![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 17, Issue 11, 4632-4644, November 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,||
*Department of Biochemistry and #McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6; Department of Biophysics and Biophysical Chemistry, John Hopkins University School of Medicine, Baltimore, MD 21205-2185; Lady Davis Institute for Medical Research, McGill University, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2; Howard Hughes Medical Institute and ||Departments of Biological Chemistry and Internal Medicine, University of Michigan, Ann Arbor, MI 48109; and ¶Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935
Submitted June 1, 2006;
Revised August 4, 2006;
Accepted August 14, 2006
Monitoring Editor: Sandra Schmid
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. This limits the availability of eIF2·GTP·Met-tRNAiMet ternary complexes, reduces formation of 43S preinitiation complexes, and blocks viral (and most cellular) mRNA translation. However, many viruses have developed counterstrategies that circumvent this cellular response. Herein, we characterize a novel class of translation initiation inhibitors that block ternary complex formation and prevent the assembly of 43S preinitiation complexes. We find that translation driven by the HCV IRES is refractory to inhibition by these compounds at concentrations that effectively block cap-dependent translation in vitro and in vivo. Analysis of initiation complexes formed on the HCV IRES in the presence of inhibitor indicates that eIF2 and Met-tRNAiMet are present, defining a tactic used by HCV to evade part of the antiviral interferon response. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 1998) and recruit 43S preinitiation complexes (Pestova et al., 1996) or to directly engage the 40S ribosomal subunit (Wilson et al., 2000; Jan and Sarnow, 2002; Pestova and Hellen, 2003). The latter case is exemplified by the cricket paralysis virus (CrPV) IRES, which recruits 40S ribosomal subunits and initiates translation from the A site, in the absence of a Met-tRNAiMet positioned in the P site (Wilson et al., 2000; Jan and Sarnow, 2002; Pestova and Hellen, 2003).
In hepatitis C virus (HCV), the IRES can recruit the 40S ribosomal subunit independently of translation factors, followed by formation of a 48S complex that contains eIF3 (Buratti et al., 1998; Sizova et al., 1998; Kolupaeva et al., 2000; Kieft et al., 2001; Otto and Puglisi, 2004). After binding of TC, a 60S subunit is recruited to generate an 80S ribosomal complex competent for elongation (Pestova et al., 1998). Although the order in which the 40S ribosomal subunit, the TC, and eIF3 are recruited to the HCV IRES remains to be defined, addition of TC to an HCV-bound 40S docks the AUG into the ribosomal P site and the presence of eIF3 allows for formation of the 80S initiation complex (Pestova et al., 1998). Recent mutational analysis suggests that the HCV IRES coordinates eIF3 and eIF2 interaction with the ribosome, leading to correct positioning of the Met-tRNAiMet in the P site (Ji et al., 2004). Whether the TC is recruited to the 40S ribosome before binding the HCV IRES or assembles once the 40S ribosome has loaded onto the HCV IRES is not known. Recently, an initiation factor-independent mode of ribosome recruitment has also been described for the HCV IRES under high Mg++ conditions in vitro (Lancaster et al., 2006).
A previous forward chemical genetic screen identified a new inhibitor of translation initiation, named NSC119889, that suppressed cap-dependent translation but did not significantly affect translation driven by the HCV IRES (Novac et al., 2004). Herein, we characterize the mode of action of NSC119889 and find that it prevents the association of Met-tRNAiMet to eIF2. We find that translation from the HCV IRES proceeds to near wild-type levels in the presence of this class of TC inhibitors in vitro and in vivo. In addition, we demonstrate that the HCV IRES is still capable of recruiting eIF2 and Met-tRNAiMet at concentrations of NSC119889 that block cap-dependent translation. Our results indicate that the HCV IRES has evolved a mechanism to facilitate recruitment of TC under conditions when these are limiting for initiation, as occurs during the cellular antiviral interferon response.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-[32P]GTP (3000 Ci/mmol), [5-3H]uridine (22 Ci/mmol), and [6-3H]thymidine (10 Ci/mmol) were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Preparation of plasmid DNA, restriction enzyme digestions, agarose gel electrophoresis of DNA and RNA, and SDS-PAGE analysis were carried out using standard methods. Salubrinal and Sal003 were purchased from ChemBridge (San Diego, CA).
43S Preinitiation Complex Formation
43S preinitiation complexes were formed essentially as described by Lorsch and Herschlag (1999). Essentially, purified eIF2 was incubated for 10 min with saturating 5'-guanylylimidodiphosphate (GMP-PNP) (1 mM final) to facilitate the exchange of eIF2-bound GDP for GMP-PNP, followed by the addition of [35S]Met-tRNAiMet and a 5-min incubation to form TC. TC was then added to 40S ribosomal subunits, eIF1A, eIF1, a minimal message (5'-GGAA[UC]7UAUG[CU]10C-3'), and NCS119889 (or other compounds). Final reaction concentrations are as follows: 38 mM HEPES-KOH, pH 7.4, 135 mM KOAc, 3.25 mM MgOAc2, 2.7 mM dithiothreitol [DTT], 25 mM sucrose, 2.5% glycerol, 1 mM GMP-PNP, 200 nM eIF2, 1 µM eIF1, 400 nM eIF1A, 200 nM 40S ribosomal subunits, and 10 or 100 µM test compound (NCS119889). The reaction was quenched via gel loading after 30 min. Samples were loaded onto a 4% polyacrylamide gel running at 25 W in gel buffer [THEM: 66 mM HEPES acid, 34 mM Tris base, 2.5 mM MgCl2, and 0.1 mM EDTA, pH 7.5]. Samples were mixed with 50% sucrose/0.02% each bromophenol blue and xylene cyanol before loading. Samples were run no more than 65 min but no <35 min to separate complexes and still retain free [35S]Met-tRNAiMet on the gel. The gel was placed on Whatman paper, covered with Plastic wrap, and exposed to a PhosphorImager (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) screen at –20°C overnight.
