title: The intergenic-junction variant (genotype 2 isolate) of hepatitis E virus restores the CREX 'stem-loop' structural integrity, essential for viral life cycle.
abstract: Among the known human HEV strains (genotypes 1, 2, 3 and 4), the genotype 2 Mexican isolate has two 'double-base' substitutions (5'U5100G5101→CU/3'C5117U5118→GG) flanking the conserved cis-reactive element (CRE) in the intergenic-junction sequences. While the 'C5100U5101' natural mutations in the upstream ORF1 coding region replace 'alanine' for the conserved 'valine', the 'G5117G5118' doublet resides in the downstream non-coding/promoter region of ORF3 gene. Though a stable 'stem-loop' structure containing CRE, critical for virus replication had been reported, the phenotypic effect of genotype 2 'CU/GG' variations were neither mentioned nor explored. In this study, the evolutionary significance of such tolerable mutations in the conserved regulatory-sequences was investigated. Multiple sequence alignment of intergenic-junction of human HEV strains showed further base conservations flanking the CRE sequences. In silico analysis of the conserved sequences (nts. 5099-5121) of the representative genotypes revealed a stable RNA 'stem-loop' structure (CREX). Of the four genotype-specific CREX, the Mexican mutant bases 'CU/GG' very interestingly, compensated and complemented themselves (5'C5100:3'G5118 and 5'U5101:3'G5117) in the 'lower-stem'. The substitution of 'GG' bases in the ORF3 promoter-region did not affect its 'optimal-context' and therefore, negated its regulatory role at 'nucleotide' level. Virtual mutations introduced to break the two base-pairings in the CREX 'lower-stem', completely destabilized the secondary structure. Further molecular characterization of the CREX mutants in HEV-SAR55 replicon background showed a drastic downregulation of viral RNA replication in S10-3 cells. Though the CREX-mutant RNA were encapsidated into trans-complemented viral capsids (ORF2), and produced virions, they were poorly infectious to naïve HepG2/C3A cells. In conclusion, the compensatory mutations in the intergenic-junction of Mexican isolate suggest strict conservation of the CREX 'stem-loop' structure, essential for HEV genome replication. This could have a greater regulatory role in viral life cycle, including RNA packaging.
authors.affiliation: Department of Pharmacognosy, King Saud University College of Pharmacy, Riyadh 11451, Saudi Arabia. Electronic address: Khalid_parvez@yahoo.com.
authors.forename: Mohammad Khalid
authors.initials: MK
authors.lastname: Parvez
mesh_terms: D001483:Base Sequence; D056945:Hep G2 Cells; D016752:Hepatitis E virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D055029:Inverted Repeat Sequences; D012367:RNA, Viral / Q000235:genetics*; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D012367:RNA, Viral
keywords: CRE; CREX; Cis-reactive element; HEV; Hepatitis E; Intergenic-junction; ORF1; ORF2; ORF3