Mutational analysis of hepatitis E virus ORF1 'Y-domain' : Effects on RNA replication and virion infectivity

AIMTo investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.METHODSSequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed 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.RESULTSIn silico analysis 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.CONCLUSIONThis 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.

AIM 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 analyzed in silico. Site-directed mutagenesis of the Y-domain(HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed m RNA(p SK-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 na?ve Hep G2/C3 A cells.RESULTS In silico analysis identified a potential palmitoylation-site(C336C337) and an α-helix segment(L410Y411S412W413L414F415E416) in the HEV Y-domain. Molecular characterization of C336 A, C337 A and W413 A 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 thedownstream 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. CONCLUSION 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.