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. 2010 Sep 1;7(5):596–605. doi: 10.4161/rna.7.5.13096

Components of the multifactor complex needed for internal initiation by the IRES of hepatitis C virus in Saccharomyces cerevisiae

Amy B Rosenfeld 1,✉, Vincent R Racaniello 1
PMCID: PMC3073256  PMID: 20935471

Abstract

Interaction between the 40S ribosomal subunit and the IRES of hepatitis C virus (HCV) is thought to be independent of initiation proteins, while joining of the 60S ribosomal subunit, and initiation of translation is dependent upon components of the translation machinery. An established in vivo functional assay for internal initiation mediated by the HCV IRES was used to identify proteins needed for IRES dependent translation in Saccharomyces cerevisiae strains possessing alterations of the translation machinery. Internal initiation dependent upon the HCV IRES was abrogated in strains lacking eIF5B, and reduced in strains with altered eIF3, either lacking the Hcr1p subunit, a component of eIF3 not previously known to interact with HCV RNA, or possessing an amino acid change in the Rpg1p subunit. The HCV RNA-induced conformational change in the 40S subunit might affect positioning of eIF3 and lead to different interactions between the ribosome, eIF3, and the multifactor complex. HCV IRES dependent initiation was unaffected in strains in which the concentration of the initiating tRNA was reduced. Alteration of the δ subunit of eIF2B, which leads to inefficient recycling or substitution of aspartic acid for serine 51 of eIF2α had no effect on internal initiation. Production of human Pkr inhibited HCV IRES dependent initiation in yeast. The synthesis of Pkr in yeast is known to result in high levels of eIF2α phosphorylation, increased Gcn4p synthesis, and reduced ribosomal protein production. These alterations may explain the effect of Pkr synthesis on HCV IRES dependent initiation in yeast.

Key words: flavivirus, translation, IRES, hepatitis, yeast, internal initiation

Introduction

In eukaryotic cells, initiation of translation occurs by two distinct mechanisms, 5′-dependent initiation and internal ribosome binding. Initiation on the majority of cytoplasmic messenger RNAs (mRNA) in the cell occurs by a 5′-end dependent mechanism. The 40S ribosomal subunit is recruited to the 5′-end of mRNA through interactions with initiation proteins. The pre-initiation complex of the 40S ribosomal subunit and associated proteins then scans to the initiating codon. In contrast, translation of some viral and cellular RNAs occurs after internal binding of the 40S ribosomal subunit. Initiation of translation also ends with recognition and placement of the initiating AUG codon in the P-site of the 40S ribosomal subunit. Internal binding may place the P-site of the 40S ribosomal subunit on or slightly upstream of the initiating codon. In the latter case scanning by the 40S ribosomal subunit is required.

Translation of hepatitis C virus (HCV) RNA initiates by internal ribosome entry.1,2 The 40S ribosomal subunit interacts directly with the viral internal ribosome entry site (IRES), placing the initiating AUG codon within the P-site. Binding of the viral RNA and the 40S ribosomal subunit in vitro is independent of initiation proteins.3,4 However, formation of the 80S ribosome and recruitment of Met-tRNAi require initiation proteins. The eIF3 complex binds the HCV IRES5,6 and promotes joining of the 60S ribosomal subunit and initiation. Alterations of the IRES that prevent eIF3 binding also inhibit initiation, suggesting that binding of the 60S ribosomal subunit is dependent upon interaction between eIF3 and the RNA.5–10 Several protein subunits of eIF3, including p170, p116, p66 and p47 bind HCV RNA5–7,10 but the function of these proteins in HCV IRES mediated initiation is not known.

eIF3 is part of the multifactor complex (MFC), which also contains of eIF2-GTP-Met tRNAi (ternary complex), eIF1, eIF1A and eIF5.11 The MFC binds the 40S ribosomal subunit and the mRNA, facilitates recognition and placement of the initiating tRNA in the P-site of the 40S ribosomal subunit and couples GTP hydrolysis to AUG recognition.12–18 Alteration of any MFC protein results in defects in 5′-end dependent initiation. It is not known if alterations in these proteins affect internal initiation dependent upon the HCV IRES.

The eIF2 complex recruits the Met-tRNAi to the 40S ribosomal subunit.19,20 All three polypeptides of eIF2, α, β and γ21 directly interact with the 40S ribosomal subunit.22,23 The γ subunit also binds the initiating Met-tRNAi and a molecule of GTP24β27 thereby promoting the formation of the eIF2-GTP-Met tRNAi ternary complex. The β polypeptide facilitates placement of the Met-tRNAi in the P-site of the 40S ribosomal subunit and recognition of the initiating AUG.28 Interaction between eIF2 and the mRNA and eIF2 and GTPase activating protein eIF5 is also mediated by the β subunit of eIF2.29–31 Alteration of the α or β polypeptides may lead to initiation at non-AUG codons.12,32,33 Therefore, the eIF2 complex is necessary for recognition of the correct anticodon-codon interaction and should be required for both 5′-end dependent and internal initiation.

