Our research activities are focused in particular on dissecting the molecular mechanisms of the interactions between the Hepatitis B Virus (HBV) and its host. At present we are investigating the early steps of hepadnaviral infection, i.e. the identification of cellular receptors, the biochemical characterization of receptor interactions and viral surface proteins, the determination of the structure of receptor-ligand complexes via X-ray and NMR analysis, the evaluation of dependencies of the infection process on cellular transport mechanisms, the visualization of particle entry into hepatocytes through high-resolution fluorescence microscopy, the identification of determinants of hepadnaviral host tropism, the characterization of the fusion process of viral and host membrane and the identification of components participating in viral morphogenesis.
Building on the knowledge acquired in basic research program we are applying our findings in translational approaches, here focusing on two lines of research. In our first approach novel entry inhibitors (Myrcludex B) are developed for the treatment of acute and chronic hepatitis B and hepatitis D infections, an approach that is presently applied in clinical settings (Phase II studies in HBV and HDV) . In our second approach hepatotropic therapeutics are established, that are generated by coupling sequences of an HBV envelope protein and various active substances.
Infections with human Hepatitis B Virus (HBV) are one of the biggest medical problems that humans face. Despite the availability of a preventative vaccine available since the 1980s about 350 million people are still chronically infected with HBV, mainly in Asian and African countries. Those affected carry a higher risk to develop liver cirrhosis or even hepatocellular carcinoma (HCC) within the next decades. Approximately 650,000 people worldwide die as a consequence of chronic hepatitis B. Although there are effective therapies, which can slow down the progress of liver disease progression, these do not cure infection and do not lead to eliminations of the virus. It is therefore vital to perform basic research on HBV in order to identify additional steps of the replication cycle, which, when addressed help to eliminate the virus from the host. My research group has been pursuing this goal for the last 20 years. Here central research aims include the molecular analysis of early steps of Hepatitis B Virus (HBV) infection.
Based on a cell culture system for HBV made available in 2002 (HepaRG cells) it was possible to identify and synthetically prepare fragments of the large HBV envelope protein, which are able to block entry of HBV into hepatocytes even at picomolar concentrations. Furthermore we could show that these peptides contain a specific recognition sequence for hepatocytes, leading to a very effective transport such peptides to the liver cell. As a result of this liver-tropism, major advantages for the therapeutic of the drug can be expected. In addition this targeted “guidance” into the liver could potentially be used in other applications (e.g. in malaria therapy, in the targeted interference with other hepatotropic viruses or in the liver specific therapy of metabolic diseases). These possibilities are currently examined extensively for their practicability.
We could provide a proof of principle for the effectiveness of the described peptides in vivo in close collaboration with the group of Dr. Petersen in Hamburg. In order to test the resulting therapeutic applications in a clinical setting, in recent years we developed the therapeutic lead substance Myrcludex B, whose pre-clinical studies were finished in June 2010. In February 2011 an application for the permission for a first clinical study was made with the „Bundesarzneimittelbehörde (BfArM)”. In the following July the BfArM granted the permission for a phase Ia trial in humans. From July 2011 until February 2012 24 healthy test subjects were treated without side effects. Currently a phase Ib trial is running in Heidelberg and two phase IIa trial in Russia. If the trials proof to be successful, an essential contribution will have been made towards future therapies of acute and chronic Hepatitis B, leading to an effective and novel treatment for HBV and HDV infection. The antiviral therapeutic agent will be the first that has been developed based on basic research followed by an application in a clinical setting. The research on Myrcludex B was supported by the German Federal Ministry for Education and Research (BMBF) and is part of the translational priority programme of the newly established German Centre for Infection Research (Deutsches Zentrum für Infektionsforschung,DZIF) Initiative.
1 Identification of host restriction factors to Hepatitis B Virus infection in mouse liver cells
Summary: Mouse liver cells reconstituted with human NTCP are susceptible towards Hepatitis Delta Virus (HDV) but not towards Hepatitis B Virus (HBV) due to an additional restriction step (Ni et al., Gastroenterology, 2013). The aim of this study is to further characterize this cellular restriction and eventually to identify the host factor(s) involved in this process. We employ cell fusion assays in combination with novel murine liver cell lines and screening strategies to evaluate the molecular mechanisms behind restriction. Identification of these mechanisms will eventually lead to the development of fully immunocompetent mouse models for both HDV and HBV infection.
Dirk Grimm (Bioquant, Heidelberg)
Thomas Baumert (INSERM, Strasbourg)
Dagmar Wirth (HZI, Hannover)
Project relevant Publications:
> Ni Y, Lempp FA, Mehrle S, et al. Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology. 2014;146(4):1070-83.
> Slijepcevic D, Kaufman C, Wichers CG, et al. Impaired uptake of conjugated bile acids and Hepatitis B Virus preS1-binding in Na(+) -taurocholate cotransporting polypeptide knockout mice. Hepatology. 2015;
> Yan H, Peng B, He W, et al. Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide. J Virol. 2013;87(14):7977-91.
2 Crystallization of the Duck Hepatitis B Virus Receptor Carboxypeptidase D
Summary: Although cellular receptors for HBV and DHBV have been identified in the past, no information allowing structural insights into hepadnaviral receptor interaction is available so far. We are addressing this question using DHBV and one of its cellular receptors, carboxypeptidase D (CPD), as a model. CPD consists of three extracellular domains (A, B and C), of which only the membrane proximal C-domain is capable of DHBV-binding. In this project, we aim to crystallize the DHBV receptor carboxypeptidase D C-domain alone as well as in combination with the viral ligand to better understand the molecular mechanisms involved in the very early steps of hepadnaviral receptor interaction.
