Replacement of the hepatitis E virus ORF3 protein PxxP motif with heterologous late domain motifs affects virus release via interaction with TSG101

•HEV viral egress is reliant on an intact PSAP motif in ORF3.•HEV ORF3 subcellular localization is not dependent on the PSAP motif.•Direct ubiquitination of ORF3 does not play a role in HEV lipid association.•Interactions between ORF3 and TSG101 can be exploited as a potential antiviral target.

The ORF3 protein of hepatitis E virus (HEV) contains a “PSAP” amino acid late domain motif, which allows for interaction with the endosomal sorting complexes required for transport (ESCRT) pathway aiding virion release. Late domain motifs are interchangeable with other viral late domain motifs in several enveloped viruses, however, it remains unknown whether HEV shares this functional interchangeability and what implications this might have on viral replication. In this study, by substituting heterologous late domain motifs (PPPY, YPDL, and PSAA) for the HEV ORF3 late domain (PSAP), we demonstrated that deviation from the PSAP motif reduces virus release as measured by viral RNA in culture media. Virus release could not be restored by insertion of a heterologous late domain motif or by supplying wild-type ORF3 in trans, suggesting that the HEV PSAP motif is required for viral exit which cannot be bypassed by the use of alternative heterologous late domains.