Marion Duriez1, Yassmina Mandouri2, Bouchra Lekbaby1, Hualin Wang1, Aurélie Schnuriger3, Francois Redelsperger1, Chiara Ida Guerrera4, Marine Lefevre5, Veronique Fauveau6, James Ahodantin1, Ivan Quetier1, Cerina Chhuon4, Samir Gourari7, Alexandre Boissonnas1, Upkar Gill8, Patrick Kennedy8, Nabil Debzi9, Delphine Sitterlin10, Mala K Maini11, Dina Kremsdorf1, Patrick Soussan12. 1. INSERM U1135, Centre d'immunologie et de maladie infectieuse, Paris, France; Université Pierre et Marie Curie, Paris, France. 2. 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. 3. INSERM U1135, Centre d'immunologie et de maladie infectieuse, Paris, France; Université Pierre et Marie Curie, Paris, France; Service de Virologie, Hôpital Trousseau, Paris, France. 4. Université Paris Descartes, Paris, France. 5. Service d'Anatomopathologie, Hôpital Tenon, Paris, France. 6. Université Pierre et Marie Curie, Paris, France; Université Paris Descartes, Paris, France. 7. Service de microbiologie CHU Mustapha Bacha, Alger, Algeria. 8. Division of Infection and Immunity, UCL, London, UK; Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine & Dentistry, QMUL, London, UK. 9. Service d'Hépatologie, CHU Mustapha Bacha, Alger, Algeria. 10. Université Versailles Saint Quentin en Yvelines, Versailles, France; Laboratoire de Génétique et Biologie Cellulaire, EA 4589/EPHE, Montigny le Bretonneux, France. 11. Division of Infection and Immunity, UCL, London, UK. 12. INSERM U1135, Centre d'immunologie et de maladie infectieuse, Paris, France; Université Pierre et Marie Curie, Paris, France; Service de Virologie, Hôpital Tenon, Paris, France. Electronic address: patrick.soussan@inserm.fr.
Abstract
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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Authors: Tobias Flecken; Marie-Anne Meier; Peter Skewes-Cox; David T Barkan; Markus H Heim; Stefan F Wieland; Meghan M Holdorf Journal: J Virol Date: 2019-04-17 Impact factor: 5.103
Authors: Chun Shen Lim; Vitina Sozzi; Margaret Littlejohn; Lilly K W Yuen; Nadia Warner; Brigid Betz-Stablein; Fabio Luciani; Peter A Revill; Chris M Brown Journal: Microb Genom Date: 2021-01