Irmgard Sinning (BZH, Heidelberg)
Project relevant Publications:
> Urban S, Breiner KM, Fehler F, Klingmüller U, Schaller H. Avian hepatitis B virus infection is initiated by the interaction of a distinct pre-S subdomain with the cellular receptor gp180. J Virol. 1998;72(10):8089-97.
> Urban S, Schwarz C, Marx UC, Zentgraf H, Schaller H, Multhaup G. Receptor recognition by a hepatitis B virus reveals a novel mode of high affinity virus-receptor interaction. EMBO J. 2000;19(6):1217-27.
> Gomis-rüth FX, Companys V, Qian Y, et al. Crystal structure of avian carboxypeptidase D domain II: a prototype for the regulatory metallocarboxypeptidase subfamily. EMBO J. 1999;18(21):5817-26.
3 The impact of signaling pathways on Hepatitis B and Hepatitis D virus infections
Summary: The entry of viruses into their target cells requires the coordinated interaction of viral and host factors. In favor infection, viruses must first bind to the cell surface, trigger internalization into the cytoplasm and transmit their genomes to sides of replication. The infection of hepatocytes with Hepatitis B virus (HBV) and Hepatitis D virus (HDV) follows a multi-step process, in which heparan sulfate proteoglycans (HSPGs) serve to bind and concentrate the viral particles on the cell surface (Schulze et al. 2007).
This nonspecific step is followed by the highly specific binding of HBV and HDV to their recently identified receptor, NTCP (Yan et al.2012, Ni et al. 2013). Virus receptor binding actively promotes entry by initiating conformational changes in the virus particle, by activating signaling pathways and promoting endocytic internalization (Marsh & Helenius 2006). Binding to NTCP triggers an endocytosis-mediated internalization depending on cavelin-1 (Macovei et al. 2010). Viruses use signaling activities to induce cellular changes that promote viral entry and early cytoplasmic events, as well as optimization of later processes in the replication cycle (Pelkmans et al 2005). This specific modification of cellular signaling events was first reported for adenoviruses which activate phosphatidyl-inositol 3-kinase (PI3K) through interaction of adenovirus pentons with cellular integrins (Nemerow and Stewart, 1999).
Based on the recently discovered HBV/HDV entry receptor NTCP and hence newly developed cell culture models, it is now possible to study the complete life cycle of both viruses in vitro. Thus, the analysis of cellular signaling pathways during the early entry steps of HBV and HDV is possible. We performed a kinase screen with clinically approved molecule inhibitors to characterize the requirement of signaling events for HBV and HDV infection, aiming to understand the prerequisites in signaling events for an effective infection. If applicable, we hope to define similarities and differences between HBV and HDV infections and to further break down which steps during the viral entry depend or require cellular signaling events. Finally, we are focusing on the identification of specific kinases as host factors, important for the establishment of infection and potential therapeutically targets.
Project relevant Publications:
> Schulze A, Gripon P, Urban S. Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans. Hepatology. 2007 Dec;46(6):1759-68.
> Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, Fu L, Song M, Chen P, Gao W, Ren B, Sun Y, Cai T, Feng X, Sui J, Li W. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife. 2012 Nov 13;1:e00049. doi: 10.7554/eLife.00049. Erratum in: Elife. 2014;3:e05570.
> Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Fälth M, Stindt J, Königer C, Nassal M, Kubitz R, Sültmann H, Urban S. Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Virus entry: open sesame. Cell. 2006 Feb 24;124(4):729-40. Marsh M1, Helenius A.
> Macovei A1, Radulescu C, Lazar C, Petrescu S, Durantel D, Dwek RA, Zitzmann N, Nichita NB. Hepatitis B virus requires intact caveolin-1 function for productive infection in HepaRG cells. J Virol. 2010 Jan;84(1):243-53. doi: 10.1128/JVI.01207-09.
> Pelkmans L1, Fava E, Grabner H, Hannus M, Habermann B, Krausz E, Zerial M. Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature. 2005 Jul 7;436(7047):78-86. Epub 2005 May 11.
> Nemerow GR, Stewart PL. Role of alpha(v) integrins in adenovirus cell entry and gene delivery. Microbiol Mol Biol Rev. 1999 Sep;63(3):725-34. Review.
4 Mechanisms of HBx-mediated regulation of cccDNA transcription
Summary: The human Hepatitis B Virus (HBV) replicates in its relaxed circular (rc) DNA genome that is converted into covalently closed circular (ccc) DNA. cccDNA, the template of gemonic RNAs, is generated from rcDNA-containing nucleocapsids, which are imported into the nucleus after de novo infection. It is still unclear how cccDNA elimination is accomplished in patients resolving acute HBV infection and how HBV clearance in chronic infections can be achieved in future curative therapies. Our previous work indicates that cccDNA-dependent viral gene expression depends on the regulatory HBV X protein (HBx). Taking advantage of long-term cell culture systems expressing NTCP receptor and specific real-time PCR quantification we will analyse the cccDNA kinetics in infected hepatocytes. We will further follow previous studies that demonstrated a crucial role of HBx on cccDNA-mediated transcription, by protein-protein interactions with host factors. These studies will answer key questions of HBV persistent infections and favor to the design of curative therapies.
Bingqian Qu, Yi Ni, Florian Lempp, Dr. Stephan Urban
Project relevant Publications:
> Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Fälth M, Stindt J, Königer C, Nassal M, Kubitz R, Sültmann H, Urban S. Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology. 2014 Apr;146(4):1070-83.
> Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M, Levrero M, Zoulim F, Hantz O, Protzer U. Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection. J Hepatol. 2011 Nov;55(5):996-1003.
> Belloni L, Pollicino T, De Nicola F, Guerrieri F, Raffa G, Fanciulli M, Raimondo G, Levrero M. Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19975-9.