title: Mutational analysis of hepatitis E virus ORF1 "Y-domain": Effects on RNA replication and virion infectivity.
abstract: OBJECTIVE
  To investigate the role of non-structural open reading frame 1 "Y-domain" sequences in the hepatitis E virus (HEV) life cycle.
  
  
  METHODS
  Sequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzedin silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model.In vitrotranscribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions' infectivity was assayed on naïve HepG2/C3A cells.
  
  
  RESULTS
  In silicoanalysis identified a potential palmitoylation-site (C336C337) and an α-helix segment (L410Y411S412W413L414F415E416) in the HEV Y-domain. Molecular characterization of C336A, C337A and W413A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.
  
  
  CONCLUSIONS
  This is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.
authors.affiliation: Mohammad Khalid Parvez, Department of Pharmacognosy, King Saud University College of Pharmacy, Riyadh 11451, Saudi Arabia.
authors.forename: Mohammad Khalid
authors.initials: MK
authors.lastname: Parvez
mesh_terms: D001665:Binding Sites; D004252:DNA Mutational Analysis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D016752:Hepatitis E virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D000072417:Protein Domains; D012367:RNA, Viral / Q000235:genetics; D012093:Replicon; D014764:Viral Proteins / Q000235:genetics; D014771:Virion / Q000235:genetics* / Q000472:pathogenicity; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D049452:Green Fluorescent Proteins
keywords: Hepatitis E virus; Open reading frame 1; Palmitoylation; Y-domain; α-helix