins constitute a family of over 100 members that share conserved tripartite motifs and exhibit diverse biological functions. Several TRIM proteins have been shown to restrict viral infections and regulate host cellular innate immune responses. In order to identify TRIM proteins that modulate the infection of hepatitis B virus (HBV), we tested 38 human TRIMs for their effects on HBV gene expression, capsid assembly and DNA synthesis in human hepatoma cells (HepG2). The study revealed that ectopic expression of 8 TRIM proteins in HepG2 cells potently reduced the amounts of secreted HBV surface and e antigens as well as intracellular capsid and capsid DNA. Mechanistic analyses further demonstrated that the 8 TRIMs not only reduced the expression of HBV mRNAs, but also inhibited HBV enhancer I and enhancer II activities. Studies focused on TRIM41 revealed that a HBV DNA segment spanning nucleotide 1638 to nucleotide 1763 was essential for TRIM41-mediated inhibition of HBV enhancer II activity and the inhibitory effect depended on the E3 ubiquitin ligase activity of TRIM41 as well as the integrity of TRIM41 C-terminal domain. Moreover, knockdown of endogenous TRIM41 in a HepG2-derived stable cell line significantly increased the level of HBV preC/C RNA, leading to an increase in viral core protein, capsid and capsid DNA. Our studies have thus identified eight TRIM proteins that are able to inhibit HBV transcription and provided strong evidences suggesting the endogenous role of TRIM41 in regulating HBV transcription in human hepatoma cells.三联体基序（tripartite motif, TRIM）蛋白是一类拥有超过100个成员的蛋白家族，它们共享保守的三联体基序，并具备多样的生物学功能。已有研究证实，多种TRIM蛋白能够限制病毒感染，并调控宿主细胞的天然免疫应答。为了鉴定可调节乙型肝炎病毒（hepatitis B virus, HBV）感染的TRIM蛋白，我们在人肝癌细胞（HepG2）中检测了38种人源TRIM蛋白对HBV基因表达、衣壳组装以及DNA合成的影响。本研究发现，在HepG2细胞中异位表达8种TRIM蛋白，可强效减少分泌型HBV表面抗原、e抗原以及细胞内衣壳和衣壳相关DNA的含量。机制分析进一步证实，这8种TRIM蛋白不仅能够降低HBV mRNA的表达水平，还可抑制HBV增强子I与增强子II的活性。针对TRIM41的专项研究显示，一段跨越核苷酸1638至1763的HBV DNA片段，是TRIM41介导的HBV增强子II活性抑制所必需的；且该抑制效应依赖于TRIM41的E3泛素连接酶活性，以及TRIM41 C端结构域的完整性。此外，在HepG2来源的稳定细胞系中敲低内源性TRIM41，可显著提升HBV preC/C RNA的水平，进而导致病毒核心蛋白、衣壳以及衣壳DNA的含量增加。综上，本研究鉴定出8种可抑制HBV转录的TRIM蛋白，并为TRIM41在人肝癌细胞中调控HBV转录的内源性作用提供了坚实的实验证据。