379 ULTRA-DEEP PYROSEQUENCING OF BASIC CORE PROMOTER, PRECORE AND CORE REGIONS OF HEPATITIS B VIRUS TO STUDY VIRAL VARIABILITY AND QUASISPECIES COMPLEXITY

Abstract

Methods: HepaRG and PHH were either infected with HBV or transfected with either purified viral nucleocapsid or recombinant HBc protein. Moreover engineered HepaRG cell lines, which express HBV proteins, were also used to analyse the role of individual proteins. Ligands of PRRs were used to engage innate receptor and analyse the inhibitory effect of HBV proteins. HBV replication was analysed with standard procedures, whereas the effect of viral proteins on the expression of various interferons (a, b, l. . . ), interferon-induced (ISG) and pro-inflammatory cytokines genes was analysed by RT-qPCR, WB and ELISA. ChIP experiments were also performed to analyse the binding of HBc to target promoters as well as to analyse the recruitment of epigenome-modifying enzymes to target promoters. Results: HBV is capable to inhibit dsRNA-mediated interferon response after only few hours of infection. This inhibition occurs also with Dane-depleted and UV-inactivated virus suggesting that neo-synthesis of HBV protein is not mandatory. Using engineered cell lines we demonstrate that HBc is responsible for this very early inhibition. The transfection of purified nucleocapsid or recombinant HBc leads to the same inhibitory phenotype. HBc needs to be located in the nucleus to inhibit the transcription of targeted genes (i.e. IFNs, ISG), as inhibition of its trafficking, revert the phenotype. ChIP analyses with anti-HBc revealed that HBc is capable to bind to target promoters and to recruit epigenome-modifying enzymes to establish a negative mark on target promoters. Conclusion: Our results demonstrate that HBc is responsible for the very early inhibition of IFN response by HBV. The precocity of this inhibition is instrumental for the establishment of a persistent infection in vitro and is possible because HBc from incoming virions is capable to inhibit gene expression in the nucleus of infected cells by either direct interference with transcriptional machinery or by recruiting epigenome-modifying enzymes leading to repressive marks.