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Published online ahead of print on 1 April 2009 as doi:10.1099/vir.0.010561-0
Journal of General Virology 2009;90:1880.

A more recent version of this article appeared on August 1, 2009 J Gen Virol (2009), DOI 10.1099/vir.0.010561-0
© 2009 Society for General Microbiology
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ORF3 protein of hepatitis E virus is essential for virion release from infected cells

Kentaro Yamada1, Masaharu Takahashi1, Yu Hoshino1, Hideyuki Takahashi1, Koji Ichiyama1, Shigeo Nagashima1, Toshinori Tanaka1 and Hiroaki Okamoto2,3

1 Division of Virology, Department of Infection and Immunity, Jichi Medical School of Medicine;
2 Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medic

3 E-mail: 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, using an infectious cDNA clone (pJE03-1760F/wt) that can replicate efficiently in PLC/PRF/5 and A549 cells and produce progenies into culture medium, we generated a derivative ORF3-deficient ({triangleup}ORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The {triangleup}ORF3 mutant in the culture medium of the 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 progenies were detectable in the culture medium of {triangleup}ORF3 mutant-infected PLC/PRF/5 cells, as compared with the wild type-infected PLC/PRF/5 cells, and the HEV RNA level in the culture medium of {triangleup}ORF3 mutant-infected A549 cells was below or near the limit of detectability. Immuno-capture PCR assay revealed that the ORF3 protein is present on the surface of cell culture-generated wild-type HEV but not on the {triangleup}ORF3 mutant. Wild-type HEV in the culture supernatant peaked at the sucrose density of 1.15-1.16 g ml-1, contrasting with the {triangleup}ORF3 mutant in the culture supernatant which banded at 1.27-1.28 g ml-1, similar to HEV in the cell lysate and faecal HEV. These results indicate that the ORF3 protein is responsible for virion egress from infected cells and is present on the surface of the released HEV particles which may be associated with lipids.

Received 29 January 2009; accepted 26 March 2009.




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