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Previous Article | Next Article 
Molecular and Cellular Biology, August 2004, p. 6861-6870, Vol. 24, No. 15
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.15.6861-6870.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6
Received 9 March 2004/ Returned for modification 2 April 2004/ Accepted 30 April 2004
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ABSTRACT |
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INTRODUCTION |
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To understand the mechanism of control of IRES function, it is imperative to study the mode by which the 40S ribosome subunit is recruited to the IRES. To this end, we and others undertook the identification and characterization of cellular proteins that interact with the poliovirus IRES. The studies of Meerovitch et al. led to the identification of the first IRES trans-acting factor (ITAF), the La autoantigen (47, 48). Subsequently, La was shown to bind to the hepatitis C virus (HCV) IRES near the initiation codon and to stimulate HCV RNA translation in a rabbit reticulocyte lysate at low concentrations (1, 2). The HCV IRES-La interaction has been recapitulated in a Saccharomyces cerevisiae three-hybrid system (57). In addition, La binds to cellular IRESs, such as X-linked inhibitor of apoptosis (XIAP) (30) and BiP (42).
Initiation of poliovirus translation from the correct initiation site in a rabbit reticulocyte lysate is feeble, and aberrant products are readily detected (8, 14), possibly due to limiting amounts of one or more translation factors. Interestingly, the amount of La protein present in reticulocyte lysate is very low compared to extracts from nucleated cells (48), and the addition of recombinant La enhances poliovirus RNA translation and eliminates the production of aberrant proteins (48, 65). It was thus concluded that La protein is a bona fide ITAF. However, the physiological significance of these studies was questioned (35, 36), because of the large amounts of recombinant La that had been used (10-fold more recombinant La than the amount of La protein present in HeLa cell extract). Thus, the possibility that La is not a physiological ITAF but rather mimics a genuine poliovirus ITAF, owing to its RNA binding properties was raised (35, 36, 72). To address this issue, we studied the requirement of La autoantigen for poliovirus translation in vivo. Using small interfering RNA (siRNA) and a dominant-negative mutant of La (LaDN), we demonstrate that La is required for optimal translation of poliovirus. In addition, we demonstrate that LaDN inhibits the initiation of formation of 48S ribosome complexes on HCV and poliovirus mRNAs.
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MATERIALS AND METHODS |
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siRNA transfection and bicistronic reporter cotransfection. Target sequences for siRNA were designed by using the Ambion web-based criteria and purchased from Dharmacon. The positions and sequences of the siRNAs used in the present study are given in Table 1. One day prior to transfection, the cells were trypsinized, resuspended in medium without antibiotics, and transferred to 24-well plates at a density of 5 x 105 cells per well in a volume of 500 µl. Transfection experiments were performed using Lipofectamine 2000. For each transfection, 3 µl of siRNA duplex (20 µM annealed duplex from Dharmacon) was mixed with 50 µl of OPTIMEM (Invitrogen). In a separate tube, 3 µl of Lipofectamine 2000 per reaction mixture was added to 50 µl of OPTIMEM and incubated for 5 min at room temperature. Both solutions were mixed and incubated for an additional 20 min at room temperature to allow the formation of complexes. The solutions were then added to the cells in 24-well plates. Cells were incubated at 37°C in the presence of the transfection solution for 24 h, washed with DMEM, and used for virus infection. For Western blotting and metabolic radiolabeling experiments, cells were lysed directly in 24-well plates with 1x Laemmli sample buffer. The lysates were incubated at 95°C for 5 min, and an equal amount of protein was resolved on sodium dodecyl sulfate (SDS)-15% polyacrylamide gels. For in vivo translation experiments, HeLa cells at 90% confluency were cotransfected in 24-well plates using Lipofectamine 2000 as described above. Briefly, 500 ng of bicistronic reporter pcDNA3-RLuc-PolioIRES-FLuc (56) and 3 µl of indicated siRNA duplex (20 µM annealed duplex from Dharmacon) were used. Cell extracts were prepared in passive lysis buffer (Promega) 20 h after transfection and assayed for Renilla reniformis luciferase (RLuc) and firefly luciferase (FLuc) activity in a Lumat LB9507 bioluminometer (EG&G Bertold) using a dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions.