Ternary Complex Analysis
The kinetic parameters of TC formation in the presence or absence of NSC119889 or NSC119893 were performed using yeast factors and ribosomes, after the method of Wong and Lohman (1993). All binding assays were performed in binding assay buffer: 25 mM HEPES-KOH, pH 7.5, 2.5 mM magnesium acetate, 80 mM potassium acetate, pH 7.5, 2 mM DTT, and 0.285 µg/µl creatine kinase. Reactions were conducted in 96-well plates that had been flushed with Sigmacote (Sigma-Aldrich, St. Louis, MO) and rinsed with double-distilled H2O. Seven twofold serial dilutions of 1 µM eIF2 were made in eIF2 storage buffer [20 mM HEPES, pH 7.5, 100 mM KOAc, pH 7.5, 0.1 mM Mg(OAc)2, 2 mM DTT, and 10% glycerol) containing 0.6 µg/ml creatine kinase (included in eIF2 dilutions for all experiments). Inclusion of creatine kinase was found to be essential to prevent nonspecific loss of eIF2 to tube walls at low concentrations of the factor, as observed previously for the mammalian factor (Benne et al., 1979). For measurements of the dissociation constant, 1 nM [35S]Met-tRNAi was incubated for 10 min at 26°C with 10 µl of a dialyzed eIF2 dilution or storage buffer alone in a total volume of 25 µl of binding assay buffer with 500 µM GTP·Mg+2 or no nucleotide. Twenty microliters of each reaction was filtered through an upper nitrocellulose membrane (HAWP; Millipore, Billerica, MA) that retains protein·tRNA complexes and a lower Nytran Supercharge membrane (Whatman Schleicher and Schuell, Keene, NH) that retains unbound tRNA. These membranes were sandwiched between the halves of a dot-blotter (Topac, Hingham, MA). Immediately after the sample passed through the filters, 200 µl of ice-cold reaction buffer was applied as a wash. Filters were air dried and exposed to PhosphorImager (GE Healthcare) screens overnight. The data produced from the exposure of the plate was processed using ImageQuant software (GE Healthcare). Values of fraction tRNA bound to eIF2 [bound/(bound + free)] were corrected for background binding of labeled ligand to the nitrocellulose filter in the absence of eIF2 (<1%).
For GTP affinity measurement, binding assays were performed in binding assay buffer described above. Reactions, 25 µl each, were conducted in 1.5-ml microcentrifuge tubes. Eight twofold serial dilutions of GTP in water were made beginning with a 50 µM sample and added to reaction mixture containing 1 µl (
10 nM) gel purified -[32P]GTP and 10 µM final concentration of NSC119889 dissolved in dimethyl sulfoxide (DMSO) when required. Control reactions without the compound received DMSO alone. To each reaction, eIF2 was added to 400 nM, and the reactions were incubated for 10 min at 26°C before 20 µl of each reaction was filtered through a 2.5-cm disk of HAWP membrane (Millipore) on a standard vacuum manifold. Reactions were immediately washed with 10 ml of ice-cold binding assay buffer. Filters were placed into scintillation vials and subjected to scintillation counting after the addition of 5 ml of Optifluor (PerkinElmer Life and Analytical Sciences) scintillation fluid. Before use, filters were presoaked for 10 min in ice-cold reaction buffer. A series of reactions with no protein were also performed and used to correct each reaction for the background sticking of [-32P]GTP to the filters in the absence of eIF2. Binding curves for Met-tRNAiMet and GTP were fit according to the equation fraction bound = Bmax[S]/(Kd + [S]) using the program Kaleidagraph, where Bmax is the maximum fraction bound at infinite [S].
Purification of Initiation Complexes Bound to the HCV IRES
Initiation complexes were formed on the HCV IRES following a modified procedure of Ji et al. (2004). Essentially, 4-ml translation reactions containing 2 ml of micrococcal nuclease-treated Krebs-2 extract were prepared in the presence of 50 µM NSC119889 or 0.5% DMSO with either GMP-PNP (1 mM final) or cycloheximide (600 µM) as indicated. The reaction was incubated 10 min at 30°C before addition of 40 µg of MS2-HCV IRES RNA followed by incubation at 37°C for 30 min. Then, 400 µg of MS2-MBP fusion protein (Ji et al., 2004) was added, and the reaction further incubated at 37°C for 30 min. The reaction was then stopped on ice and loaded onto a 2.5-ml amylose column. The resin was washed with 10 volumes of binding buffer [20 mM Tris-HCl, pH 7.5, 100 mM KCl, 2.5 mM MgCl2, and 2 mM DTT]. HCV IRES-bound complexes were eluted with binding buffer supplemented with 10 mM maltose. Eluted fractions containing ribosomes (as determined by the OD260) were pooled and loaded onto a 30-ml 10–50% sucrose gradient in binding buffer and centrifuged at 23,000 rpm in a Beckman SW28 rotor for 15 h at 4°C. In reactions containing cycloheximide (80S complex formation), the eluant was treated with micrococcal nuclease before being loaded onto the sucrose gradient. The presence of the ribosomal complexes was detected by absorbance at 254 nm. Fractions containing the 48S and the 80S ribosomes were pooled and either used to extract RNA using TRIzol following the manufacturer's recommendation (Invitrogen, Carlsbad, CA) or to isolate proteins by trichloroacetic acid (TCA) precipitation. Detection of rRNA was performed using 1 µg of the isolated RNA fractionated on a 1% agarose/formaldehyde gel and stained using ethidium bromide. Detection of Met-tRNAiMet was performed by fractionating 5 µg of isolated RNA on an 8 M urea/10% polyacrylamide gel that was transferred onto a Hybond-N+ membrane (GE Healthcare) by using a Transblot SD semidry apparatus (Bio-Rad, Hercules, CA). The RNA was UV cross-linked using a UV-Stratalinker 2400 (Stratagene, La Jolla, CA) and Met-tRNAiMet detected by Northern blotting using a 32P-labeled DNA oligonucleotide targeting mammalian Met-tRNAiMet (5'-CCATCGACCTCTGGGTTATGGG-3'). Western blot analysis for eIF2 (Abcam, Cambridge, MA) and eIF3 (p116 subunit) (Santa Cruz Biotechnology, Santa Cruz, CA) were performed from the TCA-precipitated 48S and 80S complexes, fractionated by SDS-PAGE, transferred to Immobilon P (Millipore) membrane, and revealed by chemiluminescence.
