title: Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo.
abstract: To determine the initiation strategy of the hepatitis E virus (HEV) open reading frame 3 (ORF3), we constructed five HEV mutants with desired mutations in the ORF1 and ORF2 junction region and tested their levels of in vivo infectivity in pigs. A mutant with a C-terminally truncated ORF3 is noninfectious in pigs, indicating that an intact ORF3 is required for in vivo infectivity. Mutations with substitutions in the first in-frame AUG in the junction region or with the same T insertion at the corresponding position of HEV genotype 4 did not affect the virus infectivity or rescue, although mutations with combinations of the two affected virus recovery efficiency, and a single mutation at the third in-frame AUG completely abolished virus infectivity in vivo, indicating that the third in-frame AUG in the junction region is required for virus infection and is likely the authentic initiation site for ORF3. A conserved double stem-loop RNA structure, which may be important for HEV replication, was identified in the junction region. This represents the first report of using a unique homologous pig model system to study the molecular mechanism of HEV replication and to systematically and definitively identify the authentic ORF3 initiation site.
authors.affiliation: Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, 1410 Price's Fork Road, Blacksburg, VA 24601, USA.
authors.forename: Y W
authors.initials: YW
authors.lastname: Huang
authors.forename: T
authors.initials: T
authors.lastname: Opriessnig
authors.forename: P G
authors.initials: PG
authors.lastname: Halbur
authors.forename: X J
authors.initials: XJ
authors.lastname: Meng
mesh_terms: D000818:Animals; D001483:Base Sequence; D003062:Codon; D016752:Hepatitis E virus / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D012367:RNA, Viral / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D024363:Transcription Initiation Site / Q000502:physiology*; D014774:Virulence / Q000502:physiology*
chemical_list: D003062:Codon; D012367:RNA, Viral