Liquid β-galactosidase assays of yeast transformed with plasmids encoding bicistronic mRNAs. Yeast strain W1536-8BH (wild type) was transformed with different plasmids, and transformants were assayed for β-galactosidase synthesis. Labels on the x axis indicate the structure of the plasmid that was introduced into yeast. Vector: no promoter or DNA insert; promoter: ADH1 promoter only; ADE3: ADE3 gene only; lacZ: lacZ gene only; ADE3… lacZ: ADE3 and lacZ genes with no IRES; ADE3-HCV C5-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 5 of the polyprotein, and lacZ; ADE3-HCV C120-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 120 of the polyprotein, and lacZ; Δ28-69: ADE3-HCV C120-lacZ lacking nucleotides 28 to 69 of the HCV 5′ untranslated region; G267C: ADE3-HCV C120-lacZ with a point mutation at nucleotide 267 of the HCV 5′ untranslated region; no promoter: ADE3-HCV C120-lacZ without the ADH1 promoter; ADE3, no ATG: ADE3 gene only without an initiator ATG; ADE3, no ATG…lacZ: ADE3 and lacZ genes with no IRES without an initiator ATG of ADE3; ADE3, no ATG-HCV C120: ADE3-HCV C120-lacZ without an initiator ATG of ADE3. The last plasmid was studied in three independent colonies. y axis, β-galactosidase activity in Miller units as determined by solution assay.

Liquid β-galactosidase assays of UPF1 null yeast transformed with plasmids encoding bicistronic mRNAs. Yeast strains AAY273 (panel A) and strain BY4741 (panel B) were transformed with different plasmids, and transformants were assayed for β-galactosidase synthesis. Labels on the x axis indicate the structure of the plasmid that was introduced into yeast strain. Clear bars, isogenic wild-type yeast strain; gray bars, mutant yeast strain. Vector: no promoter or DNA insert; promoter: ADH1 promoter only; ADE3: ADE3 gene only; lacZ: lacZ gene only; ADE3…lacZ: ADE3 and lacZ genes with no IRES; ADE3-HCV C120-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 120 of the polyprotein, and lacZ; no promoter: ADE3-HCV C120-lacZ without the ADH1 promoter; G267C: ADE3-HCV C120-lacZ with a point mutation at nucleotide 267 of the HCV 5′ untranslated region. y axis, β-galactosidase activity in Miller units as determined by solution assay.

Liquid β-galactosidase assays of yeast producing a temperature-sensitive eIF4E protein transformed with plasmids encoding bicistronic mRNAs. Yeast strains CW04 (wild type) and 4-2(eIF4E-ts) were transformed with different plasmids, and transformants were assayed for β-galactosidase synthesis. Labels on the x axis indicate the structure of the plasmid that was introduced into yeast strains. lacZ: lacZ gene only; ADE3-HCV C120-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 120 of the polyprotein, and lacZ; ADE3-URE2-lacZ: ADE3, nucleotides 3 to 1033 of the yeast URE2 gene, and lacZ. y axis, β-galactosidase activity in Miller units as determined by solution assay.

Effect of human La synthesis on HCV IRES-mediated translation in yeast. A. Western blot analysis of human La synthesis in yeast. Extracts from cultures of strain 1231 (wild type) and the same strain transformed with a plasmid encoding human La protein, and HeLa cells (used as a marker for La protein) were fractionated on a 10% SDS-PAGE gel. Proteins were transferred to a polyvinylidene fluoride membrane, and human La protein (52 kDa) was detected by using a rabbit antibody specific for human La protein and the LumiGlo chemiluminescent substrate system. HeLa, extract from HeLa cells; -La, extract from untransformed wild-type yeast; +La, extract from yeast transformed with a plasmid encoding human La protein. The autoradiograph was scanned and labeled using Adobe Photoshop. B and C. Liquid β-galactosidase assays of yeast producing human La protein. Yeast strains 1231 (wild type, B) and CY1 (wild type, C) were transformed with different plasmids, and transformants were assayed for β-galactosidase synthesis. White bars, strains in the absence of human La protein; gray bars, strains synthesizing human La protein. Labels on the x axis indicate the structure of the plasmid that was introduced into yeast. Vector: no promoter or DNA insert; promoter: ADH1 promoter only; ADE3: ADE3 gene only; lacZ: lacZ gene only; ADE3…lacZ: ADE3 and lacZ genes with no IRES; ADE3-HCV C120-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 120 of the polyprotein, and lacZ; no promoter: ADE3-HCV C120-lacZ without the ADH1 promoter; G267C: ADE3-HCV C120-lacZ with a point mutation at nucleotide 267 of the HCV 5′ untranslated region. y axis, β-galactosidase activity in Miller units as determined by solution assay.