In Vitro Translations
In vitro transcriptions were performed using pSP(CAG)33/FF/HCV//Ren·pA51, pKS/FF/EMCV/Ren, and pGL3/Ren/CrPV/FF (generously provided by Dr. Peter Sarnow, University of Stanford, Stanford, CA) digested with BamHI (Novac et al., 2004). The transcribed bicistronic mRNAs were used in translation reactions using Krebs-2 extracts at a final K+ concentration of 100 mM. The amount of Krebs-2 extract in the translations corresponded to 50% of the total reaction volume. We note that whereas, in general, translation of bicistronic constructs yields higher levels of cap-dependent than IRES-mediated translation (Mizuguchi et al., 2000), the opposite is seen in our preparations of Krebs-2 extracts. Firefly and Renilla luciferase activities (relative light unit, RLU) were measured on a Lumat LB 9507 luminometer Berthold Technologies (Bad Wildbad, Germany). After in vitro translations in micrococcal nuclease-treated Krebs-2 extracts performed in the presence of [35S]methionine, protein products were separated on 10% polyacrylamide/SDS gels, which were treated with EN3Hance, dried, and exposed to X-Omat (Eastman Kodak, Rochester, NY) film. For in vitro translations using [35S]Met-tRNAiMet, tRNAiMet was charged with [35S]methionine according to Svitkin et al. (1981) and 200,000 cpm of charged initiator tRNA was used per translation reaction.
Ribosome-binding Experiments
Ribosome-binding assays were performed as described previously (Novac et al., 2004; Otto and Puglisi, 2004). In brief, 32P-labeled CAT, HCV, or CrPV transcripts were added to Krebs-2 extracts and incubated at 30°C for 10 min in 25-µl reaction volume in the presence of either 1 mM GMP-PNP or 600 µM cycloheximide. Initiation complexes formed on mRNAs were resolved on 10–30% glycerol gradients by centrifuging for 39,000 rpm/3.5 h (Figure 4, A and C) or on 5–20% sucrose gradients by centrifuging for 37,000 rpm/4 h (Figure 4, B and D) in an SW40 rotor. The amount of Krebs-2 extract in the ribosome binding assays corresponded to 50% of the total reaction volume.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). To better define its mode of action, we investigated the possibility that NSC119889 acts upstream of the ribosome recruitment phase of initiation, and we assessed the possibility that it prevents loading of Met-tRNAiMet on 40S ribosomes. This was prompted by our observations that NSC119889 stimulated translation from the CrPV IRES (described below), a phenomenon associated with conditions predicted to increase 40S availability (Wilson et al., 2000; Fernandez et al., 2002b). The assembly of eIF2, GTP, Met-tRNAiMet, eIF3, eIF1, eIF1A, and mRNA into 43S preinitiation complexes was monitored using a mobility gel shift assay and found to be inhibited by NSC119889 (Figure 1B, compare lanes 2 and 3 with 1). We then investigated the possibility that NSC119889 interferes with TC formation. In vitro assays were performed using [35S]Met-tRNAiMet and revealed that eIF2 bound Met-tRNAiMet efficiently with an apparent Kd of 
10 nM, which increased to 85 nM in the absence of GTP (Figure 1C), as reported previously (Kapp and Lorsch, 2004). On addition of NSC119889, Met-tRNAiMet binding to eIF2 was significantly decreased, with a half saturation point of 300 nM (Figure 1C). In the absence of GTP, the extent of inhibition by NSC119889 was even more pronounced with little TC formed even at 400 nM eIF2. To monitor whether NSC119889 is affecting association of GTP with eIF2, a set of filter-binding experiments was performed using [-32P]GTP and eIF2 (Figure 1D). Results revealed that the Kd of eIF2 for GTP is 2 µM, as established previously (Kapp and Lorsch, 2004), and that this interaction is not significantly altered by NSC119889 (Figure 1D). Last, the effect of NSC119889 on release of Met-tRNAiMet from the TC was examined. When 30 nM preformed ternary complexes were incubated in the presence of NSC119889, the observed [35S]Met-tRNAiMet off-rate was 0.08 min–1, similar to the previously determined off-rate of 0.03 min–1 (Kapp and Lorsch, 2004) (Kapp, unpublished data). Together, these results indicate that NSC119889 acts by preventing the association of eIF2 with Met-tRNAiMet and that it has no impact on the association of GTP with eIF2 or on dissociation of the TC.
