Cellular DNA repair in cccDNA formation during hepatitits B virus infection - addressing functionally redundant repair pathways
Final Report Abstract
The project was part of DFG Research Unit "Mechanisms of Persistence of Hepatotropic Viruses" aiming to decipher how distinct hepatotropic viruses, in particular hepatitis B virus (HBV) and hepatitis C virus (HCV), establish chronic infections in humans although they employ completely different replication strategies. HBV is a reverse transcribing DNA virus, HCV an RNA-to-RNA replicating plus-strand RNA virus. In either case has persistence two fundamental aspects: (i) the virus is able to maintain functional genomes despite the host response; (ii) the host defense is disabled to an extent that it cannot clear infection. The project focused on a particular form of the HBV genome, the stable covalently closed circular (ccc) DNA deposited in the host cell nucleus upon infection, from which all viral RNAs are transcribed. A cure of chronic hepatitis B would thus require complete elimination of cccDNA from the body, yet at project start little was known on cccDNA biology, except that the relaxed-circular (rc) DNA in incoming virions must be its precursor. Our molecular structure-driven biochemical approach identified human Tyrosyl-DNA-phosphodiesterase II (TDP2), but not TDP1, as able to release the covalently bound viral polymerase from HBV and DHBV rcDNA. Based on our finding that duck HBV (DHBV) generates much more cccDNA in human hepatoma cells than HBV we established "high-copy cccDNA" cell culture models, overcoming the problems of distinguishing cccDNA from the other viral DNA forms which has plagued the field . Stable RNAi knockdown of TDP2 slowed down, but did not ablate, cccDNA formation in DHBV-transfected human hepatoma cells.Similar results obtained in TDP2 knock-out cells excluded incomplete RNAi knock-down as an explanation; rather, other functionally redundant systems seem to take over. This is not surprising as deep redundancy in cellular DNA repair safeguards this vital system from failure of individual components. Polymerase removal from rcDNA is but one aspect of cccDNA formation which led us to propose a more general involvement of the DNA damage response. Several of the respective factors have meanwhile been reported by others to have a role in cccDNA formation, yet none appeared strictly essential, again in line with underlying redundancy. Combinatorial knock-out strategies may disentangle this complexity. Alternatively, we exploited the known impact of human adenoviruses (hAdV) on host DNA repair and found the early protein E4orf6 to strongly suppress rcDNA to cccDNA conversion. E4orf6 is known to associated with hAdV E1B55k and cellular factors including elongin B/C into cullin-based E3 ubiquitin ligases that induce proteasomal degradation of host proteins. Surprisingly, E4orf6 alone was sufficient for cccDNA suppression while an elongin interaction-deficient E4orf6 mutant was not. Likely then, E4orf6 can on its own form an active E3 ligase which induces degradation of (an) unknown cccDNA relevant host factor(s). To identify such factors stable hepatoma cell lines inducibly expressing wild-type or mutant E4orf6 have been established which will be analyzed by mass-spectrometry based comparative proteomics. We also contributed to the identification of the bile acid transporter NTCP as HBV receptor which enabled establishment of HBV infectable cell lines for addressing immunological and basic issues of HBV virology, as well as for validating DHBV- and cell line-derived data in an HBV infection setting. In addition, therapeutic implications for cccDNA by host factor targeting and cell division have been discussed.
Publications
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2010. Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner. PLoS Pathog 6:e1001082
Köck J, Rösler C, Zhang JJ, Blum HE, Nassal M, Thoma C
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2014. Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology 146:1070-83
Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Falth M, Stindt J, Königer C, Nassal M, Kubitz R, Sultmann H, Urban S
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2014. Involvement of the host DNA- repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses. Proc Natl Acad Sci USA 111:E4244-53
Königer C, Wingert I, Marsmann M, Rösler, C, Beck J, Nassal M
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2015. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut; epub
Nassal M
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2015. HBV-infected HepG2 hNTCP 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
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2017. A Role for the Host DNA Damage Response in Hepatitis B Virus ccDNA Formation-and Beyond? Viruses. 2017 May 22;9(5). pii: E125
Schreiner S, Nassal M