Effect of human Ptb synthesis on HCV IRES-mediated translation in yeast. A. Western blot analysis of human Ptb synthesis in yeast. Extracts from cultures of strain 1231 (wild type) and the same strain transformed with a plasmid encoding human Ptb protein, and HeLa cells producing soluble Pvr (46) (used as a marker) were fractionated on a 10% SDS-PAGE gel. Proteins were transferred to a polyvinylidene fluoride membrane, and human Ptb protein (57 kDa) was detected by using a mouse monoclonal antibody against the His6 epitope and the LumiGlo chemiluminescent substrate system. sPvr, extract from HeLa cells producing soluble Pvr; -Ptb, extract from untransformed wild-type yeast; +Ptb, extract from yeast transformed with a plasmid encoding human Ptb protein. The autoradiograph was scanned and labeled using Adobe Photoshop. B. Liquid β-galactosidase assays of yeast producing human Ptb protein. Yeast strain 1231 (wild type) was transformed with different plasmids, and transformants were assayed for β-galactosidase synthesis. White bars, strain in the absence of human Ptb protein; gray bars, strains synthesizing human Ptb protein. Labels on the x axis indicate the structure of the plasmid that was introduced into yeast. Vector: no promoter or DNA insert; promoter: ADH1 promoter only; ADE3: ADE3 gene only; lacZ: lacZ gene only; ADE3…lacZ: ADE3 and lacZ genes with no IRES; ADE3-HCV C120-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 120 of the polyprotein, and lacZ; no promoter: ADE3-HCV C120-lacZ without the ADH1 promoter; G267C: ADE3-HCV C120-lacZ with a point mutation at nucleotide 267 of the HCV 5′ untranslated region. y axis, β-galactosidase activity in Miller units as determined by solution assay.

Structure of bicistronic mRNA. DNA encoding the HCV genotype 1a 5′ untranslated region (nucleotides 1 to 342) and nucleotides encoding the first 5 or the first 120 amino acids of the polyprotein (core) was inserted between the yeast ADE3 and the E. coli lacZ genes. The locations of genetic alterations are indicated: deletion of bases 28 to 69, and mutation of G267 to C. The secondary structure of the HCV IRES is from reference 44.

Liquid β-galactosidase assays and Southern blot hybridization analysis of yeast transformed with plasmids encoding bicistronic mRNAs. Yeast strain 1231 (wild type) was transformed with different plasmids, and transformants were assayed for β-galactosidase synthesis and analyzed by Southern blot hybridization. A. Liquid β-galactosidase assays. Labels on the x axis indicate the structure of the plasmid that was introduced into yeast. lacZ: lacZ gene only; ADE3…lacZ: ADE3 and lacZ genes with no IRES; ADE3-HCV C120-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 120 of the polyprotein, and lacZ; G267C: ADE3-HCV C120-lacZ with a point mutation at nucleotide 267 of the HCV 5′ untranslated region. B. Southern blot hybridization analysis of plasmids in yeast strains from part A. Lanes 1 to 5, DNA from strains containing plasmid ADE3-HCV C120-lacZ; lanes 6 to 10, DNA from strains containing plasmid G267C. DNA was cleaved with StuI. Membranes were hybridized with DNA specific for the ADE3 gene, the HCV IRES, or lacZ. Expected sizes in kb are indicated at left and right.

