Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 28, 2017; 23(4): 590-602
Published online Jan 28, 2017. doi: 10.3748/wjg.v23.i4.590
Mutational analysis of hepatitis E virus ORF1 "Y-domain": Effects on RNA replication and virion infectivity
Mohammad Khalid Parvez
Mohammad Khalid Parvez, Department of Pharmacognosy, King Saud University College of Pharmacy, Riyadh 11451, Saudi Arabia
Author contributions: Parvez MK conceived, designed and executed the research, and wrote the manuscript.
Supported by the Deanship of Scientific Research at King Saud University, Riyadh, No. RG-1435-053.
Institutional review board statement: There is no such committee monitoring in vitro studies, except ethical/clinical or animal study which do not pertain to this study.
Conflict-of-interest statement: To the best of my knowledge, no conflict of interest exists.
Data sharing statement: All study-related data and detailed protocols can be obtained from the corresponding author at khalid_parvez@yahoo.com.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Mohammad Khalid Parvez, PhD, Associate Professor, Department of Pharmacognosy, King Saud University College of Pharmacy, PO Box 2457, Riyadh 11451, Saudi Arabia. khalid_parvez@yahoo.com
Telephone: +96-61-4675132 Fax: +96-61-4677245
Received: October 10, 2016
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
Abstract
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 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 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.

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.

Keywords: Hepatitis E virus, Open reading frame 1, Y-domain, Palmitoylation, α-helix

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.