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.

L'infection par le virus de l'hépatite B (VHB) est un problème de santé publique majeur avec plus de 257 millions de porteurs chroniques dans le monde ayant un risque important de développer une cirrhose et/ou un hépatocarcinome. L'histoire naturelle de l'infection est très différente selon l'âge auquel l'infection est contractée. Alors que chez l'adulte l'infection est spontanément résolutive dans la majorité des cas, la contamination materno-infantile ou en bas âge aboutit le plus souvent à une infection chronique. Le cccDNA est la forme de persistance du génome viral dans les hépatocytes infectés et la base de transcription de tous les ARN viraux. La protéine virale HBx joue un rôle crucial dans le recrutement des facteurs épigénétiques sur le cccDNA et favorise son activité transcriptionnelle. Les traitements actuels à base d'interféron et d'analogues nucléos(t)idiques ne permettent pas l'éradication du cccDNA et leur interruption est presque toujours suivie d'une réactivation de la réplication du virus. De nouvelles molécules thérapeutiques ciblant le cccDNA sont donc nécessaires pour espérer obtenir une cure fonctionnelle chez les patients chroniquement infectés. Il existe des liens étroits entre l'infection par le VHB et le métabolisme des acides biliaires (AB). Ainsi, notre équipe a précédemment montré que le récepteur nucléaire des acides biliaires, le farnesoid X receptor alpha (FXRalpha) se fixe sur deux éléments de réponse présents dans la région Enhancer II - promoteur de Core du génome viral et module son activité transcriptionnelle. De plus, le VHB et les AB entrent en compétition pour le même récepteur d'entrée hépatocytaire NTCP, modifiant la concentration cellulaire des AB avec des conséquences sur la fonction et l'expression de FXRalpha. Enfin, HBx interagit avec FXRalphaet modifie son activité. Au cours de cette thèse nous avons dans un premier temps identifié une régulation réciproque existante entre la réplication du VHB et FXRalpha. Puis nous avons montré in vitro, dans des cellules HepaRG différenciées et des hépatocytes primaires humains, que FXRalphaest un facteur proviral pour le VHB et que les agonistes de FXRalpha inhibent l'expression de l'ensemble des marqueurs viraux de manière dépendante ou indépendante de la protéine virale HBx. Enfin, dans un modèle in vivo de souris C3H/HeN transduites par un vecteur recombinant AAV2/8-VHB, nous avons obtenu l'effet inhibiteur des agonistes de FXRalphamais uniquement chez les souris adultes et pas chez les souris jeunes. Compte tenu de l'évolution de la flore intestinale avec l'âge et de son importance dans le métabolisme des AB, ces résultats suggèrent que le fort taux d'évolution chronique chez les jeunes enfants pourrait être lié à l'immaturité du métabolisme des AB. La mise en évidence d'un lien entre microbiote, métabolisme des AB et infection par le VHB contribuerait grandement à une meilleure compréhension de l'histoire naturelle de cette infection. De plus, l'identification de FXRalphacomme un facteur de l'hôte favorisant l'infection et l'existence de molécules capables de moduler l'activité de FXRalphasuggèrent que FXRalphapourrait constituer une cible thérapeutique intéressante ciblant le cccDNA et permettant d'améliorer le traitement des patients infectés par le VHB / Hepatitis B virus (HBV) infection is a major global health problem with more than 257 million chronic carriers worldwide that remain at significant risk for developing cirrhosis and/or hepatocellular carcinoma. The natural history of infection is very different depending on the age at which the infection is contracted. Whereas in adults most HBV infections spontaneously resolve, in infants and young children they usually result in chronic infection. cccDNA is the molecular form of viral persistence in infected hepatocytes and serves as a transcription template for all viral RNAs. The viral protein HBx plays a crucial role in the recruitment of epigenetic factors to the cccDNA and promotes its transcriptional activity. Currently, interferon and nucleot(s)ide analogues are the first-line agents in the treatment of chronic hepatitis B without allowing eradication of cccDNA and their interruption are almost always followed by a reactivation of the replication of the virus. New therapeutic molecules targeting cccDNA are therefore needed to hope for a functional cure in chronically infected patients. HBV infection and bile acid (BA) metabolism are tightly linked. Therefore, our team has previously shown that the bile acid nuclear receptor, the farnesoid X receptor alpha (FXRalpha) bind to two response elements present in the Enhancer