|
), for their ability to inhibit protein synthesis in vivo. We identified NSC119893 (Figure 1A) as capable of inhibiting translation in vivo and showing an IC50 of 3 µM (Figure 1E). Under our experimental conditions, RNA synthesis was not significantly affected, whereas DNA synthesis was slightly inhibited (Figure 1E). In vitro, NSC119893 also inhibited TC complex formation, albeit with reduced potency in comparison to NSC119889 (Figure 1F). For example, 10 µM NSC119889 was sufficient to inhibit 90% TC formation in the presence of 200 nM eIF2, whereas 25 µM NSC119893 achieved 50% inhibition at the same concentration of eIF2 (compare Figure 1, C and E).
Consistent with NSC119893 inhibiting translation initiation, a reduction in polysomes was observed when MEFs were exposed to compound (Figure 2A). Translation inhibition by NSC119893 was reversible with protein synthesis returning to levels present in untreated cells, 6 h after washout. The kinetics of recovery was slower than those observed when cells recovered from an anisomycin block (Figure 2B). One possibility is that this may reflect the high binding constant of NSC119893 for eIF2 in vivo, resulting in a slow off rate and prolonged recovery from inhibition. We also observed that NSC119893 causes a transient phosphorylation of eIF2 that peaks 30 min after addition of compound to cells (Figure 2C) and that has completely dissipated after 2 h (Figure 2C). To determine the extent to which phosphorylation of eIF2 on Ser51 contributes to the translation inhibition observed in vivo by NSC119893, we compared protein synthesis in wild-type (wt) mouse embryonic fibroblasts (MEFs) to that of MEFs derived from homozygous eIF2S51A/S51A "knockin" mouse embryos (Scheuner et al., 2001) (Figure 2D). The IC50 for wt MEFs exposed to NSC119893 was 3 µM, whereas for eIF2S51A/S51A MEFs it was 6 µM. Hence, phosphorylation of eIF2 by NSC119893 is a minor contributor to the inhibition of protein synthesis observed with this compound in vivo and is not responsible for the inhibition observed during long-term exposure (>2 h) (Robert, unpublished data). The basis for the transient nature of the NSC119893-induced phosphorylation of eIF2 remains to be elucidated, although one speculation is that reduction of TC is itself a stress that could trigger eIF2 phosphorylation.
|
10–25 µM (Figure 3, B and C). In contrast, HCV-driven production of Renilla luciferase was only slightly reduced (25%) at the highest concentration tested (50 µM) (Figure 3B), whereas CrPV-driven translation was stimulated up to sevenfold by increasing concentrations of NSC119889 (Figure 3C). NSC119893 also inhibited cap-dependent translation, although not as potently as NSC119889 (Figure 3D) and consistent with its weaker inhibitory effect on ternary complex formation (Figure 1F). These results suggest that HCV IRES-driven translation proceeds under conditions that reduce TC availability sufficiently to inhibit cap-dependent protein synthesis (Figure 3B). This presents a conundrum, because TC is required for proper 40S positioning at the HCV AUG codon in the ribosomal P site (Pestova et al., 1998).
|
). Recruitment of 40S ribosomes to CAT mRNA, although not very efficient in Krebs-2 extracts, was inhibited by NSC119889 (Figure 4A). In the presence of GMP-PNP, the HCV IRES forms two complexes that are detectable on 5–20% sucrose gradients (Figure 4B). Unbound HCV RNA remains at the top of the gradient in this experiment, and the heavier moving complex sediments to the similar location than does 48S complexes formed on CAT mRNA (Figure 4B). The lighter complex cosediments with 40S ribosomes, as detected by monitoring the UV254 (our unpublished data). The formation of the 40S and 48S complexes on the HCV IRES is not affected by the presence of NSC119889 (Figure 4B).
|
We probed initiation complexes formed on the HCV IRES in the presence of NSC119889 to determine whether these contained components of the TC. For this purpose, an RNA fragment containing the HCV IRES fused to three MS2 binding sites was incubated in Krebs-2 extracts supplemented with MS2-MBP fusion protein (Ji et al., 2004) and either GMP-PNP or cycloheximide to trap 40S/48S or 80S complexes, respectively. Subsequent chromatography on an amylose matrix, followed by sedimentation velocity centrifugation allows for isolation of the 40S/48S and 80S complexes (Ji et al., 2004). The presence of MS2 binding sites does not interfere with formation of these complexes on the HCV IRES (Robert, unpublished data) (Ji et al., 2004). As well, use of an HCV IRES lacking the MS2 sites failed to yield any complexes after purification by amylose chromatography and sedimentation velocity centrifugation (Robert, unpublished data). The presence of the small and large ribosomal subunits in the isolated complexes was confirmed by the presence of 18S and 28S rRNA in these fractions (Figure 5A). eIF2 was present in the 48S fractions, but not in 80S complexes, isolated from vehicle treated and NSC119889 treated extracts (Figure 5B, top, compare lane 4 with 2 and 5 with 3). We also probed for the presence of the p116 subunit of eIF3 and observed that it was present in only 48S fractions (Figure 5B, bottom, compare lane 4 with 2 and 5 with 3). The presence of Met-tRNAiMet in the ribosome complexes was probed by Northern blotting (Figure 5C). We found tRNAiMet in 48S and 80S complexes, irrespective of whether these had been formed in the absence or presence of NSC119889 (Figure 5C, compare lane 7 with 5 and 8 with 6). The probe was specific for mammalian tRNAiMet (compare lane 1 with 2 and 3) and treatment of the sample with RNAse A before electrophoresis resulted in loss of the hybridization signal (Figure 5C, compare lane 4 with 3). The apparent increase in amount of tRNAiMet in fractions from 80S complexes may be a consequence of increased stabilization by cycloheximide (Boehringer et al., 2005). These results indicate that even under conditions of reduced TC availability (i.e., 50 µM NSC119889), ribosomes bound to the HCV IRES contain eIF2 and Met-tRNAiMet.