Several initiation proteins, including eIF5B, are required for the joining of the 60S ribosomal subunit and the formation of the 80S ribosome. The eukaryotic ortholog of IF2, eIF5B interacts with the IF1 ortholog eIF1A and both the 40S and 60S ribosomal subunits34,35 to catalyze the hydrolysis of a second molecule of GTP needed for 80S ribosome formation.36 Therefore eIF5B should also be required for HCV IRES dependent initiation.

The yeast Saccharomyces cerevisiae has been used as a model organism to understand 5′-end dependent initiation.37 The rapid growth, ease of genetic manipulation, completely sequenced genome and availability of strains lacking non-essential genes make this organism ideal for identifying cell proteins that participate in IRES dependent translation. The requirement for two subunits of eIF3, the eIF2-GTP-Met tRNAi ternary complex, and eIF5B in HCV mediated internal initiation has been investigated using a functional assay for IRES dependent translation in S. cerevisiae.38 Both the a and j subunits of eIF3 are needed for efficient HCV IRES dependent initiation. The absence of eIF5B abrogates internal initiation mediated by the IRES, while alteration of the eIF2-GTP-Met tRNAi ternary complex reduces translation in a concentration dependent manner.

Results

eIF3 subunits.

Several protein subunits of eIF3, including p170, p116, p66 and p47 bind HCV RNA.5–7,10 Both p170 and p116 bind domain III of the HCV IRES; p116 binds to the apical loop and p170 interacts with the stem.6 The p170 protein is the mammalian ortholog of the yeast eIF3 subunit Rpg1p. The interaction of eIF3 and the 40S ribosomal subunit depends on eIF3j, the ortholog of yeast Hcr1p.39,40 In yeast Hcr1p is also required to stabilize the multifactor complex.41

Plasmids encoding monocistronic lacZ mRNA and the bicistronic HCV C120 mRNA were introduced into yeast strains lacking Hcr1p or producing altered Rpg1p, and the effect on translation was determined. Strains lacking Hcr1p or producing altered Rpg1p have a slow growth phenotype. Introduction of plasmids encoding wild type Hcr1p or Rpg1p into the respective strains rescued the slow growth phenotype (data not shown). Synthesis of wild type Rpg1p in the strain producing altered Rpg1p also rescued the temperature sensitive phenotype of this strain. These results verify the presence of the expected mutation in each strain. The absence of Hcr1p reduced 5′-end dependent initiation 5.2 fold and HCV IRES dependent initiation 1.6 fold (Fig. 1). HCV IRES mediated initiation may be less dependent upon the direct interaction between Hcr1p and the 40S ribosomal subunit than 5′-end dependent initiation, possibly because the structure of the RNA may circumvent the need for Hcr1p for efficient 40S ribosomal subunit joining. The presence of altered Rpg1p reduced both 5′-end dependent initiation (1.6-fold reduction) and internal initiation (2.6 fold reduction) (Fig. 1). These results demonstrate that eIF3 subunits Hcr1p and Rpg1p are needed for efficient HCV IRES mediated internal initiation.

Figure 1.

Figure 1

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Liquid β-galactosidase assays of yeast lacking HCR1 or producing altered Rpg1p transformed with plasmids encoding bicistronic mRNAs. Yeast strains W1531-8BH (clear bars), YLVH13 (hcrΔ; left hatch marks) and YLVH314L (rpg1-1; right hatch marks) 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. lacZ: lacZ gene only; spacer: ADE3 and lacZ genes with no IRES; HCV C120: ADE3 gene, HCV IRES, amino acids 1–120 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. The G267C mutation blocks ribosome binding and was used as a control for IRES dependent initiation. Y axis, β-galactosidase activity in Miller units as determined by solution assay. Average Miller units for each condition are shown in the table.

Alteration of ternary complex.

An initiating tRNA acylated with methionine is thought to decode the first codon of HCV mRNA. Translation of the viral RNA occurs at wild type levels when the initiating AUG codon is changed to AUU or CUG.42 This observation suggests that translation may begin with other than a Met-tRNAi, and that reduction of Met-tRNAi concentration in the cell should not affect HCV IRES dependent initiation. The effect of reducing levels of Met-tRNAi was addressed by determining HCV IRES mediated initiation in a yeast strain that lacks two of the four genes that encode initiating tRNAs. This altered strain displays a modest growth defect43 that was complemented by the introduction of a plasmid encoding IMT4 (data not shown). Plasmids encoding monocistronic lacZ mRNA and bicistronic HCV C120 mRNA were introduced into yeast lacking the imt3 and imt4 genes. The absence of these genes did not affect internal initiation dependent upon the HCV IRES (Fig. 2).

