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

作     者：Mohammad Khalid Parvez 

作者机构：Department of PharmacognosyKing Saud University College of PharmacyRiyadh 11451Saudi Arabia 

出 版 物：《World Journal of Gastroenterology》 (世界胃肠病学杂志（英文版）)

年 卷 期：2017年第23卷第4期

页      面：590-602页

核心收录：

学科分类：1007[医学-药学(可授医学、理学学位)] 100705[医学-微生物与生化药学] 1002[医学-临床医学] 1001[医学-基础医学(可授医学、理学学位)] 100103[医学-病原生物学] 10[医学] 

基　　金：Supported by the Deanship of Scientific Research at King Saud University Riyadh No.RG-1435-053 

主　　题：肝炎 E 病毒 开的读物框架 1 Y 域 Palmitoylation α 螺旋 

摘      要：AIM To investigate the role of non-structural open reading frame 1 Y-domain sequences in the hepatitis E virus(HEV) life *** 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 *** 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.