|
The Ternary Complex Inhibitor Aurintricarboxylic Acid (ATA) Does Not Recapitulate the Effects of NSC119889 on HCV Translation Initiation
ATA (Figure 6A) is a triphenylmethane dye that has been previously reported as an inhibitor of translation initiation. Although this compound has been reported to prevent TC formation (Vazquez, 1979), it is known to be promiscuous and exerts many off-target effects, including inhibition of enzymes and protein–DNA interactions (Hallick et al., 1977; Catchpoole and Stewart, 1994). We assessed whether addition of ATA to Krebs-2 extracts programmed with FF/HCV/Ren or Ren/CrPV/FF could mimic the effects of NSC119889. ATA produced a dose-dependent inhibition of HCV-driven translation that paralleled that of the capped firefly luciferase cistron (Figure 6B). Unlike NSC119889, ATA marginally stimulated CrPV-driven translation at 10 µM (1.5-fold) and inhibited both CrPV-driven and cap-dependent translation at higher concentrations (Figure 6C). In a reconstituted system, ATA seemed to inhibit TC binding to the 40S ribosome rather than inhibit TC formation (Lorsch, unpublished data). These results demonstrate that a promiscuous inhibitor that affects TC function, like ATA, does not recapitulate the effects of NSC119889 on the HCV IRES.
|
20%) at concentrations of NSC119893 that block cap-dependent translation threefold (i.e., 50 µM).
|
by using a recently described small molecule that potentially targets the serine/threonine phosphatase PP1 and maintains eIF2 in a phosphorylated state, called salubrinal (Boyce et al., 2005). For these studies, we used a derivative of salubrinal (Sal003) that is more potent and more soluble than salubrinal (Supplemental Figure 1). We also activated the eIF2 kinase PERK by inducing a protein misfolding response in the endoplasmic reticulum with tunicamycin. Exposure of transfected cells to Sal003 or tunicamycin reduced luciferase activity from both pGL3/Ren/CrPV/FF and pcDNA3/Ren/HCV/FF by 50–60% (Figure 7C). CrPV IRES-mediated firefly expression translation was stimulated 2- to 2.5-fold, whereas expression from the HCV IRES remained unaffected (Figure 7C). Western blotting demonstrated that eIF2 phosphorylation status was increased by compound treatment in our experiments (Figure 7D).
Our finding that HCV IRES-mediated translation occurs efficiently under conditions of limiting TC availability provides a molecular mechanism that may explain why HCV translation is more resistant to inhibition by activated PKR, an interferon-induced, double-stranded RNA-activated eIF2 kinase. PKR is targeted by the virus for inhibition by the viral proteins NS5A and E2 (Gale et al., 1998; Taylor et al., 1999; Pflugheber et al., 2002) as well as by the HCV IRES itself (Vyas et al., 2003). To assess whether HCV IRES-mediated expression was more resistant to PKR activation than cap dependent mRNAs, we activated PKR by using coumermycin-mediated dimerization of a fusion between the Escherichia coli GyrB protein and the kinase domain of PKR, called GyrB-PKR (Figure 8A). As a control, we used a catalytically inactive variant of GyrB.PKR, called GyrB.PKRK296H. On exposure of HT1080 cells expressing GyrB.PKR or GyrB.PKRK296H to coumermycin, there was a time-dependent increase in phosphorylation of eIF2 (Figure 8B) (Kazemi et al., 2004). Transfection of the Ren/HCV/FF reporter into HT1080 cells resulted in a decrease of cap-dependent mediated translation without altering HCV-driven expression observed only in the presence of coumermycin (Figure 8C). These results provide a link between PKR activation, eIF2 phosphorylation, and resistance of HCV translation to reduced ternary complex availability.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and 
subunits participate in binding to Met-tRNAiMet (Hinnebusch, 2000), suggesting that NSC119893 might bind the interface between the and subunits of eIF2 to prevent association of eIF2 with the initiator tRNA. Consistent with this, in the presence of the NSC119889 the eIF2, GTP, Met-tRNAiMet binding curve becomes distinctly sigmoidal (Figure 1C). Binding of compound to the interface would destabilize trimer formation, in which case the observed curve would represent eIF2 assembly, followed by tRNA binding.
Stimulation of CrPV IRES-driven expression in response to NSC119889 (and NSC119893) is consistent with these compounds reducing TC availability (Figures 3C and 7B). This effect on the CrPV IRES is also observed when cells are treated with thapsigargin (Wilson et al., 2000; Fernandez et al., 2002b), tunicamycin (Figure 7C), or starved of amino acids (Fernandez et al., 2002b)—manipulations known to render eIF2·GTP·Met-tRNAiMet complexes limiting. Furthermore, Thompson et al. (2001) have shown that the CrPV IRES can only be efficiently translated in yeast strains with extremely reduced levels of TC (after disruption of two tRNAmet genes and a constitutively active GCN2 gene). The CrPV IRES initiates translation by interacting directly with the 40S subunit (or the 80S ribosomes) in the absence of initiation factors, and this event is decreased when 43S ribosomal complexes are present (Pestova et al., 2004). One possible model by which to explain the stimulation of translation by NSC119889 and NSC119893 on CrPV IRES-mediated translation is that by decreasing 43S preinitiation complex formation, the pool of free 40S ribosomes is increased. This hypothesis remains to be directly tested.