Figure 2.

Figure 2

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Liquid β-galactosidase assays of yeast lacking imt3 and imt4 transformed with plasmids encoding bicistronic mRNAs. Yeast strains H2545 (clear bars) and H2545 p1775 (imt3Δ, imt4Δ; left hatch marks), 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. lacZ: lacZ gene only; spacer: ADE3 and lacZ genes with no IRES; HCV C120: ADE3 gene, HCV IRES, amino acids 1–120 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. The G267C mutation blocks ribosome binding and was used as a control for IRES dependent initiation. Y axis, β-galactosidase activity in Miller units as determined by solution assay. Average Miller units for each condition are shown in the table below.

The initiating tRNA is recruited to the 40S ribosomal subunit through interaction with the γ subunit of eIF2. All three subunits of eIF2 directly interact with the 40S ribosomal subunit.22–24,26 Initiation at non-AUG codons with a Met-tRNAi can be a consequence of the presence of altered eIF2 complexes,28,32,33 indicating that eIF2 is also required for recognition of the correct anticodon-codon interaction. Therefore the eIF2 complex may be needed for internal initiation dependent upon the HCV IRES.

The requirement for eIF2 in HCV IRES dependent initiation was investigated in a yeast strain with an alteration at amino acid 268 in the β subunit of eIF2 that is believed to reduce ternary complex concentration.32,44 The yeast SUI3 gene encodes the β subunit of eIF2. Introduction of a plasmid encoding wild type eIF2β into the altered strain rescued the slow growth phenotype (data not shown), verifying the presence of the expected alteration. Plasmids encoding monocistronic lacZ mRNA and bicistronic HCV IRES mRNA were introduced into yeast producing the altered eIF2α. The presence of altered eIF2α reduced 5′-end dependent initiation six fold, while internal initiation dependent upon the HCV IRES was reduced less than two fold (Fig. 3). Internal initiation mediated by the HCV IRES may not require eIF2α because the interaction between the 40S ribosomal subunit and the HCV IRES may be sufficient for promoting proper anticodon-codon base pairing and initiation.

Figure 3.

Figure 3

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Liquid β-galactosidase assays of yeast producing altered eIF2α transformed with plasmids encoding bicistronic mRNAs. Yeast strains H4 (clear bars) and H1022 (sui3-2; left hatch marks) 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. lacZ: lacZ gene only; spacer: ADE3 and lacZ genes with no IRES; HCV C120: ADE3 gene, HCV IRES, amino acids 1–120 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. The G267C mutation blocks ribosome binding and was used as a control for IRES dependent initiation. Y axis, β-galactosidase activity in Miller units as determined by solution assay. Average Miller units for each condition are shown in the table below.

Recycling of ternary complex.

Recycling of the eIF2-GTP -Met-tRNAi ternary complex is regulated by phosphorylation of the α subunit of eIF2. Phosphorylation of eIF2α on serine 51 reduces the rate at which eIF2, GDP and Pi are released,45 increases the rate of association of eIF2B46 and may increase the affinity of eIF2B binding to the α subunit.47 Phosphorylation of eIF2α therefore reduces the quantity of eIF2-GTP available for generating eIF2-GTP-Met-tRNAi ternary complex. Substitution of aspartic acid for serine 51 can partially mimic phosphorylated eIF2α, while a change to alanine prevents phosphorylation at this position.48 No growth defect was observed in yeast strains harboring either amino alteration when grown in rich medium. The role of eIF2α phosphorylation on HCV IRES mediated initiation was determined by introducing plasmids encoding monocistronic lacZ mRNA and bicistronic HCV IRES mRNA into yeast strains producing wild type eIF2α or altered eIF2α with serine 51 changed to either alanine or aspartic acid. Internal initiation dependent upon the HCV IRES was unaffected by either alteration (Fig. 4). 5′-end dependent initiation was not affected by the alanine substitution, and slightly reduced by the change to aspartic acid (Fig. 4). Although recycling of the eIF2-GTP-Met-tRNAi ternary complex by eIF2B is inhibited by phosphorylation of serine 51, it seems likely that eIF2B is still able to catalyze nucleotide exchange, albeit at reduced efficiency, when serine 51 is changed to aspartic acid. This amino acid change may not sufficiently reduce the concentration of eIF2-GTP-Met-tRNAi ternary complex to affect internal initiation dependent upon the HCV IRES.

Figure 4.