Analysis of bicistronic RNA in yeast. A. Schematic diagram of the predicted bicistronic mRNA from plasmid ADE3-HCV C120-lacZ. The HCV IRES is shown between the open reading frames for Ade3p and β-galactosidase. The locations of oligonucleotide primers used in reverse transcription and PCRs (C) are shown. B. Northern blot hybridization analysis of total RNA from wild-type yeast W1536-5BH or from yeast containing vector, vector with ADH1 promoter, vectors that produce monocistronic ADE3 or lacZ RNA, or the bicistronic ADE3-HCV C120-lacZ RNA. For cells containing the HCV IRES, RNA from three colonies was prepared. The hybridization probe was a 300-nucleotide lacZ DNA. The arrow indicates 7.4-kb bicistronic mRNA. The sizes of RNA molecular size markers in kb are shown at left. C. Reverse transcription-PCR products fractionated in a 1% agarose gel. Primer 1 was used to prime reverse transcription of total yeast RNA from cells containing the ADE3-HCV C120-lacZ plasmid. Lane 1, DNA markers; lane 2, PCR product obtained with primers 2 and 3; lane 3, PCR product obtained with primers 1 and 4.

Liquid β-galactosidase assays of yeast deleted for genes encoding orthologs of La protein. Yeast strains CY1 (gray bars) and YSS238B (white bars) were transformed with different plasmids, and transformants were assayed for β-galactosidase synthesis. Labels on the x axis indicate the structure of the plasmid that was introduced into yeast. Vector: no promoter or DNA insert; promoter: ADH1 promoter only; ADE3: ADE3 gene only; lacZ: lacZ gene only; ADE3…lacZ: ADE3 and lacZ genes with no IRES; ADE3-HCV C5-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 5 of the polyprotein, and lacZ; ADE3-HCV C120-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 120 of the polyprotein, and lacZ; Δ28-69: ADE3-HCV C120-lacZ lacking nucleotides 28 to 69 of the HCV 5′ untranslated region; G267C: ADE3-HCV C120-lacZ with a point mutation at nucleotide 267 of the HCV 5′ untranslated region. y axis, β-galactosidase activity in Miller units as determined by solution assay.

Effect of human Pcbp2 synthesis on HCV IRES-mediated translation in yeast. A. Western blot analysis of human Pcbp2 synthesis in yeast. Extracts from cultures of strain 1231 (wild type) and the same strain transformed with a plasmid encoding human Pcbp2 protein, and HeLa cells producing soluble poliovirus receptor (46) (used as a marker) were fractionated on a 10% SDS-PAGE gel. Proteins were transferred to a polyvinylidene fluoride membrane, and human Pcbp2 protein (48 kDa) was detected by using a mouse monoclonal antibody against the His6 epitope and the LumiGlo chemiluminescent substrate system. sPvr, extract from HeLa cells producing soluble Pvr; -Pcbp2, extract from untransformed wild-type yeast; +Pcbp2, extract from yeast transformed with a plasmid encoding human Pcbp2 protein. The autoradiograph was scanned and labeled using Adobe Photoshop. B. Liquid β-galactosidase assays of yeast producing human Pcbp2 protein. Yeast strain 1231 (wild type) was transformed with different plasmids, and transformants were assayed for β-galactosidase synthesis. White bars, absence of human Pcbp2 protein; gray bars, strains synthesizing human Pcbp2 protein. Labels on the x axis indicate the structure of the plasmid that was introduced into yeast. Vector: no promoter or DNA insert; promoter: ADH1 promoter only; ADE3: ADE3 gene only; lacZ: lacZ gene only; ADE3…lacZ: ADE3 and lacZ genes with no IRES; ADE3-HCV C120-lacZ: ADE3 gene, HCV IRES, amino acids 1 to 120 of the polyprotein, and lacZ; no promoter: ADE3-HCV C120-lacZ without the ADH1 promoter. y axis, β-galactosidase activity in Miller units as determined by solution assay.