That NSC119889 had a minimal effect on HCV-mediated translation (Figure 3B) was surprising given that previous studies have demonstrated that Met-tRNAiMet is required for formation of competent initiation complexes on the HCV IRES (Pestova et al., 1996, 1998). Indeed, NSC119889 has little effect on 40S binding to the HCV IRES (Figure 4B), while causing only a slight, but reproducible decrease in 80S initiation complex formation (Figure 4D). These 48S complexes contain both eIF2 and Met-tRNAiMet (Figure 5). The presence of eIF2 in 48S, but not 80S complexes, suggests that it is present in a bona fide initiation complex (Figure 5B). We also probed for the presence of eIF2 and eIF2 in these experiments, but found these to be in both 48S and 80S complexes (Robert, unpublished data), probably a consequence of the high-intrinsic nonsequence-specific RNA binding activity of the basic carboxy-terminal domain of eIF2 (Hinnebusch, 2000). Although we make no conclusion regarding the presence of eIF2 and eIF2 in these complexes, the most likely interpretation is that they are present in native eIF2.
What is the mechanism that would maintain translation of the HCV IRES under conditions when TC formation is reduced or availability becomes limiting? A schematic representation of the HCV translation initiation pathway is presented in Figure 9. In one route (step a), the HCV IRES recruits a 40S/eIF3/TC and forms a complex that is competent for 60S ribosome joining (Pestova et al., 1998; Kieft et al., 2001). The HCV IRES can also directly recruit a 40S ribosomal subunit (Pestova et al., 1998; Kieft et al., 2001, 2002; Lytle et al., 2001, 2002) (step b). This is then followed by recruitment of eIF3 and the TC (step b1), with stabilization of the IRES initiation codon in the ribosomal P site (Pestova et al., 1998; Otto and Puglisi, 2004). By inhibiting recruitment of TC to 40S ribosomes, we propose that HCV would predominantly use pathway b to initiate. Unlike cap-dependent translation initiation where recruited 43S ribosomes are thought to scan to the initiation codon, the HCV IRES positions the AUG codon in the ribosomal P site. This aspect of HCV initiation may allow for efficient recruitment of the TC that could be driven by the codon–anticodon interactions. Thermodynamic coupling experiments have indicated that the affinity of TC for a 40S·eIF1·eIF1A complex is 50-fold higher when an AUG is positioned at the ribosomal P site (Maag et al., 2005) and could certainly drive TC recruitment by the HCV IRES. Although the affinity of the HCV IRES for the 40S ribosome has been reported to be 2 nM (Kieft et al., 2001), the affinity between the HCV IRES and the 40S/eIF2·Met-tRNAiMet·GTP complex has not been established.
|
; Boehringer et al., 2005) and could facilitate this de novo assembly process. The recruitment of eIF2 or Met-tRNAiMet to HCV-bound 40S ribosomes could be facilitated by other auxiliary factors such as La autoantigen (Ali and Siddiqui, 1997; Costa-Mattioli et al., 2004) or polypyrimidine tract-binding protein (Anwar et al., 2000). It is worthwhile to note that the yeast homologue of La, LHP1, can act as an RNA chaperone to stabilize specific conformations of tRNA (Yoo and Wolin, 1997), and a similar protein could help recruit the Met-tRNAiMet to the IRES·40S complex.
Our results demonstrating the ability of the HCV IRES to maintain translation in the presence of activated PKR and reduced ternary complexes highlights an innate feature of HCV translation initiation that can contribute to the evasion of the interferon-induced antiviral response (Figures 7 and 8). A reduced TC dependence has also been reported for two other IRESes: EMCV (Hui et al., 2003) and cat-1 (Fernandez et al., 2002a). As well, alphavirus mRNA is efficiently translated in the presence of phosphorylated eIF2 and results suggest that initiation on this transcript may use eIF2A to recruit Met-tRNAiMet in a GTP-independent manner (Komar et al., 2005; Ventoso et al., 2006). Our study does not address the issue of whether the HCV IRES can also recruit Met-tRNAiMet in an eIF2A-dependent mechanism. Our results may also have more general implications for the basic mechanism of translation initiation. That is, 40S ribosomes bound to mRNAs lacking ternary complexes likely occur during reinitiation of translation at downstream AUGs codon after translation of (an) upstream open reading frame(s) (Kozak, 1987). Whether the ribosome acquires a preformed TC, or eIF2 and Met-tRNAiMet independently assemble in the P site of the scanning 40S ribosome, remains to be established. Compounds like NSC119889 that probe the function of the TC may be useful for addressing these issues.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
The online version of this contains supplemental material at MBC Online (http://www.molbiolcell.org). 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-06-0478) on August 23, 2006.
Address correspondence to: Jerry Pelletier (jerry.pelletier{at}mcgill.ca)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Anwar, A., Ali, N., Tanveer, R., Siddiqui, A. (2000). Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation. J. Biol. Chem 275, 34231–34235.
Benne, R., Amesz, H., Hershey, J. W., Voorma, H. O. (1979). The activity of eukaryotic initiation factor eIF-2 in ternary complex formation with GTP and Met-tRNA. J. Biol. Chem 254, 3201–3205.
