%0 Generic
%A Mehnert, Ann-Kathrin
%C Heidelberg
%D 2025
%F heidok:37088
%R 10.11588/heidok.00037088
%T Dissecting Antiviral Immune Evasion by the Hepatitis E Virus ORF2 Protein in Persistent Hepatocyte Infection
%U https://archiv.ub.uni-heidelberg.de/volltextserver/37088/
%X Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide. While genotype 1 (HEV 1) and HEV-2 exclusively result in acute infections, HEV-3 and HEV-4 infections are at a high risk of becoming chronic in immunocompromised individuals. HEV is a single-stranded RNA virus encoding three viral proteins: the viral replicase, ORF1, the capsid protein, ORF2, and ORF3, a protein essential for virion release. In hepatocytes, HEV induces a cell-intrinsic antiviral response through the expression of type III interferons (IFNs) and IFN-stimulated genes (ISGs). HEV replication can persist despite this sustained antiviral signaling, suggesting the presence of immune evasion strategies. Even though all viral proteins have been proposed to antagonize antiviral and inflammatory signaling pathways, their contributions to persistent HEV replication remain unclear. To identify the determinants of HEV persistence, I aimed to perform a comprehensive characterization of the HEV-induced cell-intrinsic antiviral response across different hepatocellular systems, in bulk and at the single-cell level.     First, I evaluated the integrity of the relevant antiviral and inflammatory signaling pathways in the hepatoma cell line HepG2/C3A and pluripotent stem cell-derived hepatocyte-like cells (HLCs). I further sought to identify the pattern recognition receptors (PRRs) specifically contributing to HEV sensing. Then, I aimed to assess the viral antagonisms mediated by the HEV proteins ORF2 and ORF3. In a comprehensive side-by-side comparison, I found that ORF2 from HEV-3 and HEV-1, rather than ORF3, interferes with antiviral and inflammatory signaling downstream of PRRs. By co-immunoprecipitation, I demonstrated that ORF2 directly interacts with TANK binding kinase 1 (TBK1), a central hub of antiviral signaling, through an unidentified interaction motif. To clarify the impact of the ORF2-mediated antagonism in the context of full-length HEV infection, I exploited HEV 3 mutants lacking expression of ORF2 (ΔORF2) or ORF3 (ΔORF3). Electroporation with ΔORF2 RNA and infection with trans-complemented ΔORF2 virus particles in HepG2/C3A cells and HLCs resulted in significantly impaired viral replication. This was a direct consequence of the increased expression of antiviral response genes due to the absent TBK1 inhibition, which is mediated by intracellular ORF2. Using spatial RNA fluorescence in situ hybridization and single-cell RNA-sequencing, I demonstrated that both actively infected cells and uninfected bystanders are the sources of the ISG response in HEV infection. In both cell types, a similar ISG subset was induced, which was globally enhanced in the absence of the ORF2 protein. These findings emphasized the persistence of HEV replication in a directly antiviral environment. Moreover, I observed that ΔORF2 replication is more vulnerable to the effectors of the antiviral response, revealing an additional and hitherto unrecognized, protective function of ORF2. Using a synchronized infection approach, I found that ORF2 drives the establishment of a balance between HEV replication and the antiviral response, following a replication-limiting bottleneck early in infection. I concluded that the various identified strategies of antiviral immune evasion mediated by ORF2 are essential for enabling persistent HEV replication in the presence of a sustained yet dampened IFN and ISG response.     The results obtained during the course of my PhD, which are presented in this dissertation, contribute to elucidating the multifaceted functions of the capsid protein ORF2 within the HEV life cycle. They further identify the antiviral immune evasion strategies mediated by ORF2 as central determinants for persistent HEV replication in hepatocytes. My findings thus provide a foundation for exploring the crosstalk between HEV-infected hepatocytes and professional immune cells in the future, and for the investigation of intergenotypic differences in the antiviral response. Ultimately, these studies will provide novel insights into decisive factors for the pathogenesis of acute and chronic manifestations of HEV infection.