Figure 4

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Liquid β-galactosidase assays of yeast producing altered Sui2p (eIF2a) transformed with plasmids encoding bicistronic mRNAs. Yeast strains y908 (clear bars), y1098 (sui2 S51A; left hatch marks) and y1101 (sui2 S51D; right hatch marks), 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. lacZ: lacZ gene only; spacer: ADE3 and lacZ genes with no IRES; HCV C120: ADE3 gene, HCV IRES, amino acids 1–120 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. The G267C mutation blocks ribosome binding and was used as a control for IRES dependent initiation. Y axis, β-galactosidase activity in Miller units as determined by solution assay. Average Miller units for each condition are shown in the table below.

In yeast, over 70% of cytoplasmic eIF2α must be phosphorylated to inhibit 5′-end dependent initiation.48 To achieve high levels of eIF2α phosphorylation, the mammalian double-stranded RNA-activated protein kinase, Pkr, was synthesized in yeast.49 Plasmids encoding monocistronic lacZ mRNA and bicistronic HCV IRES mRNA were introduced into yeast strains producing wild type Pkr and an altered form (F41A) that is catalytically inactive. Synthesis of Pkr and phosphorylation of eIF2α in yeast was confirmed by western blot analysis (Fig. 5A and B). Internal initiation mediated by the HCV IRES was reduced in the presence of wild type Pkr and was unaffected by altered Pkr (Fig. 5C). Production of β-galactosidase by 5′-end dependent initiation was reduced 3 fold in the presence of wild type Pkr, and was unaffected by altered Pkr (data not shown). These results suggest that internal initiation dependent upon the HCV IRES is inhibited by the reduction in eIF2 recycling.

Figure 5.

Figure 5

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Western blot analysis and liquid β-galactosidase assays of yeast producing human double stranded RNA activated kinase, Pkr. (A) Western blot analysis of human Pkr synthesis in yeast. Extracts from cultures of strain y908 transformed with a plasmid encoding wild-type or altered Pkr protein grown in the presence of galactose were fractionated on a 10% SDS-PAGE gel. Human Pkr protein (68 kDa) was detected by using a rabbit polyclonal antibody against the protein (Santa Cruz Biotechnology, Inc.,) PkrF41A, extract from yeast transformed with a plasmid encoding altered Pkr; Pkr, extract from yeast transformed with a plasmid encoding Pkr. B. Western blot analysis of eIF2α phosphorylation in yeast. Extracts from cultures of strain y908 transformed with a plasmid encoding wild type or altered Pkr protein grown either in the presence of galactose were fractionated on a 10% SDS-PAGE gel. Phosphorylated eIF2α (36 kDa) was detected by using a rabbit polyclonal antibody against phosphorylated serine 51 (Biosource, Inc.,) PkrF41A, extract from yeast transformed with a plasmid encoding altered Pkr; Pkr, extract from yeast transformed with a plasmid encoding Pkr. (C) Liquid β-galactosidase assays of yeast producing human Pkr protein. Yeast strains y908, y908Pkr (uninduced), y908Pkr (induced), y908PkrF41A (uninduced), y908Pkr (induced) 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. HCV C120: ADE3 gene, HCV IRES, amino acids 1–120 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. The G267C mutation blocks ribosome binding and was used as a control for IRES dependent initiation. Y axis, β-galactosidase activity in Miller units as determined by solution assay. Average Miller units for each condition are shown in the table below.

When the initiation cycle ends, the GDP molecule bound to the γ subunit of eIF2 must be exchanged for GTP to begin the next cycle. This exchange is catalyzed by eIF2B50,51 and occurs efficiently when the Met-tRNAi is already bound to the γ subunit of eIF2.52 Three subunits of eIF2B, α, β and δ, irreversibly bind phosphorylated eIF2α, leading to sequestering of eIF2B and failure to exchange GDP.53 The effect on HCV IRES mediated initiation of a change in the eIF2B δ subunit that reduces the concentration of ternary complex in the cell was determined. Plasmids encoding monocistronic lacZ mRNA and bicistronic HCV C120 mRNA were introduced into yeast strains synthesizing altered Gcd2p. Internal initiation mediated by the HCV IRES was not reduced in this strain, while 5′-end dependent initiation was reduced 3 fold (Fig. 6). These results indicate that alteration of GCD2 does not reduce the eIF2-GTP-Met-tRNAi concentration sufficiently to inhibit HCV IRES mediated initiation.

Figure 6.

Figure 6

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Liquid β-galactosidase assays of yeast producing altered eIF2Bδ transformed with plasmids encoding bicistronic mRNAs. Yeast strains H4 (clear bars) and H952 (gcd2-1; left hatch marks), 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. lacZ: lacZ gene only; spacer: ADE3 and lacZ genes with no IRES; HCV C120: ADE3 gene, HCV IRES, amino acids 1–120 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. The G267C mutation blocks ribosome binding and was used as a control for IRES dependent initiation. Y axis, β-galactosidase activity in Miller units as determined by solution assay. Average Miller units for each condition are shown in the table below.

Subunit joining.

