Mechanisms of Hepatitis B virus X-protein (HBx)- and interferon (IFN)-mediated regulation of viral transcription in authentic cell culture systems
Zusammenfassung der Projektergebnisse
Insufficient innate and adaptive immune responses are considered as a major reason for the failure to resolve an acute hepatitis B virus (HBV) infection. During escape of HBV from the innate immune surveillance in infected cells, both passive and active mechanisms are considered to be involved. Besides the impairment of the recognition through cellular pattern recognition receptors (PRRs) due to the creation of a physical barrier by reverse transcription of viral pregenomic (pg) RNA within fully assembled nucleocapsids, HBV might also use further strategies to counteract surveillance by the immune system. The understanding of these mechanisms on a molecular level will improve treatment strategies. By applying primary human hepatocytes (PHHs) and HepaRG cells, we addressed two major questions: (1) Does the regulatory protein HBx modulate transcription of viral RNAs and if so, what are the mechanisms underlying this process? (2) Which role does the Interferon (IFN) response play in the establishment and chronification of an HBV infection? In the first funding period, we showed that HBx is the viral key player for regulating the transcription of HBV transcription from its authentic template, the cccDNA, while the HBx-protein is not part of the viral particle and displays a strong and selective effect on cccDNA, but not on chromosomally integrated templates. Further, we detected HBx in the nucleus of hepatocytes when expressed from ist endogenous promoter and identified phosphorylation and acetylation sites. We established a lentiviral complementation assay, which enabled us to functionally map the essential domains, which are required for the HBx regulated HBV transcription. Regarding the effect of IFN on HBV replication, we found that both, type I and type III IFNs efficiently control HBV replication, predominantly at a late replication step. However, compared to other viruses, HBV infection does not induce robust IFN-responses which indicates a mechanism to counteract the sensors of infection. In the second funding period, we improved our molecular understanding of the mechanisms of HBx-mediated regulation of cccDNA-dependent gene expression, the identification of the IFN-sensitive steps in HBV-replication and potential surveillance mechanisms used by HBV to escape innate immune responses. We established (i) a lentiviral transduction system and functionally allocated HBx subdomains required to control cccDNA-dependent transcription, (ii) investigated the role of phosphorylation and acetylation and (iii) verified the role of DDB1 as an essential cellular component for HBx-mediated transcriptional control of HBV gene expression. Based on the lack of innate immune activation by HBV, we implemented the human Hepatitis D Virus (HDV) in our studies and found a strong activation of the host IFN-response upon HDV infection. This finding provided the basis for follow-up mechanistical studies investigating the crosstalk between HDV and the IFN response, which is further pursued in the subsequent TRR179
Projektbezogene Publikationen (Auswahl)
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2010. Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction. J Virol. 84: 1989-2000
Schulze A, Schieck A, Ni Y, Mier W, Urban S
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2010. The pre-S2 domain of the hepatitis B virus is dispensable for infectivity but serves a spacer function for L-protein-connected virus assembly. J Virol. 84: 3879-3888
Ni Y, Sonnabend J, Seitz S, Urban S
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2010. The replication cycle of hepatitis B virus. J Hepatol. 2010 Feb;52(2):282-4
Urban S, Schulze A, Dandri M, Petersen J
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2011. Human chimeric uPA mouse model to study hepatitis B and D virus interactions and preclinical drug evaluation. Hepatology. 55(3):685-94
Lütgehetmann M, Mancke LV, Volz T, Helbig M, Allweiss L, Bornscheuer T, Pollok JM, Lohse AW, Petersen J, Urban S, Dandri M
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2012. Hepatocyte polarization is essential for the productive entry of the hepatitis B virus. Hepatology 55:373-383
Schulze A. Mills K, Weiss, T. Urban S.
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2014. Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor.. J Hepatol. 60(4):723-31
Nkongolo S, Ni Y, Lempp FA, Kaufman C, Lindner T, Esser-Nobis K, Lohmann V, Mier W, Mehrle S, Urban S
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2014. Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes. Gastroenterology. 146(4):1070-1083
Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Faelth M, Stindt J, Koeniger C, Nassal M, Kubitz R, Sueltmann H, Urban S
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2015. Evidence that hepatitis B virus replication in mouse cells is limited by the lack of a host cell dependency factor.. J Hepatol. 64(3):556-64
Lempp FA, Mutz P, Lipps C, Wirth D, Bartenschlager R, Urban S
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2015. HBV-infected HepG2hNTCP cells serve as a novel immunological tool to analyze the antiviral efficacy of CD8+ T cells in vitro. J Virol. 89:7433-8
Hoh A, Heeg M, Ni Y, Schuch A, Binder B, Hennecke N, Blum HE, Nassal M, Protzer U, Hofmann M, Urban S, Thimme R
