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

Abstract

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 (DORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The DORF3 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 DORF3 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 DORF3 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 DORF3 mutant. Wild-type HEV in the culture supernatant peaked at a sucrose density of 1.15-1.16 g ml , in contrast with the DORF3 mutant in culture supernatant, which banded at 1.27-1.28 g ml , 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.

A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells

“…With the advent of an infectious cDNA clone of genotype 1 HEV (the Sar55 strain), they confirmed our previous study conducted by using a genotype 3 infectious cDNA clone of the JE03-1760F strain, reporting that the ORF3 protein is essential for virion egress from infected cells, that the ORF3 protein is present on the surface of HEV particles released from infected cells, and that the HEV particles released from infected cells are lipid-associated (Yamada et al, 2009a). Genotypes 2 and 4 HEVs have not yet been examined for the function of the ORF3 protein on virion egress by using infectious cDNA clones.…”

“…In contrast, low levels of background RNA at ,10 2 copies ml 21 were seen in the culture supernatants of cells inoculated with the mutPLAP/LSAL or DORF3 virus during the course of 8-12 day experiment (Fig. 4c), probably due to cell death as described in detail in our previous paper (Yamada et al, 2009a). Notably, no significant difference in the intracellular HEV RNA level was observed upon inoculation of the wild-type virus and its ORF3 variants (Fig.…”

“…For comparison, a derivative ORF3-deficient mutant (pJE03-1760F/ DORF3, GenBank accession no. AB437317) whose initiation codon of the ORF3 gene was mutated to GCA (Ala) (Yamada et al, 2009a), was used. As a negative control, an ORF1-deficient mutant genome (pJE03-1760F/DORF1, GenBank accession no.…”

“…In addition, we constructed a full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 HEV that can grow as efficiently as the faeces-derived virus in both PLC/ PRF/5 and A549 cells (Yamada et al, 2009b). Our previous study using an ORF3-deficient cDNA clone of pJE03-1760F/ wt revealed that the ORF3 protein is essential for virion egress from infected cells (Yamada et al, 2009a). Furthermore, the ORF3-null mutant produced virus particles with the same buoyant density (1.26-1.27 g ml 21 in sucrose) as faecal HEV, which were clearly different from the cell culture-generated wild-type virions and those in the circulation of infected individuals, which banded at 1.15-1.16 g ml 21 and are intimately associated with lipids and the ORF3 protein (Yamada et al, 2009a;Takahashi et al, 2008bTakahashi et al, , 2010.…”

“…Our previous study using an ORF3-deficient cDNA clone of pJE03-1760F/ wt revealed that the ORF3 protein is essential for virion egress from infected cells (Yamada et al, 2009a). Furthermore, the ORF3-null mutant produced virus particles with the same buoyant density (1.26-1.27 g ml 21 in sucrose) as faecal HEV, which were clearly different from the cell culture-generated wild-type virions and those in the circulation of infected individuals, which banded at 1.15-1.16 g ml 21 and are intimately associated with lipids and the ORF3 protein (Yamada et al, 2009a;Takahashi et al, 2008bTakahashi et al, , 2010. These observations suggest that the acquisition of host-cell membrane on the surface of the virions is dependent on the expression of the ORF3 protein.…”