Exactly how the 60S ribosomal subunit joins the 40S ribosomal subunit bound to HCV RNA is not understood. During 5′-end dependent initiation, hydrolysis of two molecules of GTP is necessary for the release of initiation proteins, joining of the 60S ribosomal subunit and initiation. The eIF5B protein catalyzes the hydrolysis of a second GTP molecule and joining of the 60S ribosomal subunit. Rabbit reticulocyte lysates depleted of eIF5B are unable to initiate protein synthesis;54 however yeast lacking eIF5B (Fun12p) are viable but slow growing.55 The slow growth phenotype of this strain can be partially suppressed by increased production of initiating tRNA,55 suggesting that eIF5B is necessary for proper placement of the initiating tRNA on the 40S ribosomal subunit and stabilization of this interaction. The effect on HCV IRES mediated initiation of the absence of eIF5B was determined. Production of wild type eIF5B complemented the slow growth phenotype of the fun12 null mutant (data not shown) demonstrating the presence of the correct mutation. Plasmids encoding monocistronic lacZ mRNA and bicistronic HCV C120 mRNA were introduced into the fun12Δ strain. Internal initiation mediated by the HCV IRES was abolished in this strain, and 5′-end dependent initiation was reduced 8 fold (Fig. 7). This result suggests that eIF5B is required for joining the 60S ribosomal subunit to the 40S ribosomal subunit-Met tRNAi complex during internal initiation. The requirement for eIF5B may be a consequence of the change in conformation that the 40S ribosomal subunit undergoes upon binding of the HCV RNA.

Figure 7.

Figure 7

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Liquid β-galactosidase assays of yeast lacking eIF5B transformed with plasmids encoding bicistronic mRNAs. Yeast strains J132 (clear bars) and J133 (fun12Δ; left hatch marks), 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. lacZ: lacZ gene only; spacer: ADE3 and lacZ genes with no IRES; HCV C120: ADE3 gene, HCV IRES, amino acids 1–120 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. The G267C mutation blocks ribosome binding and was used as a control for IRES dependent initiation. Y axis, β-galactosidase activity in Miller units as determined by solution assay. Average Miller units for each condition are shown in the table below.

Discussion

Internal initiation dependent upon the HCV IRES is thought to begin with the direct binding of the 40S ribosomal subunit to the HCV RNA, placing the initiating codon with the P-site of the 40S ribosomal subunit.9,56 Formation of the HCV RNA-40S ribosomal binary complex is believed to be independent of initiation proteins.3,9,56 However, initiation of translation mediated by the HCV IRES is not independent of initiation proteins. Several cellular proteins including eIF2 and eIF3 are thought to be required for or to enhance the efficiency of initiation dependent upon the HCV IRES. Formation of the 80S ribosome is believed to be dependent upon eIF3 binding the 40S ribosomal subunit-HCV RNA binary complex. Abrogation of the interaction between the HCV RNA and eIF3 not only inhibits joining of the 60S ribosomal subunit, but prevents recruitment of eIF2 and the initiating tRNA and initiation mediated by the HCV IRES.3,5,6,8,56,57 Precisely how the 60S ribosomal subunit joins the 40S ribosomal subunit when bound to the HCV RNA is not known. The eIF5B protein promotes formation of the 80S ribosome during 5′-end dependent initiation and may facilitate 80S ribosome formation on the HCV RNA. Initiation mediated by the HCV IRES most likely requires the hydrolysis of two molecules of GTP; therefore eIF5 and eIF5B would be necessary.

The eIF3 complex is composed of six proteins in yeast and eleven proteins in mammalian cells. The five core proteins of eIF3, Prt1p, Nip1p, Tif32p (Rpg1p), Tif35p and Tip34p are conserved among eukaryotes.58,59 Each is an essential protein in yeast and interacts with multiple proteins. Rpg1p mediates the interaction between eIF3 and initiation proteins eIF5 and eIF1,15 and also interacts with the 40S ribosomal subunit.18 An internal domain of Rpg1p has both sequence and functional similarity to Hcr1p, the sixth protein of the eIF3 complex.60 Yeast strains that lack Hcr1p are viable but are slow growing. The slow growth phenotype may be a result of the requirement for Hcr1p to stabilize the interaction between eIF3 and the 40S ribosomal subunit.40,41 Hcr1p also stimulates binding of the Met-tRNAi and 40S ribosomal subunit to the mRNA.15,61 Absence of Hcr1p or Rpg1p in yeast reduced both HCV IRES dependent and 5′-end dependent initiation. These results suggest that Hcr1p and Rpg1p are necessary for HCV IRES mediated internal initiation. These data are consistent with the observed interaction of p170, the human ortholog of Rpg1p, with HCV RNA.5 Incorporation of altered Rpg1p in eIF3 may result in a greater decrease in HCV IRES dependent initiation than in 5′-end dependent initiation because of the dramatic conformational change in the 40S ribosomal subunit induced by binding HCV RNA. This conformational change may effect positioning of eIF3 on the 40S ribosomal subunit, resulting in different interactions between the 40S ribosomal subunit, eIF3 and the multifactor complex. Altered Rpg1p may interfere with these new interactions. These data also suggest that internal initiation dependent upon the HCV IRES requires Hcr1p, a component of eIF3 that was not previously known to interact with HCV RNA. Whether mammalian eIF3j is required for HCV IRES mediated internal initiation has not been investigated.

