title: Early secretory pathway localization and lack of processing for hepatitis E virus replication protein pORF1.
abstract: Hepatitis E virus (HEV) is a positive-strand RNA virus and a major causative agent of acute sporadic and epidemic hepatitis. HEV replication protein is encoded by ORF1 and contains the predicted domains of methyltransferase (MT), protease, macro domain, helicase (HEL) and polymerase (POL). In this study, the full-length protein pORF1 (1693 aa) and six truncated variants were expressed by in vitro translation and in human HeLa and hepatic Huh-7 cells by using several vector systems. The proteins were visualized by three specific antisera directed against the MT, HEL and POL domains. In vitro translation of full-length pORF1 yielded smaller quantities of two fragments. However, these fragments were not observed after pORF1 expression and pulse-chase studies in human cells, and their production was not dependent on the predicted protease domain in pORF1. The weight of evidence supports the proposition that pORF1 is not subjected to specific proteolytic processing, which is unusual among animal positive-strand RNA viruses but common for plant viruses. pORF1 was membrane associated in cells and localized to a perinuclear region, where it partially overlapped with localization of the endoplasmic reticulum (ER) marker BAP31 and was closely interspersed with staining of the ER-Golgi intermediate compartment marker protein ERGIC-53. Co-localization with BAP31 was enhanced by treatment with brefeldin A. Therefore, HEV may utilize modified early secretory pathway membranes for replication.
authors.affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: Julia
authors.initials: J
authors.lastname: Perttilä
authors.affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: Pirjo
authors.initials: P
authors.lastname: Spuul
authors.affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: Tero
authors.initials: T
authors.lastname: Ahola
mesh_terms: D000818:Animals; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D006056:Golgi Apparatus / Q000737:chemistry*; D016752:Hepatitis E virus / Q000502:physiology*; D006801:Humans; D050505:Mutant Proteins / Q000032:analysis / Q000235:genetics; D055571:Secretory Pathway; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics; D014779:Virus Replication
chemical_list: D050505:Mutant Proteins; D014764:Viral Proteins