Benne, R. and Hershey, J. W. (1978). The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes. J. Biol. Chem 253, 3078–3087.
Boehringer, D., Thermann, R., Ostareck-Lederer, A., Lewis, J. D., Stark, H. (2005). Structure of the hepatitis C Virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES. Structure 13, 1695–1706.[Medline]
Boyce, M., et al. (2005). A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science 307, 935–939.
Buratti, E., Tisminetzky, S., Zotti, M., Baralle, F. E. (1998). Functional analysis of the interaction between HCV 5'UTR and putative subunits of eukaryotic translation initiation factor eIF3. Nucleic Acids Res 26, 3179–3187.
Catchpoole, D. R. and Stewart, B. W. (1994). Inhibition of topoisomerase II by aurintricarboxylic acid: implications for mechanisms of apoptosis. Anticancer Res 14, 853–856.[Medline]
Costa-Mattioli, M., Svitkin, Y., Sonenberg, N. (2004). La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro. Mol. Cell. Biol 24, 6861–6870.
Fernandez, J., Yaman, I., Merrick, W. C., Koromilas, A., Wek, R. C., Sood, R., Hensold, J., Hatzoglou, M. (2002a). Regulation of internal ribosome entry site-mediated translation by eukaryotic initiation factor-2alpha phosphorylation and translation of a small upstream open reading frame. J. Biol. Chem 277, 2050–2058.
Fernandez, J., Yaman, I., Sarnow, P., Snider, M. D., Hatzoglou, M. (2002b). Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2alpha. J. Biol. Chem 277, 19198–19205.
Gale, M. Jr, Blakely, C. M., Kwieciszewski, B., Tan, S. L., Dossett, M., Tang, N. M., Korth, M. J., Polyak, S. J., Gretch, D. R., Katze, M. G. (1998). Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol. Cell. Biol 18, 5208–5218.
Hallick, R. B., Chelm, B. K., Gray, P. W., Orozco, E. M. Jr. (1977). Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation. Nucleic Acids Res 4, 3055–3064.
Hinnebusch, A. G. (2000). Mechanism and Regulation of Initiator Methionyl-tRNA Binding to Ribosomes. Cold Spring Harbor, NY: Cold Spring Harbor Press.
Hui, D. J., Bhasker, C. R., Merrick, W. C., Sen, G. C. (2003). Viral stress-inducible protein p56 inhibits translation by blocking the interaction of eIF3 with the ternary complex eIF2.GTP.Met-tRNAi. J. Biol. Chem 278, 39477–39482.
Jan, E. and Sarnow, P. (2002). Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus. J. Mol. Biol 324, 889–902.[CrossRef][Medline]
Ji, H., Fraser, C. S., Yu, Y., Leary, J., Doudna, J. A. (2004). Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA. Proc. Natl. Acad. Sci. USA 101, 16990–16995.
Kapp, L. D. and Lorsch, J. R. (2004). GTP-dependent recognition of the methionine moiety on initiator tRNA by translation factor eIF2. J. Mol. Biol 335, 923–936.[CrossRef][Medline]
Kazemi, S., Papadopoulou, S., Li, S., Su, Q., Wang, S., Yoshimura, A., Matlashewski, G., Dever, T. E., Koromilas, A. E. (2004). Control of alpha subunit of eukaryotic translation initiation factor 2 (eIF2 alpha) phosphorylation by the human papillomavirus type 18 E6 oncoprotein: implications for eIF2 alpha-dependent gene expression and cell death. Mol. Cell. Biol 24, 3415–3429.
Kieft, J. S., Zhou, K., Grech, A., Jubin, R., Doudna, J. A. (2002). Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation. Nat. Struct. Biol 9, 370–374.[Medline]
Kieft, J. S., Zhou, K., Jubin, R., Doudna, J. A. (2001). Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 7, 194–206.[Abstract]
Kolupaeva, V. G., Pestova, T. V., Hellen, C. U. (2000). An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. J. Virol 74, 6242–6250.
Komar, A. A., Gross, S. R., Barth-Baus, D., Strachan, R., Hensold, J. O., Goss Kinzy, T., Merrick, W. C. (2005). Novel characteristics of the biological properties of the yeast Saccharomyces cerevisiae eukaryotic initiation factor 2A. J. Biol. Chem 280, 15601–15611.
Kozak, M. (1987). Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol. Cell. Biol 7, 3438–3445.
Lancaster, A. M., Jan, E., Sarnow, P. (2006). Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site. RNA 1–9.
Lorsch, J. R. and Herschlag, D. (1999). Kinetic dissection of fundamental processes of eukaryotic translation initiation in vitro. EMBO J 18, 6705–6717.[CrossRef][Medline]
Lytle, J. R., Wu, L., Robertson, H. D. (2001). The ribosome binding site of hepatitis C virus mRNA. J. Virol 75, 7629–7636.
Lytle, J. R., Wu, L., Robertson, H. D. (2002). Domains on the hepatitis C virus internal ribosome entry site for 40s subunit binding. RNA 8, 1045–1055.[Abstract]
Maag, D., Fekete, C. A., Gryczynski, Z., Lorsch, J. R. (2005). A conformational change in the eukaryotic translation preinitiation complex and release of eIF1 signal recognition of the start codon. Mol. Cell 17, 265–275.[CrossRef][Medline]
Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E., Hayakawa, T. (2000). IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol. Ther 1, 376–382.[CrossRef][Medline]
Novac, O., Guenier, A. S., Pelletier, J. (2004). Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen. Nucleic Acids Res 32, 902–915.
