INSERM U1135, Centre d'immunologie et de maladie infectieuse, Paris, France; Université Pierre et Marie Curie, Paris, France.
INSERM U1135, Centre d'immunologie et de maladie infectieuse, Paris, France; Université Pierre et Marie Curie, Paris, France; Université Versailles Saint Quentin en Yvelines, Versailles, France.
J Hepatol. 2017 Oct;67(4):687-699. doi: 10.1016/j.jhep.2017.05.025. Epub 2017 Jun 7.
BACKGROUND & AIMS: Hepatitis B virus (HBV) RNA can undergo alternative splicing, but the relevance of this post-transcriptional regulation remains elusive. The mechanism of HBV alternative splicing regulation and its impact on liver pathogenesis were investigated.
HBV RNA-interacting proteins were identified by RNA pull-down, combined with mass spectrometry analysis. HBV splicing regulation was investigated in chemically and surgically induced liver damage, in whole HBV genome transgenic mice and in hepatoma cells. Viral and endogenous gene expression were quantified by quantitative reverse transcription polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. Resident liver immune cells were studied by fluorescence-activated cell sorting.
HBV pregenomic RNA-interacting proteins were identified and 15% were directly related to the splicing machinery. Expression of these splicing factors was modulated in HBV transgenic mice with liver injuries and contributed to an increase of the HBV spliced RNA encoding for HBV splicing-generated protein (HBSP). HBSP transgenic mice with chemically induced liver fibrosis exhibited attenuated hepatic damage. The protective effect of HBSP resulted from a decrease of inflammatory monocyte/macrophage recruitment through downregulation of C-C motif chemokine ligand 2 (CCL2) expression in hepatocytes. In human hepatoma cells, the ability of HBSP to control CCL2 expression was confirmed and maintained in a whole HBV context. Finally, viral spliced RNA detection related to a decrease of CCL2 expression in the livers of HBV chronic carriers underscored this mechanism.
The microenvironment, modified by liver injury, increased HBSP RNA expression through splicing factor regulation, which in turn controlled hepatocyte chemokine synthesis. This feedback mechanism provides a novel insight into liver immunopathogenesis during HBV infection. Lay summary: Hepatitis B virus persists for decades in the liver of chronically infected patients. Immune escape is one of the main mechanisms developed by this virus to survive. Our study highlights how the crosstalk between virus and liver infected cells may contribute to this immune escape.
乙型肝炎病毒（HBV）RNA 可发生选择性剪接，但这种转录后调控的相关性仍不清楚。本研究旨在探索 HBV 选择性剪接调控的机制及其对肝脏发病机制的影响。
通过 RNA 下拉结合质谱分析鉴定 HBV RNA 相互作用蛋白。采用化学诱导和手术诱导的肝损伤、全 HBV 基因组转基因小鼠和肝癌细胞研究 HBV 剪接调控。采用定量逆转录聚合酶链反应、Western blot 和酶联免疫吸附试验定量检测病毒和内源性基因表达。通过荧光激活细胞分选研究驻留肝免疫细胞。
鉴定出 HBV 前基因组 RNA 相互作用蛋白，其中 15%直接与剪接机制相关。这些剪接因子在肝损伤的 HBV 转基因小鼠中表达被调节，并导致 HBV 剪接 RNA 增加，编码 HBV 剪接生成蛋白（HBSP）。化学诱导肝纤维化的 HBSP 转基因小鼠表现出肝损伤减轻。HBSP 的保护作用源自通过下调肝细胞中 C-C 基序趋化因子配体 2（CCL2）表达减少炎症性单核细胞/巨噬细胞募集。在人肝癌细胞中，证实了 HBSP 控制 CCL2 表达的能力，并在全 HBV 背景下得以维持。最后，慢性 HBV 携带者肝脏中病毒剪接 RNA 的检测与 CCL2 表达的降低相关，强调了这种机制。
肝损伤引起的微环境通过剪接因子调节增加了 HBSP RNA 的表达，进而控制了肝细胞趋化因子的合成。这种反馈机制为 HBV 感染期间肝脏免疫发病机制提供了新的见解。
乙型肝炎病毒在慢性感染患者的肝脏中持续存在数十年。免疫逃逸是该病毒为生存而开发的主要机制之一。我们的研究强调了病毒与受感染的肝细胞之间的相互作用如何有助于这种免疫逃逸。