II - Core promoter region of HBV genome and modulate its transcriptional activity. Moreover, HBV and BA compete for the same entry receptor of hepatocytes NTCP and modify BA cell concentration with consequences on the function and expression of FXRalpha. Finally, HBx interacts with FXRalpha and modify its activity. During my PhD. we have first identified a reciprocal regulation between HBV replication and FXRalpha. Second, we have showed in vitro, in HepaRG differentiated cells and in primary human hepatocytes, that FXRalpha is a proviral factor for HBV and that FXRalpha agonists inhibit the expression of all HBV markers in a dependent or independent manner of the viral protein HBx. Finally, in an in vivo model of C3H/HeN mice transduced with a recombinant AAV2/8-HBV vector, we obtained the inhibitory effect of FXRalpha agonists but only in adult and not in young mice. Considering the evolution of the gut flora with age and its importance in the metabolism of BA, these results suggest that the high rate of chronic progression in young children might be related to the immaturity of BA metabolism. The identification of a link between BA metabolism, gut microbiome composition and evolution of HBV infection will represent a big step toward the understanding of HBV natural history. Moreover, the identification of FXRalpha as a proviral factor for HBV and the capacity of FXRalpha ligands to modulate the transcriptional activity of cccDNA suggest that FXR ligands might represent a new class of molecules with the aim to obtain functional cure for HBV infected patients

The Hepatitis B virus (HBV) chronically infects 350 million individuals worldwide, leading to mortality by end-stage liver disease, liver cirrhosis, and hepatocellular carcinoma. The vaccine to prevent HBV infection is highly effective but is not extensively available in endemic areas, resulting in high infection rates. Nucleoside analogue treatment of HBV has allowed for higher rates of viral clearance in infected individuals, but most patients must remain on therapy long term and viral resistance to the drugs is growing. The HBV viral genome is an episome in the nucleus of infected hepatocytes. It is called covalently closed circular (ccc) DNA and is highly stable, has a long half-life, and is the template for all viral transcription and progeny production. Nucleoside analogues do not directly target cccDNA, therefore many patients experience rebound when antiviral therapy is stopped. I have designed novel DNA binding proteins called zinc finger proteins (ZFPs) to specifically bind to the cccDNA in infected cells and inhibit viral transcription. Seven ZFPs targeting the model duck HBV (DHBV) and ten ZFPs targeting HBV were developed. Kinetic analyses of the purified ZFPs were performed, characterizing their specificity and binding properties. Using the DHBV tissue culture model system, I have demonstrated that the DHBV-specific ZFPs can specifically inhibit transcription from the viral template, resulting in reduced viral RNA, protein products and progeny virions. The DHBV-specific ZFPs were tested in primary duck hepatocytes (PDH) and in vivo in the Pekin duck model. ZFPs failed to express in PDH transduced by baculovirus vectors when DHBV was present in the cells. In vivo gene delivery of the ZFPs was carried out by portal vein injection of chitosan-based nanospheres. Unfortunately, non-specific reductions in viral levels masked any direct effect by the ZFPs. Testing of the HBV-specific ZFPs in tissue culture was hindered by a lack of transfectable cell culture model. A number of different transfection methods were tested to express the HBV-specific ZFPs, all without success. Further work is being carried out using baculovirus vectors to deliver the HBV-specific ZFPs to HBV-harbouring cell lines and HBV-infected scid-Alb/uPA chimeric mice with human liver cells. / Virology

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Despite the lifecycle of the hepatitis B virus (HBV) being extensively investigated and described, there remains a significant gap in our knowledge of arguably one of the most crucial steps in the HBV lifecycle, the formation and maintenance of a covalently closed circular DNA (cccDNA) reservoir. Advancements in our understanding of host factors and pathways involved in cccDNA formation have been made through hypothesis driven studies and shRNA/siRNA screenings. We sought to create a targeted-unbiased assay to directly observe host factor-rcDNA interactions. This was achieved through an rcDNA Co-Immunoprecipitation paired Mass Spectrometry (rcDNA-CoIP/MS) assay. We created a DNA oligo complimentary to the open portion of the HBV rcDNA, labeled