Otto, G. A. and Puglisi, J. D. (2004). The pathway of HCV IRES-mediated translation initiation. Cell 119, 369–380.[CrossRef][Medline]
Pestova, T. V. and Hellen, C. U. (2003). Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA. Genes Dev 17, 181–186.
Pestova, T. V., Hellen, C. U., Shatsky, I. N. (1996). Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol. Cell. Biol 16, 6859–6869.[Abstract]
Pestova, T. V., Lomakin, I. B., Hellen, C. U. (2004). Position of the CrPV IRES on the 40S subunit and factor dependence of IRES/80S ribosome assembly. EMBO Rep 5, 906–913.[CrossRef][Medline]
Pestova, T. V., Shatsky, I. N., Fletcher, S. P., Jackson, R. J., Hellen, C. U. (1998). A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev 12, 67–83.
Pflugheber, J., Fredericksen, B., Sumpter, R. Jr, Wang, C., Ware, F., Sodora, D. L., Gale, M. Jr. (2002). Regulation of PKR and IRF-1 during hepatitis C virus RNA replication. Proc. Natl. Acad. Sci. USA 99, 4650–4655.
Scheuner, D., Song, B., McEwen, E., Liu, C., Laybutt, R., Gillespie, P., Saunders, T., Bonner-Weir, S., Kaufman, R. J. (2001). Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol. Cell 7, 1165–1176.[CrossRef][Medline]
Sizova, D. V., Kolupaeva, V. G., Pestova, T. V., Shatsky, I. N., Hellen, C. U. (1998). Specific interaction of eukaryotic translation initiation factor 3 with the 5' nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J. Virol 72, 4775–4782.
Spahn, C. M., Kieft, J. S., Grassucci, R. A., Penczek, P. A., Zhou, K., Doudna, J. A., Frank, J. (2001). Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit. Science 291, 1959–1962.
Svitkin, Y. V., Ugarova, T. Y., Chernovskaya, T. V., Lyapustin, V. N., Lashkevich, V. A., Agol, V. I. (1981). Translation of tick-borne encephalitis virus (flavivirus) genome in vitro: synthesis of two structural polypeptides. Virology 110, 26–34.[CrossRef][Medline]
Taylor, D. R., Shi, S. T., Romano, P. R., Barber, G. N., Lai, M. M. (1999). Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 285, 107–110.
Thompson, S. R., Gulyas, K. D., Sarnow, P. (2001). Internal initiation in Saccharomyces cerevisiae mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element. Proc. Natl. Acad. Sci. USA 98, 12972–12977.
Vazquez, D. (1979). Inhibitors of protein biosynthesis. Mol. Biol. Biochem. Biophys 30, 1–312.
Ventoso, I., Sanz, M. A., Molina, S., Berlanga, J. J., Carrasco, L., Esteban, M. (2006). Translational resistance of late alphavirus mRNA to eIF2[alpha] phosphorylation: a strategy to overcome the antiviral effect of protein kinase PKR. Genes Dev 20, 87–100.
Vyas, J., Elia, A., Clemens, M. J. (2003). Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA. RNA 9, 858–870.
Wilson, J. E., Pestova, T. V., Hellen, C. U., Sarnow, P. (2000). Initiation of protein synthesis from the A site of the ribosome. Cell 102, 511–520.[CrossRef][Medline]
Wong, I. and Lohman, T. M. (1993). A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions. Proc. Natl. Acad. Sci. USA 90, 5428–5432.
Yoo, C. J. and Wolin, S. L. (1997). The yeast La protein is required for the 3' endonucleolytic cleavage that matures tRNA precursors. Cell 89, 393–402.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  S. Mokas, J. R. Mills, C. Garreau, M.-J. Fournier, F. Robert, P. Arya, R. J. Kaufman, J. Pelletier, and R. Mazroui Uncoupling Stress Granule Assembly and Translation Initiation Inhibition Mol. Biol. Cell, June 1, 2009; 20(11): 2673 - 2683. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Bechill, Z. Chen, J. W. Brewer, and S. C. Baker Coronavirus Infection Modulates the Unfolded Protein Response and Mediates Sustained Translational Repression J. Virol., May 1, 2008; 82(9): 4492 - 4501. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. Raven, D. Baltzis, S. Wang, Z. Mounir, A. I. Papadakis, H. Q. Gao, and A. E. Koromilas PKR and PKR-like Endoplasmic Reticulum Kinase Induce the Proteasome-dependent Degradation of Cyclin D1 via a Mechanism Requiring Eukaryotic Initiation Factor 2{alpha} Phosphorylation J. Biol. Chem., February 8, 2008; 283(6): 3097 - 3108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Baltzis, O. Pluquet, A. I. Papadakis, S. Kazemi, L.-K. Qu, and A. E. Koromilas The eIF2{alpha} Kinases PERK and PKR Activate Glycogen Synthase Kinase 3 to Promote the Proteasomal Degradation of p53 J. Biol. Chem., October 26, 2007; 282(43): 31675 - 31687. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kazemi, Z. Mounir, D. Baltzis, J. F. Raven, S. Wang, J.-L. Krishnamoorthy, O. Pluquet, J. Pelletier, and A. E. Koromilas A Novel Function of eIF2{alpha} Kinases as Inducers of the Phosphoinositide-3 Kinase Signaling Pathway Mol. Biol. Cell, September 1, 2007; 18(9): 3635 - 3644. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Pelletier Exploring Translation Initiation as a Chemotherapeutic Target Am. Assoc. Cancer Res. Educ. Book, April 14, 2007; 2007(1): 147 - 152. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
