Untersuchungen zur Proteom-basierten Aufklärung einer durch Adenovirus 5 E4orf6 Protein vermittelten Interferenz mit der Hepadnavirus Replikation

Abstract: Hepatitis B virus (HBV), the prototypic hepadnavirus, can establish chronic infections of the liver as a result of viral genomes that persist in the cell nucleus as covalently closed circular (ccc) DNA molecules. They serve as template for all viral transcripts and thus in consequence for new virions. Until now therapeutic approaches can rarely eliminate the cccDNA, often requiring lifelong medication. The hepadnaviral genome in infectious virions is a relaxed circular (rc)DNA that is converted to cccDNA in the host cell nucleus. The mechanism is poorly understood but accumulating evidence suggests an essential role for factors of the cellular DNA damage response (DDR). Most viruses either exploit or avoid the DDR for their own replication. Adenoviruses such as human adenovirus 5 (hAdV5) are amongst the best-known examples. The hAdV5 early gene products E4orf6 (E4wt) and E1B55k cooperate to induce the proteasomal degradation of several DDR components, counteracting a DDR-mediated replication interference. In preliminary studies with human hepatoma cells (HepG2) coexpression of the two hAdV5 early proteins strongly interfered with replication and cccDNA formation of duck hepatitis B virus (DHBV) which accumulates much higher cccDNA copy numbers per cell compared to HBV and thus serves as a preferred model system. Unexpectedly, DHBV interference was already seen with E4wt alone whereas the known anti-DDR effects of hAdV5 require E1B55k as well. There, E4wt associates with cellular proteins Cullin 5 (Cul5), RING-box protein 1 (RBX1) and Elongins B and C (EloB, EloC) into a functional E3-ubiquitin ligase whose target specificity is determined by E1B55k. We therefore hypothesized that E4wt alone guides at least one unknown hepadnavirus-relevant host factor into proteasomal degradation. To identify appropriate candidates a mass-spectrometric (MS) proteome comparison was performed (lab of Prof. Dr. A. Pichlmair, TU Munich) between newly established HepG2 cell lines which inducibly express either E4wt protein or an E3-ubiquitin ligase-deficient variant (BCmut). Generation of the stable HepG2 cell lines was accomplished by either CRISPR/Cas9-mediated site-specific integration of appropriate expression cassettes into the chromosomal AAVS1 locus, or by way of the stable episomal vector pMEP4. Higher level expression in the pMEP4 cell lines allowed to confirm functionality of the E4wt protein via its ability to complement infection by an E4orf6-deficient hAdV5. In addition, the pMEP4-encoded E4wt protein formed a functional Cul5 E3-ubiquitin ligase which in the presence of E1B55k induced degradation of the known target proteins Mre11 and the tumor suppressor p53, accompanied by an E4orf6 plus E1B55k dependent Neddylation of Cul5. In line with the preliminary results DHBV replication and cccDNA levels were markedly reduced in the pMEP4-E4wt cell lines. The proteome comparison between the pMEP4-E4wt and pMEP4-BCmut cell lines revealed over 100 proteins that were significantly up- or downregulated in the E4wt expressing cells. For most of them a direct connection to DDR was not obvious. An exception was the replication protein A (RPA), a complex whose subunits RPA1, RPA2 and RPA3 were all significantly reduced in E4wt expressing cells. As the main single-stranded DNA binding protein in eukaryotic cells RPA stabilizes single-stranded DNA intermediates, especially during DNA repair processes. The potential relevance of these and the other candidate factor for hepadnaviral replication was to be tested by knockdown (KD) via RNA interference and/or knockout (KO) via CRSIPR/Cas-meditated genome editing. To this end, the TetOFF reporter cell lines DHA2/3 and DHA_Cas9 (containing an integrated SpCas9 expression cassette in the AAVS1 locus) were constructed. Upon induction of DHBV replication the cells secrete an HA-tagged eAg (HAeAg) in a cccDNA dependent manner. Complemented by direct rcDNA and cccDNA detection via Southern blotting, this enables the non-invasive monitoring of the slow (1- to 2 weeks) kinetics of cccDNA formation and the impact of host-factor KD or KO events on the process. For efficient generation of appropriate effector RNAs in the reporter cell lines, shRNA and gRNA encoding recombinant adenovirus-associated virus (rAAV) vectors and lentiviral vectors (LV) were compared. rAAV-mediated shRNA expression resulted in an only transient KD for a few days, whereby these vectors appear more suited for CRISPR/Cas genome editing. The integrating LVs, in contrast, induced a long-term shRNA KD. In this way, for 4 of the 11 preselected candidates from the proteome-derived short-list, i.e. ANP32A, HSP27, RPA1 und TAGLN2, a role in hepadnaviral replication could be made likely by decelerated and/or diminished HAeAg secretion and altered cccDNA formation kinetics. Altogether this thesis established a set of new test systems and tools for the identification of new host factors with relevance for the hepadnaviral replication cycle. Evidence for four such factors has already been obtained, while the new reporter cell lines should greatly facilitate more detailed, including mechanistic studies, of additional factors. This should shed new light on the hepadnavirus-host interaction and contribute to a better understanding of cccDNA formation and maintenance as well as to new therapeutic approaches

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Deutsch
• Anmerkungen: Universität Freiburg, Dissertation, 2020

Klassifikation und Themen

• Schlagwort: Replikation ; Hepatitis-B-Virus ; Viren ; Proteine ; Adenovirus 5 ; Zelle ; Hepatitis-B-Virus

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2020
• Urheber: Miller, Julija
• Beteiligte Personen und Organisationen: Nassal, Michael ; Albert-Ludwigs-Universität Freiburg. Medizinische Fakultät

Weitere Informationen

• DOI: 10.6094/UNIFR/167228
• URN: urn:nbn:de:bsz:25-freidok-1672285
• Rechteinformation: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 25.03.2025, 13:45 MEZ