with biotin, to facilitate easy precipitation of nuclear rcDNA and complexed proteins. Proteins precipitated were analyzed through liquid chromatography paired mass spectrometry (LC/MS). Along with previously reported host factors, several factors of DNA damage repair pathways/complexes were also identified. A component of the UV-DDB complex, DDB1, surfaced as a hit. UV-DDB/rcDNA binding was confirmed through ChIP-qPCR. DDB2, the DNA damage binding component of the UV-DDB complex was knocked out in HepG2-NTCP and HepAD38 cells. This resulted in a significant decrease in the formation of cccDNA in DDB2 knockout cell lines following infection or induction. The subsequent reduction of downstream indicators of cccDNA formation such as viral RNA and proteins, HBcAg and HBeAg, showed a consistent decrease with cccDNA levels. Ectopic expression of DDB2 in the knockout cell lines rescued HBV phenotypes of cccDNA levels and its downstream indicators. Inactive mutant DDB2 plasmids were also transfected into the DDB2 K/O cell lines and failed to rescue cccDNA indicators. We therefore showed through a novel assay that we can discover novel viral rcDNA-host interactions, such as the UV-DDB complex recruiting DNA repair pathways to “repair” rcDNA to cccDNA.

Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the availability of anti-viral agents, there is currently no cure. This double stranded DNA virus is hepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA contains a partially synthesised positive DNA stand and a gap region within the negative strand. After infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An important objective of HBV therapy is the elimination of cccDNA, as its persistence within hepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this replication intermediate is crucial. The objective of this study was to develop a method based on real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap were designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV DNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV transgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse livers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA profile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells, only 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results were validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA pools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA detection as it allows for the rapid simultaneous amplification and quantification of a specific DNA target through either non-specific or specific DNA detection chemistries. In conclusion, this HBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy

La particularité de ce virus de l'hépatite B est la synthèse d'un ADN circulaire clos covalent (ADNccc) qui est la forme de persistance du virus dans la cellule. Cet ADN est maintenu à une copie par cellule en moyenne chez l'homme grâce à un recyclage des nucléocapsides dans le noyau. En effet, durant le cycle viral, les nucléocapsides sont soit redirigées dans le noyau pour former de l'ADNccc, soit enveloppées puis sécrétées pour former de nouveaux virions. Du fait de son maintien au sein de l'hépatocyte, la formation et la régulation de l'ADNccc restent des éléments clés du traitement antiviral. Il a été montré in vitro que l'accumulation de cet ADN était régulée par les protéines d'enveloppe. Lors de l'étude du taux d'ADNccc dans des biopsies de foie de patients coinfectés HIV-HBV chronique, il a été découvert un patient présentant 300 fois plus d'ADNccc intrahépatique que la moyenne de la cohorte. L'objectif de ma thèse a été de comprendre quel était le mécanisme conduisant à cette accumulation d'ADNccc in vivo. Cela nous a permis de mettre en évidence le rôle du gène core dans l'accumulation de l'ADNccc / The feature of hepatitis B virus is the synthesis of a covalently closed circular DNA (cccDNA) which is the persistence form of the virus in cell. cccDNA is maintained to 1 copy per human cell thanks to the recycling of capsids into the nucleus. Indeed, during the viral cycle, capsids are either transported into the nucleus to form cccDNA or enveloped and secreted to form new infectious virions. Because of its maintenance in the hepatocyte, cccDNA formation and regulation are still key elements of antiviral treatment. It has been shown that, in vitro, cccDNA accumulation was regulated by envelope proteins. Upon the study of cccDNA levels in liver biopsies of HIV-HBV co-infected patients, an individual with a cccDNA level 300 fold higher than the average of the cohort was identified. My thesis objective was to understand which is the mechanism leading to the cccDNA accumulation observed in vivo. This allowed us to highlight the role of core gene in cccDNA accumulation

