Project Details
Projekt
Mechanisms of Hepatitis B virus X-protein (HBx)- and interferon (IFN)-mediated regulation of viral transcription in authentic cell culture systems
Applicant
Professor Dr. Stephan Urban
Subject Area
Virology
Term
from 2009 to 2017
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 99161398
The failure to resolve an acute HBV infection can be regarded as the consequence of insufficient innate and adaptive immune responses resulting in lifelong persistence of a tightly regulated replication competent cccDNA template in hepatocytes. To escape innate immune surveillance of HBV in infected cells, passive and active mechanisms are supposed to be involved. Reverse transcription of viral pregenomic (pg)RNA within fully assembled nucleocapsids creates a physical barrier for the recognition by cellular pattern recognition receptors (PRRs). In addition although minimalistic in its genetic repertoire, HBV might use strategies to counteract surveillance by the immune system. Understanding of such mechanisms in molecular terms will have profound implications for more successful treatment strategies. We applied primary human hepatocytes (PHHs) and HepaRG cells to investigate two questions related to the control of HBV infection: (1) Does the regulatory protein HBx modulate transcription of viral RNAs and if so, what is the mechanism underlying this process? (2) Which role does the Interferon (IFN) response play in the establishment and chronification of HBV infection? We showed that HBx is the viral key player for regulating HBV transcription from its authentic template, the cccDNA. HBx-protein is not part of the viral particle and displays a strong and selective effect on histone-associated cccDNA, but not on artificial templates. Through biochemical analyses we detected HBx expressed from its endogenous promoter in the nucleus of hepatocytes and identified phosphorylation and acetylation sites. We established a lentiviral complementation assay, which will allow the functional mapping of the essential domains required for HBxregulated 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. Compared to other viruses, HBV infection does not induce robust IFN-responses arguing for a mechanism to counteract the sensors of infection. The overall goal of this follow up application is the molecular understanding of the mechanism of HBx-mediated regulation of cccDNA-dependent gene expression, identification of the IFNsensitive steps in HBV-replication and the analysis of potential surveillance mechanisms used by HBV to escape innate immune responses. Particularly we will (i) use the previously established lentiviral transduction system to functionally allocate HBx subdomains required to control cccDNA-dependent transcription, (ii) investigate the role of phosphorylation and acetylation, (iii) attempt to identify the participating cellular enzymes (iv) assess the pathway(s) involved in shutting down HBx-dependent HBV gene transcription (v) analyze which of the late steps in HBV replication is affected by the IFN-response, (vi) investigate how - contrariwise- HBV might manage to circumvent the induction of robust IFN-responses and finally, resolve the potential connection of HBx-and IFN-pathways. The employment of the most authentic infection systems will provide us with the most relevant molecular details of the early steps in the establishment of persistent HBV infection.
DFG Programme
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