Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway

Abstract

Our previous studies demonstrated that membrane-associated hepatitis E virus (HEV) particles-now considered "quasi-enveloped particles"-are present in the multivesicular body with intraluminal vesicles (exosomes) in infected cells and that the release of HEV virions is related to the exosomal pathway. In this study, we characterized exosomes purified from the culture supernatants of HEV-infected PLC/PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatants of HEV-infected cells that had been cultivated in serum-free medium were found to contain HEV RNA and the viral capsid (ORF2) and ORF3 proteins, as determined by reverse transcription-PCR (RT-PCR) and Western blotting, respectively. Furthermore, immunoelectron microscopy, with or without prior detergent and protease treatment, revealed the presence of virus-like particles in the exosome fraction. These particles were 39.6 ± 1.0 nm in diameter and were covered with a lipid membrane. After treatment with detergent and protease, the diameter of these virus-like particles was 26.9 ± 0.9 nm, and the treated particles became accessible with an anti-HEV ORF2 monoclonal antibody (MAb). The HEV particles in the exosome fraction were capable of infecting naive PLC/PRF/5 cells but were not neutralized by an anti-HEV ORF2 MAb which efficiently neutralizes nonenveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are copurified with the exosomes in the exosome fraction and suggest that the capsids of HEV particles are individually covered by lipid membranes resembling those of exosomes, similar to enveloped viruses.IMPORTANCE Hepatitis E, caused by HEV, is an important infectious disease that is spreading worldwide. HEV infection can cause acute or fulminant hepatitis and can become chronic in immunocompromised hosts, including patients after organ transplantation. The HEV particles present in feces and bile are nonenveloped, while those in circulating blood and culture supernatants are covered with a cellular membrane, similar to enveloped viruses. Furthermore, these membrane-associated and -unassociated HEV particles can be propagated in cultured cells. The significance of our research is that the capsids of HEV particles are individually covered by a lipid membrane that resembles the membrane of exosomes, similar to enveloped viruses, and are released from infected cells via the exosomal pathway. These data will help to elucidate the entry mechanisms and receptors for HEV infection in the future. This is the first report to characterize the detailed morphological features of membrane-associated HEV particles.

Keywords: cell culture; cell membranes; electron microscopy; exosome; hepatitis E virus; morphology; multivesicular body.

FIG 1

Exosomes purified from culture supernatants of HEV-infected PLC/PRF/5 cells harbor viral proteins and RNA. (A) Western blotting of CD63-, CD9-, or CD81-positive exosomes purified from the culture supernatant of HEV-infected or uninfected PLC/PRF/5 cells. The cells were incubated in serum-free medium, and the exosomes were purified from culture supernatants by ultracentrifugation and then immunoprecipitated (IP) with anti-CD63, anti-CD9, or anti-CD81 antibody. The precipitated exosomes were subjected to Western blotting with anti-CD63, anti-CD9, or anti-CD81 antibody (upper panels). Viral ORF2 and ORF3 proteins were detected with an anti-ORF2 MAb (middle panels) and an anti-ORF3 MAb (lower panels), respectively. (B) Amplification of HEV RNA from exosomes purified from the culture supernatant of HEV-infected or uninfected cells by long-distance RT-PCR. (C) Quantitative RT-PCR of HEV RNA in exosomes purified from the culture supernatant of HEV-infected cells. The culture supernatant of HEV-infected cells, containing 6.0 × 10⁸ copies of HEV RNA (input), was ultracentrifuged under the conditions used for exosome purification. A total of 4.2 × 10⁸ copies of HEV RNA were recovered in the exosome fraction by ultracentrifugation. The data are presented as means ± SD for two independent experiments.

FIG 2

Transmission electron microscopic images of exosomes purified from the culture supernatant of uninfected (A) or HEV-infected (B) PLC/PRF/5 cells, with negative staining. Bars, 100 nm. (C) High-magnification image of virus-like particles found in the exosome fraction purified from the culture supernatant of HEV-infected cells. Bars, 50 nm. The results of one of three experiments are shown.

FIG 3

Sucrose density gradient fractionation of exosomes purified from the culture supernatant of HEV-infected cells with or without prior treatment with 1.5% digitonin.

