Fig. 7: Viral determinants for HEV replication in- and outside the HVR.

a Depicted is the Kernow-C1-p1 (p1) ORF1 encoding sequences with mutations that differentiate it from Kernow-C1-p6 (p6) ORF1. Additionally, the RPS17 insertion site is indicated on the genome. The mutations over the genome were cloned separately into p1, while the thirteen variants near the RPS17 (see Fig. 5b) were cloned together with the RPS17 RNA and were termed RPS17+flanking regions (RPS17/FR). b Replication kinetics were measured with p1 and p6 as reference (grey triangles) while constructs of interest are depicted in red. Plotted are mean relative light units (RLU) normalized to the four-hour value over time (hours post electroporation) of n = 3 biologically independent experiments, n = 6 for A220T and RPS17/FR constructs. c, f The 72-hour replication values were plotted as a column diagram. The left red/green area depicts the p1 replication, while the right red/green area depicts the p6 replication level. d The HEV full-length system was used to produce infectious particles with indicated constructs, which were titrated onto HepG2/C3A cells to determine viral titers as FFU/mL via immunofluorescence. A representative picture of a whole 96-well infected with non-enveloped HEVcc stained for the ORF2 protein (black) is shown above each column (mean, +/- SD). Dots represent individual data points of n = 5 individual experiments. e The RPS17 insertion alone or with a flanking region was cloned into the HEV3-83-2-27-Gluc replicon. Replication kinetics were measured with p1 and p6 as reference (grey triangles) while constructs of interest are depicted in green. Depicted are mean (+/- SD) relative light units (RLU) normalized to the four-hour value over time (hours post electroporation) of n = 3 biologically independent experiments for p1 and p6, n = 6 for other constructs. g The HEV full-length system was used to produce infectious particles with indicated constructs, which were titrated onto HepG2/C3A cells to determine viral titers as FFU/mL via immunofluorescence. A representative picture of a whole 96-well infected with non-enveloped HEVcc stained for the ORF2 protein (black) is shown above each column (mean, +/- SD). Dots represent individual data points of n = 3 individual experiments. Source data are provided in the Source Data file.