Hepatitis E virus RNA‐dependent RNA polymerase is involved in RNA replication and infectious particle production

Figures

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  FIGURE 1. A conserved α‐helix is predicted in the thumb subdomain of the HEV RDRP. Sequence analysis of the RDRP domain of the HEV ORF1 protein. aa sequence alignment of the RDRP domains from a broad range of Hepeviridae family members (see details in Figure S2). A consensus secondary structure prediction (Sec. Cons.) for all sequences was determined on the basis of the algorithms DSC, GOR IV, HNN SIMPA96, PREDATOR, SOPM, and PHD available at https://npsa‐prabi.ibcp.fr, as shown above the alignment (h, α‐helix; e, extended strand). Random coil or discrepant predictions are not indicated. The degree of aa physicochemical conservation at each position is shown on the bottom line and can be inferred from the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar). The fingers, palm, and thumb subdomains are denoted by light yellow, gray, and blue backgrounds, respectively. The conserved RDRP motifs A–D in the palm subdomain are indicated by colored boxes, and the fully conserved residues are highlighted in the same color. The predicted α‐helix in the thumb subdomain is highlighted by a black square. Numbering of aa is relative to the ORF1…

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  FIGURE 2. ORF1 protein aa 1628–1644 fold into an amphipathic α‐helix. (A) 13C secondary NMR chemical shifts of ORF1[1622–1647]. The secondary NMR chemical shifts (dCA‐dCB) are shown along the peptide sequence corresponding to ORF1 protein aa 1622–1647, with dCA and dCB corresponding to the difference between the experimental and the random coil chemical shift values for 13Cα and 13Cβ, respectively. Positive and negative values indicate the presence of α‐helix and β‐sheet, respectively. (B) Secondary structure propensity for ORF1[1622–1647]. Positive (black bars) and negative (white bars) values correspond to α‐helix and β‐sheet, respectively. (C) Superimposition of the 20 lowest energy conformers (Cα traces) of ORF1[1622–1647] that have been generated by CS‐Rosetta using 1H and 13C NMR chemical shifts. (D) Local root‐mean‐square deviation (RMSD) values (Å) for the backbone atoms (N, Cα, and C’) of each residue in the final bundle of structures in (C). (E) The amphipathic feature of the α‐helix in ORF1[1622–1647] is highlighted on the lowest energy structure, which is colored according to the Wimley and White hydrophobicity scale[ 33 ] ranging from hydrophilic (blue) to hydrophobic (orange)

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  FIGURE 3. The conserved hydrophobic face of ORF1 protein α‐helix 1628–1644 is involved in HEV RNA replication. (A) Replication of subgenomic HEV replicons harboring mutations in α‐helix 1628–1644. S10‐3 human hepatocellular carcinoma cells were transfected with subgenomic HEV replicons harboring a Gaussia luciferase reporter and the mutations indicated in the sequences and the captions. The bars indicate relative light units (RLU; mean ± SD) determined at day 1, 2, and 3 posttransfection. GAD denotes a replication‐defective control replicon harboring an alanine substitution in the GDD motif of the RDRP. wt denotes the parental wild‐type replicon. Helix projections drawn on the basis of the NMR structure are shown on the right, with the mutated residues circled in color. The hydrophobic moment, calculated by HeliQuest (https://heliquest.ipmc.cnrs.fr),[ 34 ] is indicated by an arrow. Asterisks denote residues that are conserved among all Hepeviridae. (B) Ribbon representation of the NMR structure model of ORF1 segment 1627–1644. Residues investigated in the replication assay are shown with their lateral chain and are color‐coded, i.e., hydrophobic in black, polar in orange, basic in blue,…

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  FIGURE 4. ORF1 protein α‐helix 1628–1644 is involved in virus production. S10‐3 human hepatocellular carcinoma cells were transfected with full‐length HEV RNA harboring replication‐competent mutations Mut D_A, Mut G_R, Chim_moose, and Chim_ferret (see Figure 3). A parental wild‐type (wt) and a replication‐defective (GAD) genome served as positive and negative controls, respectively. ΔORF3 denotes a full‐length HEV RNA that does not produce ORF3 protein. Total intracellular and extracellular infectivities were determined at day 5 posttransfection by focus formation assay. The bars indicate focus forming units (ffu; mean ± SD) determined in at least two titrations of three independent experiments (left panel). The ratio of extracellular versus intracellular infectivity is represented on the right, with total infectivity normalized to 1 for each construct. Statistical differences (Mann‐Whitney test) compared with wt are denoted by * and ** for p < 0.05 and <0.001, respectively

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  FIGURE 5. Reverse genetics indicate a functional interaction between the HEV RDRP thumb and palm subdomains. (A) Schematic representation of the subgenomic HEV replicon used to select for second‐site adaptive changes. In vitro–transcribed replicon RNA was electroporated into S10‐3 cells and maintained under selection with 500 µg/ml G418 for at least 13 days. The selected G418‐resistant cells were then lysed for total RNA extraction, and sequence analysis was performed to identify potential adaptive changes in the RDRP sequence. (B) Selection of G418‐resistant cells replicating the HEV Mut R_A replicon. S10‐3 cells were electroporated with Mut R_A replicon RNA or GAD or wild‐type (wt) parental replicon RNAs as negative and positive controls, respectively, followed by G418 selection for 10 days and crystal violet staining. The number of electroporated cells plated in a 100‐mm dish is indicated on the left. Results of a representative experiment are shown. (C) Replication of subgenomic HEV replicons harboring mutations in α‐helix 1628–1644. S10‐3 cells were transfected with subgenomic HEV replicons harboring a Gaussia luciferase reporter and single mutations Mut R_A or Palm E_K or the double…

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  FIGURE 6. The amphipathic α‐helix of the RDRP thumb subdomain is located in close proximity to the first α‐helix of the palm subdomain. RDRP crystal structure model (3BSN) of Norwalk virus strain Ast6139/01/Sp bound to primer‐template RNA.[ 35 ] Fingers, palm, and thumb subdomains are shown in yellow, gray, and cyan, respectively. The position of the compensatory change identified in the HEV RDRP palm subdomain is highlighted in red, and the predicted location of amphipathic α‐helix 1628–1644 in the thumb subdomain is indicated by a dashed black circle