The hepatitis E virus capsid protein ORF2 counteracts cell-intrinsic antiviral responses to enable persistence in hepatocytes

We used scRNA-seq to study the cell-intrinsic antiviral response to hepatitis E virus infection in the immunocompetent hepatoma cell line HepG2/C3A. In this study, we identified a replication-limiting bottleneck at approximately 56 h post-infection at which the presence of the viral capsid protein ORF2 is decisive for establishment of an equilibrium between viral replication and the antiviral response. ORF2 antagonizes antiviral signaling downstream of pattern recognition receptors, at least partly through direct interaction with the central adaptor protein TBK1. By scRNA-seq, we confirmed that ORF2 dampens the antiviral response directly within infected cells, thereby also affecting the antiviral response in uninfected bystanders. In the presence of ORF2, the antiviral response was globally dampened at the early, replication-limiting bottleneck. This further substantiated that the equlibirium between viral replication and the antiviral response is a crucial mechanism of HEV persistence in hepatocytes. Overall design: We infected HepG2/C3A cells with HEV-3 Kernow/C1-p6 wild-type (WT) virus particles or derived ?ORF2 virus particles, which do not encode the ORF2 protein and were produced by trans-complementation. Virus particles were bound at 4 °C for 2 h, internalized for 8 h, and harvested for scRNA-seq analysis by microfluidics-based 3'-targeted 10x Genomics 56 h and 7 days post-internalization. Uninfected samples and WT infection were analyzed at both time points, while ?ORF2 infection was only analyzed at 56 h post-internalization.