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21976号 / 生博第416号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 朝長 啓造, 教授 野田 岳志 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Le virus de l'hépatite B (HBV) infecte de manière chronique 240 millions de personnes dans le monde, et est la principale cause de carcinome hépatocellulaire. Actuellement, les traitements standards permettent une suppression virale à long terme, mais ne sont pas capables d'éliminer complètement le virus, en raison de la persistance de l'ADN circulaire et clos de façon covalente (ADNccc). Ce minichromosome viral réside dans le noyau des hépatocytes infectés, grâce à sa structure chromatinienne. En effet, lors de l'infection d'un hépatocyte, l'ADN viral partiellement double brin (ADN relâché circulaire (rc)) est libéré dans le noyau, où il est réparé et enveloppé par des protéines histones, afin de former une structure d'épisome chromatinisé. Les mécanismes conduisant à la formation ainsi qu'à la chromatinisation de l'ADNccc sont encore largement inconnus, et leur élucidation constituerait une première étape vers l'identification de nouvelles cibles thérapeutiques, susceptibles d'altérer la persistance de l'ADNccc. Dans ce but, nous avons étudié le rôle des facteurs hôtes de réparation de l'ADN, et des voies d'assemblage des nucléosomes, dans la formation de l'ADNccc, à des stades précoces (entre 30 minutes et 72 heures) de l'infection, dans des lignées cellulaires d'HepG2-NTCP, ainsi que dans des hépatocytes primaires humains. Nous nous sommes particulièrement concentrés sur la protéine chaperone d'histones, HIRA, qui est connue pour déposer le variant d'histone 3.3 (H3.3) sur l'ADN cellulaire d'une manière indépendante de la réplication et en association avec le remaniement des nucléosomes pendant la transcription et la réparation de l'ADN. Nous avons été capables de détecter l'ADNccc dans la fraction nucléaire des hépatocytes dès 30 minutes et 24 heures post-infection, par qPCR et Southern Blotting (SB), respectivement. L'extinction de HIRA par ARN interférent (siARN) avant l'inoculation du virus, a conduit à une forte diminution de l'accumulation de l'ADNccc (à la fois par qPCR et Southern Blot), qui était indépendante de la protéine HBx (en utilisant un virus HBx-défectueux). Les niveaux d'ADNrc sont restés stables, indiquant soit une éventuelle transition de l'ADNrc en ADNccc incomplète, ou retardée. L'analyse par immunoprécipitation de la chromatine a montré que HIRA était liée à l'ADNccc dès 30 minutes après infection, et que son recrutement était concomitant avec le dépôt de l'histone H3.3, ainsi que la liaison de la protéine de capside du HBV (HBc). Après 24 heures d'infection, une augmentation de la liaison de H3.3 et de l'ARN polymérase II sur l'ADNccc a été observée, en corrélation avec l'initiation de la transcription de l'ARN viral de 3.5 kb. Par des expériences de co-immunoprécipitation et de test de proximité entre protéines (PLA), nous avons montré que HIRA était capable d'interagir avec HBc dans des hépatocytes infectés et dans une lignée cellulaire HepaRG exprimant HBc de manière inductible. En conclusion, nos résultats suggèrent que la chromatinisation de l'ADN viral entrant est un événement très précoce, nécessitant l'histone chaperone HIRA. Bien que HBx ne soit pas requis pour ce processus, HBc pourrait jouer un rôle majeur, suggérant que l'interaction entre HIRA et HBc pourrait représenter une nouvelle cible thérapeutique à étudier / Hepatitis B virus (HBV) chronically infects 240 million people worldwide and is the major cause of hepatocellular carcinoma (HCC). Currently standard-of-care treatments can achieve longterm viral suppression, but are not able to completely eliminate the virus, due to the persistence of the covalently closed circular DNA (cccDNA). cccDNA, the viral minichromosome, resides in the nucleus of infected hepatocytes by virtue of its chromatin structure. Indeed, upon entry into hepatocytes, the partially double stranded viral DNA (relaxed circular (rc)DNA) is released into the nucleus, where it is repaired and wrapped by histones to form an episomal chromatinized structure. The mechanisms leading to cccDNA formation and chromatinization are still largely unknown and their elucidation would be a first step toward the identification of new therapeutic targets to impair cccDNA persistence. To this aim, we investigated the role of host factors belonging to DNA repair and nucleosome assembly pathways in cccDNA formation at early time points (i.e. between 30 minutes and 72 hours) post-infection