RNA-Protein Interactome at the Hepatitis E Virus Internal Ribosome Entry Site

Figures

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  FIG 1. Identification of host interaction partners of the HEV IRESl element by RaPID and yeast three-hybrid assays. (A) Schematic of the predicted secondary structure of HEV IRESl RNA (highlighted in blue) fused to the BirA-binding RNA motifs (RMB-SL1 and RMB-SL2). Stem-loops of HEV IRESl are denoted as SL A, SL B, and SL C. (B) Schematic of the predicted secondary structure of the HEV IRESl RNA (highlighted in blue) fused to the MS2 coat protein-binding RNA motifs (MS2-SL1 and MS2-SL2). Stem-loops of the HEV IRESl are denoted as SL A, SL B, and SL C. (C) Workflow to identify the HEV IRESl-binding host proteins; B, biotin; A, agarose; S, streptavidin; AD, GAL4 activation domain; BD, GAL4 DNA-binding domain; L, leucine; U, uracil; H, histidine; 3-AT, 3-amino-1,2,4-triazole; −, deficiency in the medium; +, supplemented in the medium.

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  FIG 2. Identification of HEV IRESl RNA-binding proteins by RaPID and Y3H assays. (A) Venny analysis of the HEV IRESl RNA-binding proteins identified by the RaPID assay. Blue- and green-colored ovals represent the numbers of host proteins identified by RaPID assay using HEV 618-738 RNA (blue) and HEV IRESl RNA (green) in the absence of biotin treatment. HEV 618-738-biotin (yellow) and HEV-IRESl-biotin (pink) represent the number of host proteins identified by RaPID assay using HEV 618 to 738 RNA and HEV IRESl RNA in biotin-treated cells. Proteins overlapping between different data sets are shown in respective merged color, and unique proteins identified in each data set are shown in single color. (B) Summary of HEV IRESl RNA-binding proteins identified by the RaPID and Y3H assays. (C) Y3H assay-mediated confirmation of the interactions between HEV IRESl RNA and host proteins identified by screening of the human liver cDNA library. The YBZ1 strain was transformed in the indicated combinations and plated on medium lacking leucine and uracil (LU−). Four random colonies from each cotransformant plate were replica plated onto medium lacking leucine, uracil, and histidine (LUH−) and…

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  FIG 3. Assessment of the ability of the HEV IRESl-binding host proteins (identified by the human liver cDNA library screening) to interact with the HCV IRES and FMDV IRES. The YBZ1 strain was transformed in the indicated combinations and plated on medium lacking leucine and uracil (LU−). Four random colonies from each cotransformant plate were replica plated onto medium lacking leucine, uracil, and histidine (LUH−) and supplemented with 5 mM or 10 mM 3-amino-1,2,4-triazole (3-AT). The same colonies were used in liquid β-galactosidase assays. Relative β-galactosidase units are plotted as mean ± SEM.

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  FIG 4. Confirmation of HEV IRESl RNA-protein interactions by biotinylated RNA pulldown assay and construction of the RNA-protein interaction network. (A) Formaldehyde-agarose gel electrophoresis and ethidium bromide staining-mediated visualization of in vitro-transcribed and purified nonbiotinylated IRESl RNA (IRESl RNA [B−]), biotinylated IRESl RNA (IRESl RNA [B+]), and biotinylated control RNA (control RNA [B+]), as indicated. An RNA size marker was resolved in parallel (lane 1). (B) Formaldehyde-agarose gel electrophoresis and ethidium bromide staining-mediated visualization of eluates from streptavidin pulldown of samples containing control RNA (B+), IRESl RNA (B−), and IRESl RNA (B+), as indicated. (C) Western blotting of the indicated proteins using aliquots of eluates from biotinylated RNA pulldown of samples containing control RNA (B+), IRESl RNA (B−), and IRESl RNA (B+), as indicated; 5% input indicates that 5% of the cell lysate was used for incubation with the RNAs during the pulldown assay. (D) Schematic of the HEV IRESl RNA-protein interaction network. The black node denotes HEV IRESl RNA, and blue and red nodes denote host proteins identified by the RaPID and Y3H assays,…

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  FIG 5. In silico mapping of the interaction between HEV IRESl RNA and ribosomal proteins. (A) The interaction between HEV IRESl (cyan) and RPS3A (lime) is shown in the cartoon. The interacting nucleotides and amino acids are rendered in licorice and colored in atom-wise mode; C, cyan/white; O, red; N, blue; S, yellow. (B) The interaction between HEV IRESl (cyan) and RPS7 (lime) is shown in the cartoon. The interacting nucleotides and amino acids are rendered as shown in A. (C) The interaction between HEV IRESl (cyan) and RPL26 (lime) is shown in the cartoon. The interacting nucleotides and amino acids are rendered as shown in A. (D) The interaction between HEV IRESl (cyan) and RPL5 (lime) is shown in the cartoon. The interacting nucleotides and amino acids are rendered as shown in A. (E) The interaction between HEV IRESl (cyan) and GAPDH (green) is shown in the cartoon. The interacting nucleotides and amino acids are rendered as shown in A. (F to J) The quantitative polar contacts between nucleotide and amino acids are measured for the indicated complexes and are shown in bar graphs (y axis, hydrogen bond distance; x axis, interacting pair of nucleotides@amino acids). (K) Binding free…

