I. Cell biology of hepatitis B virus infection: towards curative approaches of chronic hepatitis B

HBV is a pararetrovirus replicating its DNA genome via reverse transcription of an overlength RNA precursor (the pregenome). Hallmarks of HBV are the compact genome organization, the use of internal promoters and the persistence of the viral DNA genome as an episome in the nucleus of infected cells. This covalently closed circular DNA (cccDNA) serves as persistence reservoir and is not affected by existing antiviral therapy. Consequently, these treatments are not curative, but only viro-suppressive and have to be applied life-long.

Schematic of the HBV replication cycle. Upon infection of the hepatocyte, the nucleocapsid is transported into the nucleus, the viral DNA genome is converted into the cccDNA form and serves as template for the synthesis of all viral RNAs. These are translated in the cytoplasm where viral capsids form in conjunction with the reverse transcriptase, the RNA pregenome and the core protein. Virions form by budding into the ER. SVPs bud into the same compartment but lack the inner viral components. Courtesy of Dr. Katrin Schöneweis, Department of Infectious Diseases, Molecular Virology, Heidelberg.
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Most HBV infections are asymptomatic and can persist, with the risk of persistence increasing with decreasing age. While more than 90% of perinatal HBV infections persist, this number drops to 5-10% in case of adults, arguing that immunological competence of the infected individual is critical to control virus infection. Although the underlying mechanisms of persistence are not understood, there is increasing evidence that HBV is a “stealth” virus, neither inducing an interferon response, nor being profoundly affected by the antiviral program triggered by this cytokine. In addition, there is solid evidence that an insufficient adaptive immune response accounts for chronicity, most notably a lack of T cell response against the viral surface protein(s). This is due, at least in part, to the massive production of subviral particles (SVPs), which are “empty” virus envelopes containing viral surface glycoproteins, but lacking a capsid and the viral genome. Of note, SVPs are released in ~1,000-fold excess over virus particles. SVPs are non-infectious, but the excessive amount of HBV surface proteins contained therein probably is blunting especially T cell immunity.
To overcome this limitation we attempt to develop strategies to block excessive production of SVPs. To this end, we study the cell biology of SVP production and release by using state-of-the-art infectable cell culture models. By using RNA interference-based screens we want to identify host cell factors and pathways in SVP formation and secretion with the aim to exploit these factors and pathways as targets for the development of curative therapeutic approaches of chronic hepatitis B.

Seitz S, Iancu C, Volz T, Mier W, Dandri M, Urban S, Bartenschlager R. 2016. A slow maturation process renders hepatitis B virus infectious. Cell Host & Microbe, 20(1):25-35. doi: 10.1016/j.chom.2016.05.013,
Bartenschlager R, Schaller H. 1992. Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. EMBO J. 1992 Sep;11(9):3413-20.


II. Establishment of immune-competent mouse models for HBV and HCV infection and their use to study liver tumor formation

Understanding HBV and HCV persistence and pathogenesis is only possible by using fully immune-competent small animal models. Although some progress has been made for HCV and HBV, thus far such mouse models are not available. In this project we are seeking to develop immune-competent mouse models by using various approaches, including hydrodynamic injection of viral genomes and the establishment of related viruses sharing immunological features with HBV and HCV. By using these models we aim to understand the mechanisms responsible for the failure of the immune system to control virus infections and to map viral factors contributing to immune escape. Moreover, these animals will provide the basis to study how persistent hepatitis virus infection will promote liver tumor formation. As complementary approach we are using mice transiently or stably expressing viral proteins in the liver. To mimic tumor-promoting factors these mice are given specific diets or have a genetically defined liver inflammation. In this way the contribution of continuous expression of distinct viral proteins to liver tumor formation can be studied.

Bühler S, Bartenschlager R. 2012. Promotion of hepatocellular carcinoma by hepatitis C virus. Dig Dis. 2012;30(5):445-52. doi: 10.1159/000341688.



III. Induction of antiviral immune responses against hepatitis viruses and viral countermeasures

As part of our research activities that are ongoing in our sister unit at the Department of Infectious Diseases, Molecular Virology, in this subproject we aim to understand the strategies how HCV infection is sensed by pattern recognition receptors and how the virus escapes this pathway. We have earlier shown that HCV blocks signaling pathways by cleaving the adaptor protein MAVS and obtained evidence that HCV persistence might be facilitated by the stochastic nature of the interferon response. We now want to dissect further the predominant sensor of HCV RNA and how, in addition to MAVS cleavage, the virus counteracts the interferon response. Moreover, we want to characterize the ISGs responsible for control of HCV infection. Finally, by using co-culture systems we want to study the cross-talk between innate and adaptive immune response. Here we focus on T cell exhaustion, which is a hallmark of HCV and HBV infection and that appears to be caused by mechanisms differing between these two viruses.

Schematic how HCV activates the interferon (IFN) response and points of viral countermeasure that are based on proteolytic cleavage of signaling molecules by the viral NS3/4A protease. IFN released from infected cells induces an antiviral state in infected and bystander cells. It is unclear to what extent HCV blocks Jak/Stat signaling as contradicting reports exist. The important role of IL28 SNPs in the outcome of HCV infection (acute self-limited versus persistence) is indicated. It is also unclear whether sustained IFN production as induced by HCV infection has an effect on T cell response. Figure adapted from Metz et al., 2013.
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Meylan, E., J. Curran, K. Hofmann, D. Moradpour, M. Binder, R. Bartenschlager and J. Tschopp. 2005. Cardif is a novel adaptor protein in RIG-I-mediated antiviral responses targeted by hepatitis C virus. Nature, 437:1167-1172.

Bauhofer O, Ruggieri A, Schmid B, Schirmacher P, Bartenschlager R. 2012. Persistance of HCV in Quiescent Hepatic Cells during an Interferon-Induced Antiviral Response. Gastroenterology. 143:429-38.

Jo J, Aichele U, Kersting N, Klein R, Aichele P, Bisse E, Sewell AK, Blum HE, Bartenschlager R, Lohmann V, Thimme R. 2009.Analysis of CD8+ T-cell mediated inhibition of hepatitis C virus replication using a novel immunological model. Gastroenterology. 136:1391-401.

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