The first amino acid of the viral polyprotein is a methionine, which implies that the eIF2-GTP-Met-tRNAi ternary complex is required for internal initiation. The concentration of the initiating tRNA within the cell, and the proteins that regulate recycling of the eIF2-GTP-Met-tRNAi ternary complex, should also affect the efficiency of internal initiation. However, reducing the concentration of the initiating tRNA did not affect HCV IRES mediated initiation. This result may not be surprising since the efficiency of internal initiation dependent upon the HCV IRES was unaffected in mammalian cells when the initiating codon was altered from AUG to CUG or AUU,42 suggesting that initiation may not begin with a Met-tRNAi. It has not been determined if the first amino acid of the altered polypeptides was a methionine, leucine or isoleucine. Alternatively, because transcription of the four genes which encode initiating tRNA is thought to of unequal efficiency,43 initiating tRNA concentrations in the IMT3 IMT4 null strain might not be sufficiently reduced to affect HCV initiation.

Reducing the concentration of the eIF2-GTP-Met-tRNAi ternary complex can be a consequence of inefficient recycling of GDP. Alteration of the δ subunit of eIF2B, which leads to inefficient recycling, had no affect on internal initiation, but reduced 5′-end dependent initiation. This finding was unanticipated as cleavage of the mRNA encoding the γ subunit of eIF2B in HeLa cells inhibited internal initiation dependent upon the HCV IRES but not 5′-end dependent initiation,62 suggesting that at least a catalytically active eIF2B complex is required for internal initiation. These observations suggest that there are different requirements for eIF2B during internal initiation in yeast and mammalian cells.

It is not known how the initiating tRNA is placed in the P-site of the 40S ribosomal subunit bound to HCV RNA. No direct interaction between the HCV RNA and eIF2-GTP-Met-tRNAi ternary complex has been observed. However, targeted cleavage of mRNAs encoding the γ subunit of eIF2 in HeLa cells reduces internal initiation dependent upon the HCV IRES.62 This observation implies that the eIF2-GTP-Met-tRNAi ternary complex is required for HCV IRES mediated initiation. Yet, 5′-end dependent initiation was not inhibited in these cells, suggesting that internal initiation mediated by the HCV IRES is much more sensitive to alterations in eIF2.

Phosphorylation of serine 51 of the α subunit of eIF2 regulates recycling of the eIF2-GTP-Met-tRNAi ternary complex.47,63–65 Substitution of aspartic acid for serine 51 can partially mimic phosphorylated eIF2α, resulting in incomplete induction of the yeast general control response.48 This alteration of eIF2α did not affect HCV IRES mediated initiation. However, production of the human cytoplasmic protein Pkr in yeast abrogated internal initiation dependent upon the HCV IRES. Pkr has no affect on HCV IRES dependent initiation in the presence of the S51A alteration of eIF2α (data not shown). Furthermore, the HCV IRES was still able to mediate internal initiation in the presence of a catalytically inactive Pkr (Pkr F41A). These results suggest that Pkr phosphorylates serine 51 of eIF2α in yeast, reducing the concentration of eIF2-GTP-Met-tRNAi sufficiently to inhibit HCV IRES dependent initiation. These data imply that internal initiation dependent upon the HCV IRES is inhibited by the presence of eIF2-GDP. This finding was unexpected. In mammalian cells, internal initiation mediated by HCV IRES is stimulated when Pkr is activated66 suggesting that eIF2α phosphorylation may confer an advantage to HCV IRES dependent initiation. The reason for the different findings in yeast and mammalian cells is not known. Production of Pkr in yeast may result in a greater amount of eIF2α phosphorylation, dramatically reducing eIF2-GTP-Met-tRNAi ternary complex concentration and stimulating production of Gcn4p and Gcn4p regulated genes. One consequence of increased Gcn4p production is reduction of ribosomal proteins, which could inhibit internal initiation mediated by the HCV IRES. Although it is believed that the RNA of the ribosome is catalytic, it is possible that HCV IRES dependent initiation is more reliant upon ribosomal proteins than 5′-end dependent initiation. Internal initiation mediated the HCV IRES may be more sensitive to changes in ribosomal proteins as a consequence of the necessary conformational change induced in the 40S ribosomal subunit upon binding to the HCV RNA. This induced conformation change results in the head and shoulder of the 40S ribosomal subunit being brought closer together, stabilizing the HCV RNA within the mRNA binding groove of the subunit.4 During 5′-end dependent initiation this function may require eIF3. The eIF2-GTP-Met-tRNAi ternary complex may also be more tightly associated with the HCV RNA-40S ribosomal subunit-eIF3-complex than with the multifactor complex of eIF1-eIF5 and eIF3; therefore, small reductions of eIF2-GTP-Met-tRNAi ternary complex concentration caused by the S51D change in eIF2α may not affect HCV IRES mediated initiation. Furthermore, production of Pkr in yeast might affect an unknown protein that is required for internal ribosome binding.

