The intergenic-junction variant (genotype 2 isolate) of hepatitis E virus restores the CREX ‘stem-loop’ structural integrity, essential for viral life cycle

• 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.