Release of genotype 1 hepatitis E virus from cultured hepatoma and polarized intestinal cells depends on open reading frame 3 protein and requires an intact PXXP motif

Abstract

Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.

FIG. 1.

Infectious virus is released into the medium of cultured Caco-2 cells. One half of the medium from cells transfected with Sar55 genomes was collected on the days shown, and duplicate samples were plated on HepG2/C3A cells. Foci were detected by immunofluorescence microscopy with anti-ORF2 and manually counted. FFU, focus-forming unit.

FIG. 2.

ORF3 protein is needed for efficient release of genotype 1 HEV from cells. Intestinal (C25j) or hepatoma (S10-3) cells were transfected with Sar55 wild-type (WT) and mutant genomes. Real-time RT-PCR was performed to quantify viral genomes in the medium; points represent the mean from two independent, parallel cultures. GAD, nonreplicating polymerase mutant; DEL3, ORF3 null mutant; 5426T and 5426A, ORF3 point mutants.

FIG. 3.

Infection of rhesus macaques by 5426T ORF3 point mutant. Animals were transfected intrahepatically with in vitro transcripts of an HEV mutant. Serum samples were collected weekly, and virus genomes were quantified by HEV-specific real-time RT-PCR.

FIG. 4.

Representative confocal images taken along the z axis of transfected cells. ORF2 protein (green) and ORF3 protein (red) in a confluent monolayer of C25j cells (upper panel) and S10-3 cells (lower panel) on a transwell membrane are shown. Nuclei (blue ovals) and the transwell membrane (blue bottom layer) are stained with DAPI. The three wavelengths were collected separately and superimposed. All images were taken with a 63× oil objective. Viruses from left to right in each row are wild-type, 5426A, 5426T.

FIG. 5.

Iodixanol gradients of Sar55 HEV. Wild-type (WT) or ORF3 null mutant (DEL3) virions released into the medium of cultured C25j cells or into feces of an infected rhesus macaque were layered over preformed gradients and centrifuged to determine buoyant density. Hepatitis C virions were included as an internal marker. Fractions were weighed, and density is plotted as a dotted line; viral genomes were quantified by virus-specific RT-PCR. HCV values are not shown, but the HCV peak is indicated by an arrow. The buoyant density is shown above the HEV peak.

FIG. 6.

Immunoprecipitation of Sar55 in the absence (solid bars) or presence (open bars) of nonionic detergent. Equal amounts of Sar55 wild-type (WT) or ORF3 null mutant (DEL3) virions in the medium of cultured C25j cells (A and B) and virions shed into the feces of an infected macaque (C) were incubated with an irrelevant, anti-ORF2, or anti-ORF3 antibody, and immune complexes were captured on GammaBind beads. Virions bound to beads were quantified by real-time RT-PCR. WT, wild type. Values are the mean of triplicates with standard deviations shown as error bars.

FIG. 7.

Immunoprecipitation of Sar55 wild-type (WT) virions released into the medium of two different cell lines (A and B) or intracellular virions recovered by lysing cells (C). See the legend of Fig. 6 for details of immunoprecipitation.

FIG. 8.

Immunoprecipitation of semipurified intracellular virions from detergent-treated cell lysates. S10-3 cells were transfected with genomes of wild-type (WT), an ORF3 null mutant (DEL3), or an ORF3 point mutant (5426A or 5426T). See the legend of Fig. 6 for details of immunoprecipitation.