The last steps of initiation are the positioning of the initiating tRNA in the P-site of the 40S ribosomal subunit, base pairing of the anticodon with the codon, and the formation of the 80S ribosome. The β subunit of eIF2 is critical for correct positioning of the initiating Met-tRNAi in the P-site of the 40S ribosomal subunit. Alterations of the β subunit of eIF2 allow for initiation at non-AUG codons.12,44 Internal initiation mediated by the HCV IRES was minimally affected by the presence of altered eIF2α within the eIF2-GTP-Met-tRNAi ternary complex. The interaction between the 40S ribosomal subunit and the HCV RNA may stabilize base pairing between the anticodon and codon and positioning of the initiating Met-tRNAi, reducing the requirement for eIF2β. The lower dependence on eIF2β may also explain the observation that initiation can occur at wild type efficiency at CUG or AUU codons.42

The eIF5B protein catalyzes hydrolysis of the second GTP molecule and joining of the 60S ribosomal subunit. Through interactions with both the 40S and 60S ribosomal subunit and eIF1A, eIF5B also aids in stabilizing the Met-tRNAi-40S ribosomal subunit interaction during 80S formation.67 Internal initiation mediated by the HCV IRES was abolished in a yeast strain lacking eIF5B, and 5′-end dependent initiation was reduced 8 fold. During internal initiation, placement of the initiating tRNA within the P-site and joining of the 60S ribosomal subunit may be more dependent upon eIF5B than in 5′-end dependent initiation. Increased dependence upon eIF5B provides further support the hypothesis that internal initiation dependent upon the HCV IRES occurs through a prokaryotic-like mode of initiation. The bacterial ortholog of eIF5B, IF2, is an essential protein68 that binds GTP, the initiating formyl-methionine-tRNAfmet and the 30S and 50S ribosomal subunits. IF2 aids in binding and positioning of the initiating tRNA in the P-site of the 30S and subsequent joining of the 50S ribosomal subunit.

Materials and Methods

Yeast.

Strains YLVH13 (MATa hcr1Δ LEU2 ade2-1 trp1-1 can100 leu2-3,112 his3-11, 15 ura3) and YLVH314L (MATa leu2-3,112::LEU2::rpg1-1 rpg1Δ2::TRP1 ade2-1 can1-100 his3-11,115 ura3) were courtesy of Leos Valasek, Academy of Sciences of the Czech Republic, Prague. Strains H13-3774 and 314L-3914 were generated by introducing plasmids encoding Hcr1p or Rpg1p (courtesy of Alan Hinnebusch, NIH) by standard cation transformation.69 Strains H2545 (MATa IMT1 IMT2 imt3::TRP1 imt4::TRP1 trp1-GAL+ ura3-52 i leu2::HISG), H2546 (MATa IMT1 IMT2 imt3::TRP1 imt4::TRP1 trp1-GAL+ ura3-52 i leu2::HISG gcn2Δ), H4 (MATa leu2-3,112 ura3-52), H952 (MATα gcd2-1 ura3-52 leu2-3,112) and H1022 (MATα leu2-3,11 ino1-13 ura3-52 his4-303 sui3-2) were courtesy of Alan Hinnebusch, NIH. To verify the expected phenotypes, strains H2545 and H2546 were complemented with B1775 (pRS425 IMT4) and strain H1022 was complemented with B921 (pRS316 SUI3).

Strains J132 (ura3-52 leu2-3,112) and J133 (ura3-52 leu2-3,112 fun12::LEU2) were courtesy of Thomas Dever, NIH. Strain J133-pC1105 was generated by introducing a plasmid encoding wild type eIF5B (Fun12p) (courtesy of Thomas Dever, NIH) into J133 by standard cation transformation.69 The strains y908, y1098 and y1101 were generated by introducing plasmids encoding eIF2α (Sui2p) wild-type, eIF2α S51A and eIF2α S51D (courtesy of Alan Hinnebusch, NIH) into strain H1642 (courtesy of Alan Hinnebusch, NIH) by standard cation transformation.69 The strains y908-Pkr, y908-PkrF41A, y1098-Pkr and y1098-PkrF41A were generated by introducing plasmids encoding wild type human Pkr or altered F41A Pkr into strains y908 and y1098 by standard cation transformation.69

Plasmids.

