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

Abstract

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.

Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions

“…The PSAP motif between amino acid residues 95 and 98 of the ORF3 protein was found to be conserved among all known HEV strains of genotypes 1-4 and avian HEV strains (Nagashima et al, 2011 As shown in Fig. 1(a), the expression of Tsg101-Myc was detectable by Western blotting analysis using anti-Myc mAb (left panel) and anti-Tsg101 mAb (right panel).…”

“…The ORF2 protein is the viral capsid protein of 660 aa, while the ORF3 protein is a small protein of only 113 or 114 aa, which has been suggested to act as an adaptor to link the intracellular transduction pathways, reduce the host inflammatory response and protect virus-infected cells (Chandra et al, 2008). Recently, it was demonstrated that the ORF3 protein is important for egress from cultured cells (Emerson et al, 2010;Nagashima et al, 2011;Yamada et al, 2009a).…”

“…Small interfering RNA (siRNA)-mediated Tsg101 depletion potently blocks HIV-1 release (Garrus et al, 2001). Moreover, all late domain (L-domain) types require a subset of class E Vps factors, and their function is blocked by dominant-negative (DN) forms of ESCRT-III components (Martin-Serrano et al, 2003a;Strack et al, 2003;von Schwedler et al, 2003) or Vps4 (Garrus et al, 2001;Martin-Serrano et al, 2003b;Tanzi et al, 2003).The PSAP motif between amino acid residues 95 and 98 of the ORF3 protein was found to be conserved among all known HEV strains of genotypes 1-4 and avian HEV strains (Nagashima et al, 2011 As shown in Fig. 1(a), the expression of Tsg101-Myc was detectable by Western blotting analysis using anti-Myc mAb (left panel) and anti-Tsg101 mAb (right panel).…”

“…The ORF2 protein is the viral capsid protein of 660 aa, while the ORF3 protein is a small protein of only 113 or 114 aa, which has been suggested to act as an adaptor to link the intracellular transduction pathways, reduce the host inflammatory response and protect virus-infected cells (Chandra et al, 2008). Recently, it was demonstrated that the ORF3 protein is important for egress from cultured cells (Emerson et al, 2010;Nagashima et al, 2011;Yamada et al, 2009a).At least four major genotypes (1-4) have been identified in mammals. While HEV genotypes 1 and 2 have only been found in humans and are associated with epidemics in developing countries, HEV genotypes 3 and 4 are zoonotic Known late domains consist of the amino acid sequence P(T/ S)AP, PPxY or YxxL, where 'x' represents any amino acid (Göttlinger et al, 1991;Huang et al, 1995;Wills & Craven, 1991;Yasuda & Hunter, 1998).…”

Hepatitis E: Molecular Virology and Pathogenesis

“…This suggests that the PSAP motif plays an important role as a functional domain for HEV budding. 79 Similar role of the PSAP motif in virus release have been shown in vivo in chickens and not in chicken liver cell line expressing pORF2. 80 pORF3 was initially predicted to express as 123 aa long polypeptide.…”