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

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; Palmitoylation; Y-domain; α-helix.

Figure 1

Schematic representation of hepatitis E virus nonstructural polyprotein (ORF1) domain organization, showing the undefined Y-domain. Saturation mutations covering the entire Y-domain (nts 650-1339; 10 constructs of 68 bases each) as well as specific amino acid (C₃₃₆, C₃₃₇ and W₄₁₃) mutations within the predicted membrane-binding motif are shown. MTase: Methyltransferase; Y: Undefined; PCP: Papin-like cysteine protease; P/HVR: Proline-rich/hypervariable region; X: Macro; Hel/NTPase: Helicase/nucleotide triphosphatase; RdRp: RNA-dependent RNA polymerase.

Figure 2

Multiple sequence analysis of ORF1 Y-domain of hepatitis E virus strains (GenBank; n = 206) showing the conservation of predicted palmitoylation-site residues (C₃₃₆C₃₃₇).

Figure 3

In silico analysis of Y-domain of hepatitis E virus and closely-related viruses. A: Residue and positional conservation of L₃₁₀, S₃₁₂ and W₃₁₃ (highlighted in red) in the predicted membrane-binding α-helix among HEV, EEV, SFV and SINV; B: Predicted HEV α-helix LYSWLFE counterpart of SINV; C: Secondary structure prediction of α-helix in the HEV Y-domain.

Figure 4

Multiple sequence analysis of human hepatitis E virus strains (GenBank; n = 206), showing the highly conserved segment (L₃₁₀Y₃₁₁S₃₁₂W₃₁₃L₃₁₄F₃₁₅E₃₁₆) of predicted membrane-binding helix (α1) within the ORF1 Y-domain.

Figure 5

Molecular characterization of hepatitis E virus Y-domain sequences. A: Agarose-gel electropherograms showing the gross RNA yield of pSK-GFP saturation mutants: Ydom1 to Ydom10 (left panel) and specific amino acid mutants: YdomC₃₃₆A, YdomC₃₃₇A and YdomW₄₁₃A (right panel), compared to wild-type (WT); B: Flow cytometry analysis of GFP-positive S10-3 cells, showing the replication competence of the Y-domain mutant replicons.

Figure 6

Analysis of Y-domain mutant virions’ infectivity. A: In silico prediction of stable RNA hairpin/stem-loop structures (wild-type) of three consecutive regions (Ydom3: nts 788-856, Ydom4: nts 857-925 and Ydom5: nts 926-994); B: Flow cytometry analysis of naïve HepG2/C3A cell infectivity by trans-encapsidated virions harboring the three saturation mutant RNAs (Ydom3, Ydom4 and Ydom5).