Plasmids encoding the lacZ gene of Escherichia coli (monocistronic plasmid) or the bicistronic transcript of the ADE3 gene of S. cerevisiase, the HCV IRES including sequence encoding the first 120 amino acids of the HCV polyprotein fused with the second amino acid of β-galactosidase were previously described38 and introduced into all stains by standard cation transformation.69

Transformation.

Yeast were transformed with different plasmids using standard cation transformation69 and plated on selective media (synthetic complete lacking uracil, uracil and leucine or uracil, tryptophan and leucine).

β-galactosidase assays.

Transformants were assayed by colony filter assay.70 Colonies were transferred to a nitrocellulose filter and frozen for 25 min at −80°C. The frozen filter was placed with the colonies face up on top of a Whatman 3 MM filter presoaked in Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 50 mM MgSO4, 50 mM β-mercaptoethanol, 2.7 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside dissolved in N,N-dimethylformamide) and incubated 8 hrs at 30°C until blue color develops. The Miller solution assay was used to quantitate β-galactosidase expression.71 Five randomly picked transformants from three individual transformations were grown in broth under selection (synthetic complete minus uracil, minus uracil and leucine or minus uracil, tryptophan and leucine) for 40 hours, diluted and harvested during mid-logarithmic growth. An OD600 of 1 (Spectonic Genesys 5) was collected and assayed for β-galactosidase activity, the amount of substrate, ONPG, hydrolyzed per minute (Miller units).

Western blot analysis.

Cultures of wild type yeast or yeast transformed with the plasmid encoding human Pkr were grown in rich broth (YEPD) or under selection (synthetic complete minus leucine and tryptophan) for 40 hours. Strains producing human Pkr were diluted into synthetic complete plus 2% raffinose minus leucine and tryptophan, grown overnight and diluted into synthetic complete plus 2% galactose minus leucine and harvested during mid-logarithmic growth. The entire culture was collected, washed with water, resuspended in pretreatment buffer (20 mM Tris, pH 7.8, 0.1 M β-mercaptoethanol, 1 M sorbitol) and incubated at 30°C for 10 minutes. Following pretreatment, cultures were collected, resuspended in treatment buffer (1 M sorbitol, 10 mM potassium phosphate, pH 6.0) containing 0.1 mg/ml zymolase (zymolase 20T Seikagaku, Inc., Tokyo, Japan) and incubated at 30°C for 1 hour. Approximately 90% of the cells were converted to spheroplasts as judged by microscopy. All cells were collected, resuspended in 50 µl of hypotonic solution (100 mM HEPES, 10 mM NaCl, 1 mM DTT) containing protease inhibitors (Roche Applied Science) and 0.1 g glass beads (Sigma), transferred to disposable glass tubes and vortexed for 30 seconds 3 times. Extracts were transferred to cold microcentrifuge tubes and the glass beads were washed with an additional 50 µl of hypotonic solution containing protease inhibitors. Protein concentration was determined by Bradford assay and equal amounts of protein were loaded on a 10% SDS-PAGE gel and fractionated by electrophoresis at 80 volts for 2 hours. Proteins were transferred to a polyvinylidene fluoride membrane (Immobilon-P, Millipore) by semi-dry transfer at 20 volts for 1 hour. The membrane was then incubated at room temperature in 5% non-fat milk dissolved in blot buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 hour. To detect synthesis of human Pkr, a rabbit polyclonal anti-Pkr antibody (Santa Cruz Biotechnology, Inc.,) was diluted 1 to 3,000 in blot buffer containing 5% non-fat milk and incubated with the membrane at 4°C overnight. To detect phosphorylation of eIF2α, a rabbit polyclonal antibody specific to phosphorylated serine 51 of eIF2α (BioSource, Inc.,) was diluted 1 to 3,000 in blot buffer containing 5% non-fat milk and incubated with the membrane at 4°C overnight. After incubation with antibody, membranes were washed 3 times in blot buffer. Mouse anti-rabbit IgG conjugated to horseradish peroxidase (Promega, Inc.,) was diluted 1 to 3,000 in blot buffer containing 2.5% non-fat milk and incubated with the membrane at room temperature for 1 hour. Membranes were washed 3 times in blot buffer and proteins were visualized by LumiGlo chemiluminescent substrate system (Kirkegaard and Perry Laboratories Inc.,).

Acknowledgements

This work was supported in part by NIH grants R01AI50016 and T32AI07161.

We thank Thomas Dever and Alan Hinnebusch, National Institutes of Health, for yeast strains and plasmids and Aaron Mitchell, Columbia University, for advice and discussion.

Footnotes

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