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Interactions of HBV capsid involved in nuclear transport

Abstract : The Hepatitis B Virus (HBV) is an enveloped virus containing a partially double stranded DNA genome (rcDNA). HBV causes acute and chronic infections. HBV is not cytotoxic but chronic inflammation leads to liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV replicates via an RNA intermediate, which is transcribed from a covalently closed circular form of the viral DNA (cccDNA). This pregenomic RNA is specifically encapsidated into the capsid by interaction with the viral polymerase, which also interacts with the core protein (Cp), forming the capsid. The polymerase retrotranscribes the pregenomic RNA into single stranded DNA and subsequently partially double stranded DNA resulting in mature capsids (MatC). Cp is an 185 aa long polypeptide comprising a N-terminal assembly domain, and a flexible C-terminal domain (CTD). The CTD includes two overlapping nuclear localization signals (NLS) of eight aa and an Importin ß Binding Domain (IBB) of 34 aa. The CTD is fixed in the interior of the capsid by interacting with single stranded nucleic acids but translocates to the exterior in MatC and empty capsids (EmpC). Cp is over expressed leading to assembly of EmpC. The virus has to deliver its genome into the nucleus of infected cells for replication. Nuclear transport is mediated by the capsid that interacts with nuclear import receptors. The group has recently shown that MatC need either both, importin  (Imp.) and importin ß (Imp.ß), or Imp.ß alone for transport of the capsids into the nuclear basket. In this structure where genome liberation likely occurs, the transport of the capsid is arrested by interaction between the capsid and the nucleoporin Nup153. In the thesis we demonstrate that MatC binds to Imp.α but not Imp.ß, suggesting that only the part of the CTD, which contains the NLSs is exposed on capsids’ surface. In collaboration with the Adam Zlotnick (Indiana University, U.S.A.) we showed that EmpC, in contrast, bind Imp.β directly without Imp.α acting as an adaptor. This interaction, which is stronger than the one of Imp. occurs needs IBB exposure, meaning that the entire CTD becomes externalized. Furthermore, exposure to very high Imp.ß concentration led to EmpC destabilization. The genome release within the nuclear basket implies that Nup153 is involved in genome liberation from MatC. To verify this hypothesis we used MatC with a radioactively labeled genome, which were exposed to the capsid binding-Nup153 fragment. Investigating the accessibility of the genome to nucleases we found that the Nup153 fragment had no impact on capsids stability, suggesting the need of cellular factors driving disassembly. This conclusion is in agreement with our observation that MatC added to isolated nuclei resulted in nuclear capsid entry, which requires disassembly. To further study the disassembly step and the consequent release of the viral genome, we developed a system to directly visualize the viral genome allowing investigations of genome uncoating in real time. The system is based on the cooperative binding of a fluorescent fusion protein between the bacterial protein OR with GFP to a double stranded DNA sequence called Anch. Using this model we showed that infection of OR-GFP-expressing hepatoma cells with HBV containing a modified Anch genome allowed monitoring genome release into the nucleus. In future, this system may help identifying factors involved in genome release and repair and to decipher their molecular interactions.
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Lara Gallucci. Interactions of HBV capsid involved in nuclear transport. Human health and pathology. Université de Bordeaux, 2018. English. ⟨NNT : 2018BORD0130⟩. ⟨tel-01993716⟩

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