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ORF3 protein of hepatitis E virus is essential for virion release from infected cells

Kentaro Yamada

Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi-Ken 329-0498, Japan

Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp

The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein remains unclear. To elucidate the role of the ORF3 protein in the virus life cycle, an infectious cDNA clone (pJE03-1760F/wt) that can replicate efficiently in PLC/PRF/5 and A549 cells and release progeny into the culture medium was used to generate a derivative ORF3-deficient (ORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The ORF3 mutant in the culture medium of mutant RNA-transfected PLC/PRF/5 cells was able to infect and replicate within PLC/PRF/5 and A549 cells as efficiently as the wild-type pJE03-1760F/wt virus. However, less than 1/100 of the number of progeny was detectable in the culture medium of ORF3 mutant-infected PLC/PRF/5 cells compared with wild-type-infected PLC/PRF/5 cells, and the HEV RNA level in the culture medium of ORF3 mutant-infected A549 cells was below or near the limit of detection. An immunocapture PCR assay revealed that the ORF3 protein is present on the surface of cell-culture-generated wild-type HEV but not on the ORF3 mutant. Wild-type HEV in the culture supernatant peaked at a sucrose density of 1.15–1.16 g ml^–1, in contrast with the ORF3 mutant in culture supernatant, which banded at 1.27–1.28 g ml^–1, similar to HEV in cell lysate and faecal HEV. These results suggest that the ORF3 protein is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which may be associated with lipids.

Present address: Division of Infectious Diseases, Department of Social and Environmental Medicine, Institute of Scientific Research, Oita University, Oita-Ken 879-5593, Japan.

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this study are AB437317 (pJE03-1760F/ORF3) and AB437318 (pJE03-1760F/fs).

Primer and probe sequences are available with the online version of this paper.