FIG 4

Transmission electron microscopic images of negatively stained eHEV or neHEV particles. (A) neHEV particles generated from eHEV particles in culture supernatant treated with DOC-Na and trypsin, shown at two different magnifications. (B) Immunogold labeling with a mouse anti-ORF2 MAb (H6225; IgG), shown at two different magnifications. neHEV particles were labeled with the anti-ORF2 MAb and 12-nm colloidal gold-conjugated goat anti-mouse IgG. (C) Immunogold labeling with a mouse anti-ORF3 MAb (TA0536; IgG), shown at two different magnifications. neHEV particles were incubated with the anti-ORF3 MAb and a 12-nm colloidal gold-conjugated goat anti-mouse IgG. (D) Virus-like particles in purified exosomes without (upper panel) or with (lower panel) prior treatment with detergent and protease. (E) Immunogold labeling with anti-ORF2 MAb (H6225), shown at two different magnifications. eHEV particles treated with detergent and protease were labeled with the anti-ORF2 MAb and a 12-nm colloidal gold-conjugated goat anti-mouse IgG. (F) Immunogold labeling with anti-ORF3 MAb (TA0536), shown at two different magnifications. eHEV particles treated with digitonin were labeled with the anti-ORF3 MAb and a 12-nm colloidal gold-conjugated goat anti-mouse IgG. Bars, 50 nm. Results representative of one of three experiments are shown.

FIG 5

Transmission electron microscopic images of negatively stained genotype 3 (A to D) or 4 (E to G) eHEV and neHEV particles. (A) Genotype 3 eHEV particles in the exosome fraction (open arrows) and neHEV particles treated with detergent and protease (closed allows) were mixed and then observed by TEM at two different magnifications. (B) Immunogold labeling with anti-ORF2 MAb (H6225). Genotype 3 neHEV and eHEV particles were mixed and then labeled with the anti-ORF2 MAb and a 12-nm colloidal gold-conjugated goat anti-mouse IgG. (C) Immunogold labeling with anti-ORF3 MAb (TA0536). neHEV particles were not labeled with the anti-ORF3 MAb and a 12-nm colloidal gold-conjugated goat anti-mouse IgG. (D) Immunogold labeling with mouse MAb TA1708 (IgM) against eHEV particles. The purified exosomes separated from the culture supernatants of HEV-infected cells were labeled with MAb TA1708 and a 12-nm colloidal gold-conjugated goat anti-mouse IgM. (E) Genotype 4 eHEV particles in the exosome fraction, shown at two different magnifications. (F) eHEV particles in the exosome fraction (open arrows) and neHEV particles treated with detergent and protease (closed allows) were mixed and then observed by TEM at two different magnifications. (G) neHEV particles generated from genotype 4 eHEV particles in culture supernatant treated with DOC-Na and trypsin, shown at two different magnifications. Bars, 50 nm. Results representative of one of three experiments are shown.

FIG 6

Inoculation of exosomes purified from culture supernatants of HEV-infected PLC/PRF/5 cells by ultracentrifugation. (A) Quantitation of HEV RNA in culture supernatants of PLC/PRF/5 cells that were inoculated with purified exosomes or in culture supernatants from primary infection for cells cultured for up to 20 days. The data presented are means ± SD for three wells each. (B) Immunofluorescence staining of PLC/PRF/5 cells inoculated with exosomes purified from culture supernatants of HEV-infected cells. At 10 days postinoculation, the cells were incubated with an anti-ORF2 MAb (H6225) (upper panels) or anti-ORF3 MAb (TA0536) (lower panels) and then stained with Alexa Fluor 488-conjugated anti-mouse IgG. The nuclei were stained with DAPI. (C) Expression of viral proteins in culture supernatants of cells inoculated with exosomes purified from HEV-infected or uninfected cells. At 16 days postinoculation, ORF2 or ORF3 protein in the culture supernatant was detected by Western blotting with an anti-ORF2 MAb (H6210) (left) or anti-ORF3 MAb (TA0536) (right), respectively.

FIG 7

Quantitation of HEV RNA in the culture supernatant of PLC/PRF/5 cells inoculated with neHEV particles (A) or eHEV particles included in the exosome fraction (B) that had been mixed with anti-ORF2 MAb (H6225; 1 mg/ml) or a negative-control MAb (905; 1 mg/ml) and cultured for up to 28 days. The data presented are means ± SD for three wells each.