| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2815879 | Gene | 2015 | 6 Pages |
•The HEV genotype-2 has 5′UG → CU/3′CU → GG substitutions flanking the conserved CRE region.•In silico, 5′CU/3′GG complemented to form 5′C5100:3′G5118/5′U5101:3′G5117 pairings, critical in CREX ‘stem-loop’ structure.•Analysis of CREX mutants showed drastic downregulation of HEV RNA replication in S10-3 cells.•The mutant RNA were efficiently encapsidated but produced poorly infectious virions.•The data therefore, suggests strict conservation of the CREX structure, essential for HEV life cycle.
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.
