Indiana University-Purdue University Indianapolis (IUPUI) / The function(s) of the intracellular form of HBeAg, previously reported as the preCore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we propose to elucidate the translocation of p22 during its formation from endoplasmic reticulum (ER) to cytosol, how it differs from core in its inability to form a capsid and the biological functions of cytoplasmic and nuclear p22. Firstly, we have identified that a portion of p22, after the cleavage of its signal peptide in ER, is released back into the cytosol through an ERAD-independent mechanism, as neither wildtype nor dominant-negative p97 affected the ER-to-cytosol translocation of p22 or ER-Golgi secretion of HBeAg. Secondly, despite sharing the same sequence with core protein except for the extended 10 amino acid precore region at the N-terminus, we observed that p22 wildtype and C-7Q mutant are unable to form a capsid. Thirdly, we report that p22 but not the secreted HBeAg significantly reduced interferon stimulated response element (ISRE) activity and expression of interferon stimulated genes (ISGs) upon interferon-alpha (IFN- α) stimulation. Furthermore, in line with this, RNA-seq analysis of ISG induction profile from IFN-α treated patients showed that HBeAg(+) patients exhibited reduced and weak antiviral ISG upregulations compared to HBeAg(-) patients. Further, mechanistic study indicated that while p22 did not alter the total STAT1 or p-STAT1 levels in IFN-α treated cells, it blocked the nuclear translocation of p-STAT1 by interacting with karyopherin α1, indicating that the cytoplasmic p22 may impede JAK-STAT signaling to help the virus evade host innate immune response and cause resistance to IFN therapy in patients. Additionally, nuclear p22 and nuclear core were found to interact with the promoter regions (ISRE – containing) of ISGs, suggesting a new mechanism of inhibition of ISG expression upon stimulation. Finally, we found that the nuclear p22 can bind to cccDNA minichromosome and affects cccDNA maintenance and/or transcription. Thus, our results indicate that there is a novel ER sorting mechanism for the distribution of the intracellular and secretory HBeAg, and the intracellular HBeAg may contribute to HBV persistence by interfering with IFN-α elicited JAK-STAT signaling and regulating cccDNA metabolism.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19593号 / 医博第4100号 / 新制||医||1014(附属図書館) / 32629 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松岡 雅雄, 教授 朝長 啓造, 教授 西渕 光昭 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’infection par le virus de l’hépatite B (VHB) reste un problème de santé publique avec plus de 880 000 décès chaque année dans le monde. Au stade chronique de l’infection virale B, des complications peuvent survenir comme la fibrose, la cirrhose et le carcinome hépatocellulaire. L'implication du VHB, de ses protéines ou de sa variabilité génétique dans la fibrose hépatique reste à élucider. Toutefois, des méta-analyses semblent montrer un lien statistiquement significatif entre la présence de la double mutation A1762T/G1764A dans le promoteur basal du core (PBC) du VHB et la fibrose sévère. C’est pourquoi nous avons orienté nos travaux de recherche selon deux axes : l’implication des mutations du PBC et de la région précore (PC) dans la sévérité des lésions hépatiques ainsi que le rôle des protéines HBc et HBe du VHB dans l’induction de la fibrogénèse. Dans un premier temps, deux études cliniques ont permis de confirmer l’association significative du double mutant PBC avec la fibrose sévère indépendamment du génotype viral. Nous avons également démontré un effet antagoniste de la mutation G1899A située dans la région PC vis-à-vis du double mutant PBC dans la sévérité de la fibrose. La deuxième partie de nos travaux a consisté à développer une technique innovante de production de protéines en utilisant la transduction de la lignée HepaRG par des vecteurs lentiviraux contenant la séquence de la protéine GFP (green fluorescent protein). En parallèle, nous avons synthétisé des particules lentivirales contenant les séquences sauvages et mutées du PBC et de la région PC, avec pour objectif de produire les protéines HBc et HBe dans les HepaRG. / The hepatitis B virus (HBV) infection remains a significant public health problem with more than 880 000 deaths every year worldwide. At the stage of chronic HBV infection, complications can occur such as fibrosis, cirrhosis and hepatocellular carcinoma. The role of HBV, its protein and its genomic variability in fibrosis are still unclear. Meta-analysis seems to indicate a strong link between the double mutation A1762T/G1764A detection in the basal core promotor (BCP) of HBV and the development of fibrosis. In this context, our work aimed to explore: i. the implication of BCP or precore (PC) regions mutations on the severity of fibrosis, and ii. the role of HBc and HBe proteins in fibrosis induction. For the first approach, our studies confirmed the association between the presence of the BCP double mutation and severe fibrosis, independently of the viral genotype. We also showed that the G1899A mutation in the PC region presents an antagonist effect regarding the double BCP mutant for fibrosis severity. In the second part of our work, we developed an innovative technology to produce protein via the transduction of HepaRG cells using lentiviral technology with a plasmid vector containing GFP (greenfluorescent protein) sequence. We also obtained lentiviral particles containing the wild and mutated sequences for the BCP and PC regions, in order to produce HBc and HBeprotein in HepaRG cells and to explore of the pathogenic role of BCP and PC mutants.