in both HepG2-NTCP cell line and Primary Human Hepatocytes (PHH). We particularly focused on the histone chaperone Hira, which is known to deposit histone variant 3.3 (H3.3) onto cellular DNA in a replication-independent manner and in association to nucleosome reshuffling during transcription and DNA repair. We were able to detect cccDNA in the nuclear fraction of hepatocytes as early as 30 minutes and 24h post-infection, by qPCR and Southern Blotting (SB), respectively. Knock-down of Hira by RNA interference before virus inoculation led to a strong decrease in cccDNA accumulation (both in qPCR and SB) which was independent from HBx protein expression (using an HBx defective virus). rcDNA levels remained stable, indicating either a possible incomplete or delayed rcDNA to cccDNA transition. Chromatin Immunoprecipitation analysis showed that Hira was bound to cccDNA already at 2 hours post-infection and that its recruitment was concomitant with the deposition of histone H3.3 and the binding of HBV capsid protein (HBc). After 24 hours of infection, an increase of H3.3 and Pol2 binding on cccDNA was observed, correlating with the initiation of the transcription of the 3.5 kb RNA. By Co-Immunoprecipitation and Proximity Ligation Assay experiments, we showed that Hira was able to interact with HBc in infected hepatocytes and in a HepaRG cell line expressing HBc in an inducible manner. Altogether, our results suggest that chromatinization of incoming viral DNA is a very early event, requiring the histone chaperone Hira. While HBx is not required for this process, HBc could play a major role, suggesting that the interaction between Hira and HBc could represent a new therapeutic target to be investigated

Malgré l’existence d’un vaccin préventif efficace, l’infection chronique par le virus de l’hépatite B (HBV) demeure un problème majeur de santé publique. La persistance de l’infection par HBV étant clairement associée à des réponses immunitaires insuffisantes, l’immunothérapie par le vaccin à base d’ADN nu, visant à stimuler les réponses humorales et cellulaires, apparaît comme particulièrement pertinente pour la thérapie des hépatites B chroniques. Toutefois, l’efficacité thérapeutique d’une telle stratégie reste limitée chez l’homme, d’où la nécessité d’optimiser cette approche vaccinale pour une utilisation ultérieure en clinique. Ainsi, l’objectif général de ce travail de thèse était d’explorer, avec le modèle du DHBV (« Duck Hepatitis B Virus »), étroitement apparenté au HBV humain, si l’administration du vaccin à ADN par électroporation (EP) pouvait davantage améliorer son efficacité prophylactique et thérapeutique. Nous avons montré, dans un 1er temps chez des canards naïfs, que l’administration du vaccin à ADN par EP permet de potentialiser le pouvoir neutralisant et d’élargir le répertoire épitopique de la réponse humorale dirigée contre la protéine d’enveloppe du DHBV, même avec des doses d’ADN relativement faibles. Dans un 2ème temps, nous avons montré chez des animaux chroniquement infectés par le DHBV, que l’administration par EP du vaccin à ADN ciblant les protéines structurales du DHBV et le DuIFN-γ améliore considérablement l’efficacité thérapeutique du vaccin, notamment au regard de la séroconversion et de la clairance virale. Les résultats ainsi obtenus confirment l’intérêt majeur de cette approche vaccinale pour la thérapie des hépatites B chroniques / Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Since persistence of HBV infection is mostly associated with insufficient immune responses, therefore DNA vaccination capable of activating both humoral and cellular immune responses appears as a pertinent strategy for chronic hepatitis B therapy. However, the efficacy of such therapeutic approach remains limited in humans. Improvement of DNA vaccine efficacy is therefore needed for future therapeutic applications in clinic. The main objective of this thesis was to investigate in the duck hepatitis B virus (DHBV) model, whether the protective and therapeutic efficacy of DNA vaccine can be enhanced using EP-based delivery system. Firstly, we showed in naïve ducks that EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response even with relatively low DNA doses, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose. Secondly, we showed in chronic DHBV-carriers that in vivo EP is able to dramatically enhance the therapeutic potency of DNA vaccine targeting hepadnaviral proteins. Indeed, this approach was able to consistently restore humoral immune response and to sustainably decrease and even clear viral infection. Thus, these data strongly support the use of this approach for chronic hepatitis B therapy in humans