Project TP 6

(M. Nassal)

Cellular DNA repair in cccDNA formation during hepatitis B virus infection - addressing functionally redundant repair pathways

Acting as template for all viral transcripts, the covalently closed circular (ccc) DNA form of the hepatitis B virus (HBV) genome is crucial for persistence. None of the current drugs directly targets or even eradicates cccDNA; a few persisting cccDNA molecules can reactivate infection. Improving this situation requires cccDNA biogenesis to be understood. Currently, little is known on how the P protein- and RNA-linked relaxed circular (RC) DNA genome from virions is converted into cccDNA, except that this requires several distinct enzymatic steps. Shared features between RC-DNA and damaged cellular DNA (strand-breaks, non-DNA adducts) suggested that these activities are provided by the cell´s DNA repair machinery. Indeed, results obtained so far strongly imply tyrosyl-DNA-phosphodiesterase 2 (TDP2) as one cellular repair factor in cccDNA formation but they also suggest an involvement of mechanistically distinct alternative pathways. Such redundancy is a hallmark of cellular DNA repair. The overall aim of the project is to elucidate the molecular pathway of cccDNA biogenesis. Focusing on P protein removal from RC-DNA we obtained compelling biochemical evidence that TDP2, involved in cellular protein-DNA adduct repair, can perform this function. However, in-cell TDP2 knockdown by conventional RNAi techniques revealed only modest reductions in cccDNA. Such phenotypes are well known from cellular DNA repair where endonucleolytic pathways can substitute for TDP. Beyond final proof for a physiological role of TDP2, the proposal therefore aims at defining whether, and if so which, of these alternative pathways operate in cccDNA formation. Furthermore, nucleolysis is the only way to remove the RNA primer from RC-DNA, hence this step will be also be analyzed. As before, we will combine in vitro plus in-cell approaches to identify additional repair factors, in particular structure-specific endonucleases, that can act on RC-DNA and then evaluate the consequences of modulating their cellular levels on cccDNA formation. Numerous new tools including in vitro assays for repair factor activity and human cell lines producing well detectable cccDNA are now available. However, we also realized that the limited silencing efficiency of the RNAi techniques used so far hampered definite conclusions on whether a cell factor is decisive for, or contributing to, cccDNA biogenesis. Hence we will exploit advanced technologies, including inducibly shRNA expressing lentivirus vectors and genome-editing by designer nucleases, to increase knockdown efficiency or completely knockout individual repair factors. In case of factors where this approach is not applicable, small compounds and adenovirus early proteins will be used for inhibition. In combination, this repertoire of methods will enable inactivation of more than one repair factor at a time, and thus identify at least some of the key cell factors involved in hepadnaviral cccDNA biogenesis.
© Dep. of Infectious Diseases, Molecular Virology. University of Heidelberg
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