Published online Jan 28, 2017. doi: 10.3748/wjg.v23.i4.590
Peer-review started: October 11, 2016
First decision: December 1, 2016
Revised: December 5, 2016
Accepted: January 4, 2017
Article in press: January 4, 2017
Published online: January 28, 2017
Processing time: 100 Days and 15.9 Hours
To investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.
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 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.
In 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.
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.
Core tip: The function of hepatitis E virus (HEV) Y-domain remains elusive. In silico analysis of closely-related virus sequences mapped a potential palmitoylation-site (CC) and α-helix segment (LYSWLFE) in the Y-domain. Mutant replicons of the universally conserved residues C336, C337 and W413 showed non-viability. Saturation mutations in the Y-domain (nucleotide sequences 788-994) severely affected RNA replication, revealing their post-transcriptional indispensability. Notably, the three residues corresponded to the non-conserved codons, indicating their post-translational essentiality. RNA secondary structure prediction showed hairpin formations by the critical bases (788-994), where mutations drastically affected virion infectivity. This is the first demonstration of the critical role of Y-domain sequences in HEV life cycle that warrants further molecular/biochemical studies.