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  FIG 6. Effects of siRNAs against selected ribosomal proteins and RNA helicase DHX9 on cell viability and global protein translation. (A) Western blotting of the indicated proteins after 48 and 72 h of treatment with the indicated siRNAs (lanes 2 and 3). Lane 1 shows protein from cells treated for 72 h with a nontarget (NT) siRNA. (B) Percent viability of Huh7 cells treated with the indicated siRNAs for 72 h. The value for the nontarget siRNA-treated cells was considered to be 100%, and other values were calculated relative to that. Values are shown as mean ± SEM of triplicate samples. (C) Top, Western blotting of AHA-labeled and biotinylated total cellular protein after 72 h of treatment with the indicated siRNAs (lanes 3 to 8). Lane 1 shows protein from mock-transfected cells, and lane 2 shows protein from cells transfected with nontargeting siRNA. Bottom, Ponceau staining of the blot shown on top.

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  FIG 7. Essential roles of the ribosomal protein RPL5 and RNA helicase DHX9 in mediating the function of the HEV IRESl element. (A) Schematic of the pRL-FF/Luc bicistronic reporter plasmid. (B) Firefly-Luc activity in Huh7 cells transfected with the indicated siRNAs and the pRL-FF/Luc plasmid for 72 h. The Firefly-Luc values were divided by cell viability and are represented as mean ± SEM of triplicate samples. (C) Renilla-Luc activity in Huh7 cell lysates used in B. The Renilla-Luc values were divided by cell viability and are represented as mean ± SEM of triplicate samples. (D) Gaussia-Luc activity in Huh7 cells transfected with the indicated siRNAs and the HCV-IRES-Gaussia-Luc cRNA for 72 h. The Gaussia-Luc values were divided by cell viability and are represented as mean ± SEM of triplicate samples. (E) RT-qPCR measurement of intracellular levels of g1-HEV genomic RNA in Huh7 cells transfected with the indicated siRNAs for 72 h. Values for g1-HEV RNA were normalized to GAPDH RNA and are represented as mean ± SEM of triplicate samples.

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  FIG 8. The HEV IRESl element associates with actively translating ribosomes. (A) Schematic of the ribosome fractionation assay; CHX, cycloheximide; Puro, puromycin. (B) Measurement of Firefly-Luc and Renilla-Luc activities in Huh7 cells cotransfected with the pSuper-HEV IRESl and the pRL-TK plasmids. Mock shows pSuper vector-transfected Huh7 cells processed in parallel. Values are represented as mean ± SEM of triplicate samples. (C) RT-qPCR analysis of Firefly-Luc and Renilla-Luc RNAs obtained by the RNA immunoprecipitation assay using anti-m7G-cap antibody; Mock, RNA from the pSuper vector-transfected Huh7 cells; FF+Renilla, RNA from the Huh7 cells cotransfected with the pSuper-HEV IRESl and pRL-TK plasmids; Input, amount of RNA used for RNA immunoprecipitation; Unbound, RNA isolated from the supernatant of immunoprecipitation samples before washing; Bound, RNA isolated from the elution fraction of the immunoprecipitation samples. Values are shown as mean ± SEM of triplicate samples. (D) RT-qPCR analysis of control RNA, Firefly-Luc RNA, and Renilla-Luc RNA levels in aliquots of total RNA isolated from sucrose density gradient fractions of Huh7 cells cotransfected with the…

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  FIG 9. RPL5, RPL26, and RNA helicase A are important for association of the HEV IRESl element with the polysome. (A) RT-qPCR analysis of the Firefly-Luc RNA level in density gradient fractions 1, 5, 9, and 13 of Huh7 cells transfected with the indicated siRNAs and the pSuper-HEV IRESl and pRL-TK plasmids for 72 h and treated with cycloheximide for 30 min. Values are shown as mean ± SEM of triplicate samples. (B) RT-qPCR analysis of Renilla-Luc RNA levels in density gradient fractions 1, 5, 9, and 13 of Huh7 cells transfected with the indicated siRNAs and the pSuper-HEV IRESl and pRL-TK plasmids for 72 h and treated with cycloheximide for 30 min. Values are shown as mean ± SEM of triplicate samples. (C) Western blotting of RPL5 protein levels in the indicated density gradient fractions of Huh7 cells transfected with the indicated siRNAs and the pSuper-HEV IRESl and pRL-TK plasmids for 72 h and treated with cycloheximide for 30 min.