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

We have previously demonstrated that the release of hepatitis E virus (HEV) from infected cells depended on ORF3 protein, which harbours one or two PSAP motifs. To elucidate the PSAP motif(s) in the ORF3 protein during virion egress, five PSAP mutants derived from an infectious genotype 3 cDNA clone of pJE03-1760F/wt that can grow efficiently in PLC/PRF/5 cells were analysed. Four mutants, including mutLSAP, mutPSAL, mutLSAL (the substituted amino acids in the authentic PSAP motif are underlined) and mutPLAP/PSAP (the changed amino acid in the additional PSAP motif is underlined) generated progenies as efficiently as the wild-type virus. Conversely, the HEV RNA level in the culture supernatant of mutPLAP/LSAL RNA-transfected cells was significantly lower than in cells transfected with the wild-type RNA, similar to an ORF3-null mutant. Consistent with the ORF3-deficient mutant, the mutPLAP/LSAL mutant with no intact PSAP motifs banded at 1.26-1.27 g ml(-1) in sucrose, and was captured by anti-ORF2, but not by anti-ORF3, with or without prior treatment with detergent (0.1 % sodium deoxycholate). The absence of the ORF3 protein on the mutant particles in the culture supernatant was confirmed by Western blotting, despite the expression of ORF3 protein in the RNA-transfected cells, as detected by immunofluorescence and Western blotting. Therefore, at least one of the two intact PSAP motifs in the ORF3 protein is required for the formation of membrane-associated HEV particles possessing ORF3 proteins on their surface, thus suggesting that the PSAP motif plays a role as a functional domain for HEV budding.