Viral helicase and methyltransferase promote genotype one-hepatitis e virus internal ribosome entry site-like element activity

Figures

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  Fig. 1. Effect of HEV-encoded proteins on HEV IRESl mediated translation of Firefly-luciferase reporter. a Schematic of the pRL-FF/Luc bicistronic reporter plasmid. b Schematic of the g1-HEV genome and the proteins encoded by it. Numbers indicate amino acid position from the N-terminal end. UTR: Untranslated region; MeT: Methyltransferase; Y: Y-domain; PCP: Papain like cysteine protease; V: V-domain; X: X-domain; Hel: Helicase; RdRp: RNA dependent RNA polymerase; ORF1-4: Open Reading Frame 1-4. Numbers above the schematic indicate predicted boundaries of the protein domains. Numbers below the schematic indicate boundary of protein domains used in our experiments. c Firefly-Luciferase activities were evaluated in Huh7 cells cotransfected with different viral proteins and pRL-FF/Luc bicistronic reporter plasmid. Firefly activities were measured after 48 hours of transfection and values were plotted after division with readings for respective Renilla-Luciferase and represented as mean (±SD) of three independent experiments. d Firefly-Luciferase activities were evaluated in Huh7 cells transfected with capped-genomic RNA of g1-HEV or g1-HEV mut (g1-HEV-GAA mutant) for 48 hours, followed by…

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  Fig. 2. Identification of HEV-encoded proteins that interact with the HEV IRES RNA by Yeast three hybrid assay. Yeast Three Hybrid analyses of the interaction between HEV-encoded proteins and the HEV IRESl RNA on lucine, uracile and histidine deficient (LUH−) media. Respective quantitative β-galactosidase units of YBZ1 cotransformants are shown. Normalized β-galactosidase units are plotted as ± SEM of triplicate samples. L: Leucine, U: Uracil, H: Histidine, “−”: Deficiency in the medium, “+”: Supplemented in the medium.

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  Fig. 3. HEV MeT, Y-domain and Helicase do not interact with the HCV IRES or FMDV IRES. Yeast Three Hybrid analysis of the interaction between HCV IRES, FMDV IRES, HEV IRESl and HEV MeT, Y-domain and Helicase proteins. Respective quantitative β-galactosidase units of YBZ1 cotransformants are shown. Normalized β-galactosidase units are plotted as ± SEM of triplicate samples. L: Leucine, U: Uracil, H: Histidine, “−”: Deficiency in the medium, “+”: Supplemented in the medium.

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  Fig. 4. Assessment of HEV IRESl RNA-viral-protein interactions by biotinylated-RNA pull down assay. a Formaldehyde-agarose gel electrophoresis and ethidium bromide staining mediated visualization of in vitro synthesized HEV IRESl RNA (B−) and HEV IRESl RNA (B+). RNA size marker was loaded in lane 1. B− and B+ denote biotinylated and non-biotinylated, respectively. b Assessment of quality and purity of the purified MeT-His and Helicase-His proteins on SDS-PAGE by staining with Coomassie brilliant blue. c Western blot analysis of the purified MeT-His and Helicase-His proteins using anti-His tag (upper panel), anti-MeT (middle panel) and anti-Helicase (lower panel) antibodies. d Western blot analysis of the indicated proteins using aliquots of elutes from biotinylated-RNA pull down of samples containing the control RNA (B+), HEV IRESl RNA (B−) and HEV IRESl RNA (B+), are as indicated. Input denotes 5% of the respective proteins used for incubation with the RNAs during the pull-down assay. e Formaldehyde-agarose gel electrophoresis and ethidium bromide staining mediated visualization of RNAs eluted from the streptavidin beads after the pull-down assay, mentioned in (D). RNA size marker was…

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  Fig. 5. Mapping the minimal region of MeT-Y that mediates its interaction with the HEV IRESl RNA. a Yeast Three Hybrid (Y3H) analyses of the interaction potential of MeT-Y and its deletion mutants with the HEV IRESl RNA. Protein region retained in the deletion mutants is graphically represented as solid lines and N-terminal and C-terminal amino acid positions are indicated. Respective β-galactosidase units of YBZ1 cotransformants are shown. Normalized β-galactosidase units are plotted as ± SEM of triplicate samples. L: Leucine, U: Uracil, H: Histidine, “−”: Deficiency in the medium, “+”: Supplemented in the medium. b Western blot analysis of the indicated samples using anti-HA (upper image) and anti-GAPDH (lower image) antibodies. c Amino acid sequence analysis of 260-330 aa of MeT-Y of the indicated HEV isolates.

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  Fig. 6. Mapping the minimal region of Helicase that mediates its interaction with the HEV IRESl RNA. a Yeast Three Hybrid (Y3H) analysis of the interaction potential of Helicase and its deletion mutants with the HEV IRESl RNA. Protein region retained in the deletion mutants is graphically represented as solid lines and N-terminal and C-terminal amino acid positions are indicated. Respective quantitative β-galactosidase units of YBZ1 cotransformants are shown. Normalized β-galactosidase units are plotted as ± SEM of triplicate samples. L: Leucine, U: Uracil, H: Histidine, “−”: Deficiency in the medium, “+”: Supplemented in the medium. b Western blot analysis of the indicated samples using anti-HA (upper image) and anti-GAPDH (lower image) antibodies. c Amino acid sequence analysis of 103–199 aa of helicase of the indicated HEV isolates.

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  Fig. 7. Identification of the stem loops in the HEV IRESl RNA that mediates its interaction with the HEV MeT-Y and Helicase. a Schematic of HEV IRESl and its stem loops (dotted boxes). Nucleotide residues mutated to dissolve individual stem loops are indicated. b Yeast Three Hybrid (Y3H) analyses of the interaction potential of HEV IRESl RNA and its mutants with the MeT-Y and MeT-Y (262–330) proteins. c Yeast Three Hybrid (Y3H) analyses of the interaction potential of HEV IRESl RNA and its mutants with the Helicase and Helicase (103–199) proteins. Respective quantitative β-galactosidase units of YBZ1 cotransformants are shown. Normalized β-galactosidase units are plotted as mean ± SEM of triplicate samples. L: Leucine, U: Uracil, H: Histidine, “−”: Deficiency in the medium, “+”: Supplemented in the medium.