title: Genetic code expansion as a tool for the  visualisation of Hepatitis B and Delta viruses
creator: Zehnder, Benno
description: Hepatitis B virus (HBV) and hepatitis delta virus (HDV) are widespread human pathogens that cause acute and chronic infections of the liver associated with inflammation, cirrhosis, and hepatocellular carcinoma. Despite the medical burden caused by HBV and HDV, therapeutic options are limited, and curative therapy is not available yet. The identification of the bile acid transporter sodium taurocholate cotransporting polypeptide (NTCP) as functional receptor for both viruses has provided new opportunities to study virus and host factors involved in virus entry and maintenance of viral persistence in vitro. The present study aimed to investigate the contribution of de novo synthesised HBV core protein (HBc) to the maintenance of HBV persistence, and to generate fluorescently labelled HBV and HDV for the study of viral entry and trafficking.  We engineered an infectious HBV mutant with a genome encoding a stop codon in the HBc ORF (ΔHBc HBV), which could not initiate nucleocapsid production upon infection. In vitro infection with wild-type (WT) and ΔHBc HBV resulted in comparable numbers of covalently closed circular (ccc)DNA, which is the transcriptionally active template for all viral transcripts and is the determinant factor of HBV persistence. During long-term infection in HepG2-NTCP cells, cccDNA levels, transcription of viral RNA, and secretion of viral proteins remained stable in WT and ΔHBc HBV-infected cells. These findings suggested that HBV cccDNA remained stable without requiring reimport of newly synthesised nucleocapsids in vitro. Furthermore, lack of de novo produced HBc did not affect the transcriptional activity of cccDNA.  The generation of fluorescently labelled HBV and HDV has been hampered by the complex genome organisation of both viruses, structural restriction of these small virions, and the large excess of non-infectious subviral particles. For this study, the amber suppression technology was chosen as labelling strategy. This method expands the genetic code by site-specific incorporation of non-canonical amino acids (ncAA) in response to amber stop codons. Combined with click chemistry fluorophores, this is currently the smallest fluorescent tag that can be genetically encoded into proteins. Amber stop codons were inserted at 11 positions in the HBc ORF, and the incorporation of ncAA in response to these stop codons could rescue the expression of full-length HBc. Secretion of infectious HBV particles was observed after genetic encoding of N-Propargyl-L-lysine in HBc, which enabled copper-catalysed click labelling and visualisation of incoming nucleocapsids during in vitro infection.  For the fluorescent labelling of HDV, we capitalised on the naturally present amber stop codon which terminates translation of the small hepatitis delta antigen (S-HDAg). During HDV replication, this amber stop codon is edited to a tryptophan codon, resulting in expression of a large HDAg (L-HDAg). Amber suppression mediated incorporation of ncAA at the amber stop could induce the production of L-HDAg with ncAA instead of tryptophan. Interestingly, the early expression of L-HDAg increased secretion and infectivity of HDV particles. L-HDAg could be labelled with a click chemistry functionalised fluorophore in transfected cells and in purified virions.  This study highlights the persistence of HBV cccDNA in infected cells without requiring continuous replenishment by nucleocapsid reimport, suggesting that therapeutic targeting of capsid reimport is likely not sufficient to eliminate cccDNA. Furthermore, genetic code expansion mediated labelling of HBV and HDV provides a tool to visualise viral entry processes.
date: 2021
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/30712/1/ThesisBennoZehnderForBib.pdf
identifier: DOI:10.11588/heidok.00030712
identifier: urn:nbn:de:bsz:16-heidok-307124
identifier:   Zehnder, Benno  (2021) Genetic code expansion as a tool for the visualisation of Hepatitis B and Delta viruses.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/30712/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng