FIG 1.

ORF3 is required for efficient HEV growth in polarized human hepatocytes. (A) Western blots of HEV ORF2 and ORF3 proteins in HepG2/C3A cells 5 days after transfection with wild-type (WT) or ORF3null HEV RNA. β-Actin served as the loading control. (B) Specific infectivity of WT and ORF3null virions recovered from HEV RNA-transfected cells. HEV genome equivalents (GE) were determined by qRT-PCR. ORF2-positive focus-forming units (FFU) were determined in F2 cells (a subclone of HepG2/C3A cells) by immunofluorescence assays at 5 days postinoculation. n.s., not significant. (C and D) Transepithelial electrical resistance (TEER) (C) and permeability (D) of F2 or 293T cells grown on a porous membrane (pore size, 3 μm) for 18 days. Data are shown as means ± standard errors of the means (SEM) from 2 independent experiments, each with duplicate wells. (E) Polarized F2 cells were inoculated with WT or ORF3null HEV (10³ GE per cell). Lactate dehydrogenase (LDH) levels in the basolateral culture medium were measured at 5 and 10 days postinoculation (dpi). Data are shown as means ± SEM from triplicate wells. (F) Immunofluorescence images showing HEV ORF2-positive cells (green) in polarized F2 cell cultures 5 and 10 days after infection with WT or ORF3null HEV (10³ GE per cell) in either the presence or absence of a JAK inhibitor (JAKi) (0.5 μM). Nuclei were counterstained with DAPI. Bars, 100 μm. (G) HEV-positive areas at 5 and 10 dpi were quantified using ImageJ software (NIH). Data are shown as means ± SEM from at least 2 independent experiments, each with duplicate wells. (H and I) Polarized F2 cells were inoculated with WT or ORF3null HEV (10³ GE per cell), and the levels of HEV RNA (H) and interferon-stimulated genes (ISGs) (I) were quantified by qRT-PCR at the indicated times. Data are shown as means ± SEM from 2 independent experiments, each with duplicate wells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.