Tanggis, Takahashi M, Kobayashi T, Okamoto H, Nishiyama T, Nishizawa T, Nagashima S, Primadharsini PP
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 culture supernatant of HEV-infected PLC/PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatant 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 a 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 native PLC/PRF/5 cells, but were not neutralized by an anti-HEV ORF2 mAb, which can efficiently neutralize the non-enveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are co-purified 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, similarly 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 non-enveloped, while those in circulating blood and culture supernatant 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 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.
