Fig 1.
Identification of functional cis-acting RNA elements by ORF1 transcomplementation system.
(A) Schematic representation of how cis-acting RNA elements were identified by utilizing an HEV replicon and ORF1 transcomplementation system. (B) and (C) Replication of HEV RNA mutants in ORF1-transcomplemented HepG2C3A cells. A series of truncated HEV RNA replicons harboring a secretory Gaussia luciferase (Gluc) reporter were transfected into HepG2C3A-ORF1 cells. Cell culture supernatants from each group were collected and Gluc activity measured two days post-transfection. The data are presented as the percentage of Gluc activity relative to that of the full-length rHEV-Gluc GAD. #27 and #31 mutants’ replication were enhanced, as indicated by increased Gluc activities (red). Values are means plus standard deviations (SD) (error bars) (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significance assessed by one-way ANOVA.
Fig 2.
The synonymous mutations in the cis-acting RNA elements severely impair HEV replication.
Synonymous mutations were introduced in the (A-B) ORF1 (102-131nt) or (C-D) ORF2 (7309-7340nt) coding regions. rHEV-Gluc WT, synonymous mutants, or GAD mutant replicon RNA were transfected into HepG2C3A cells. Cell culture medium was collected two days after transfection and Gaussia luciferase activity quantified. The data are presented as the percentage of Gaussia luciferase activity relative to that of the WT rHEV-Gluc replicon. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significantly different by one-way ANOVA.
Fig 3.
Localization of functional cis-acting elements in the HEV genome.
The effect of sequences in the HEV genome on HEV replication capability is summarized based on the data in Figs 1 and 2, S1 and S2 Figs in this study (highlighted as purple) and published results (green) [35]. The Y axis shows the relative level of viral replication as determined in the viral replicon system when the corresponding region (shown on the X axis) in the HEV genome was deleted.
Fig 4.
The cis-acting RNA elements are conserved and functional in multiple HEV genotypes.
(A-B) Consensus sequences of the cis-acting RNA elements in ORF1 (A) and ORF2 (B) across genotypes 1 to 8 were aligned by MEGA6 software. The consensus sequences are shaded. G113 and G7335 are highlighted with a red star. (C) WT, synonymous mutant (G113C, G113T or G7335A) or GAD mutant RNA of SAR55 (gt1), pSHEV3 (gt3) or TW6196E (gt4) were transfected into HepG2C3A cells. Cell culture medium was collected two days after transfection, and Gaussia luciferase activity quantified. The data are presented as the percentage of Gaussia luciferase activity relative to that of the WT rHEV-Gluc. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD (n = 4). ***, P < 0.001 by one-way ANOVA.
Fig 5.
Full-length HEV genome with synonymous mutations in the cis-acting RNA elements is significantly impaired in its ability to produce infectious virus.
(A) Schematic diagrams of the protocol to determine the production of infectious virus. For the cis-acting element mutants, synonymous mutations were introduced in the ORF1 coding region (G113C or G113T) or ORF2 coding region (G7335A). (B-C) Transfection of in vitro-transcribed WT, synonymous mutants or GAD (Pol-) RNA of full-length Kernow C1/p6 (GT 3) into S10-3 cells. Cell lysate supernatant was collected 7 days after transfection, and virus was titrated by infecting HepG2C3A cells. Cells were stained with anti-HEV ORF2 mAbs at 5 days post of infection. (B) to quantify infectious viral particles by foci-forming assay (C). Values are means plus SD (n = 3). IF, immunofluorescence; FFU, foci-forming units; LOD, limit of detection.
Fig 6.
The cis-acting RNA elements interact with HEV ORF1.
(A) A schematic representation of the RNA immunoprecipitation (RIP) assay used to examine the interaction of the HEV RNA genome with the ORF1 protein. (B) The 293T cells overexpressing ORF1 (GAD)-Flag were transfected with GAD or synonymous mutants of the viral genome. The cells were lysed and then incubated with Flag antibody to perform RIP with lgG as the negative control. The immunoprecipitated complex was subjected to RNA purification, and the ORF1-associated RNA were detected by RT-qPCR analysis. Enrichment of RNA binding to ORF1 is shown as fold normalized to input. Immunoblotting analysis was performed to confirm the efficacy of ORF1 (GAD)-Flag immunoprecipitation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) RNA structure prediction of the secondary structures of cis-acting RNA elements. RNAalifold [45] (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAalifold.cgi) was utilized to predict the secondary structures, suggesting that the G7335 would match C7408 to form the stem structure. (D) C7408T mutation restored the replication of the G7335A mutant. To verify the role of the stem structure in HEV replication based on this analysis, C7408T would restore the stem structure impaired in the G7335T mutant. Therefore, the C7408T mutation was introduced into G7335T mutant and the viral replicon RNA harboring the two mutations was transfected into HepG2C3A cells. The Gluc activity was measured as described previously. (E) RIP assay to analyze the association of G7335A mutant or G7335A/C7408T double mutant with HEV ORF1. This assay was performed as described in (B). Values are means plus SD (n = 3). **, P < 0.01; ***, P < 0.001; n.s., not significantly different by Student’s t test or one-way ANOVA.