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LaDN mutant expression. DNA transfection was performed using Lipofectamine Plus reagent (Gibco) according to the protocol provided by the manufacturer. Briefly, cells were seeded at a density of 5 x 104 cells/ml in 24-well plates and transfected 24 h later in serum-free Opti-MEM medium (Gibco) with 500 ng of pcDNA3-myc-La226-348 (kindly provided by Martin Holcik) or 500 ng of control plasmid (pcDNA3-myc), 4 µl of Lipofectamine Plus reagent, and 2 µl of Lipofectamine 2000 per well. The transfection mixture was replaced 3 h later with fresh DMEM supplemented with 10% fetal bovine serum. Twenty-four hours after transfection, cells were washed with DMEM and then infected with poliovirus. Proteins were metabolically labeled as described above.
[3H]uridine labeling of viral RNA. HeLa cells previously transfected with pcDNA3-myc-La226-348 and control plasmids were infected with poliovirus at a MOI of 20. At various times postinfection, cells were treated with 5 µg of actinomycin D per ml for 1 h prior to the addition of [3H]uridine for 1 h. Ten microliters of cytoplasmic extract was spotted onto Whatman 3MM filter paper, dried for 20 min at room temperature, incubated for 5 min with cold 10% trichloroacetic acid (TCA), and washed three times with cold 5% TCA. Radioactivity was counted using a Beckman scintillation counter.
In vitro translation. Poliovirus RNA was translated in vitro using micrococcal nuclease-treated HeLa S3 cell extracts that had not been subjected to dialysis (4, 50). The composition of the in vitro translation reaction mixture was similar to that described previously for the Krebs-2 cell-free system (67). Translation reaction mixtures (30 µl) contained 15 µl of extract and the following components (final concentrations are given) as follows: 1 mM ATP, 0.2 mM GTP, 0.2 mM CTP, 0.2 mM UTP, 10 mM creatine phosphate, 0.1 mg of creatine phosphokinase per ml, 20 µM concentrations of all L-amino acids except for L-methionine, 0.5 mCi of [35S]methionine per ml, 25 mM HEPES-KOH (pH 7.3), 15 mM KCl, 125 mM potassium acetate, 3 mM MgCl2, 250 µM spermidine, and 15 µg of poliovirus (type 1, Mahoney) RNA per ml. Following incubation (34°C, 4 h), reactions were stopped by the addition of 2 volumes of 1.5x Laemmli sample buffer. Translation products were detected following SDS-15% PAGE and autoradiography. To assay for poliovirus replication, reaction mixtures were supplemented with 20 µM unlabeled methionine and incubated at 34°C for 16 h. Reaction mixtures were treated with a mixture of RNase A and RNase T1 (50), diluted with phosphate-buffered saline, and assayed for plaque formation on HeLa cell monolayers (61).
Plaque assay. The plaque assay was performed as previously described (61). Two hundred fifty microliters of cell extract in which poliovirus was produced was used to infect HeLa monolayer cells (2 x 106 cells in 60-mm-diameter plates). After 3 days of incubation at 37°C, plaques were detected by staining the cells with 1% crystal violet.
Ribosome binding assays.
HCV IRES-containing RNA was transcribed in vitro with T7 RNA polymerase using plasmid pHCV(40-372) NS' (60) that had been linearized at the junction of the HCV and nonstructural (NS') sequence by BamHI. Poliovirus RNA was obtained as described previously (64), and rabbit globin mRNA was purchased commercially (Invitrogen). RNAs were 3' end labeled to specific activities of about 107 cpm/µg for HCV IRES and 5 x 105 for globin and poliovirus mRNAs using [
-32P]cordycepin-5'-triphosphate (Perkin-Elmer) and yeast poly(A) polymerase as recommended by the manufacturer (Amersham Biosciences). Ribosome binding experiments were performed using a cycloheximide-supplemented HeLa S10 cell extract (41) in a total volume of 40 µl for 15 min at 34°C. Reaction mixtures contained 20 µl of micrococcal nuclease-treated HeLa S10 cell extract, 0.6 mM cycloheximide, 4 µl of a 10x master mix (10 mM ATP, 2 mM GTP, 100 mM creatine phosphate, 1 mg of creatine phosphokinase per ml, 19 unlabeled L-amino acids [lacking L-methionine; 0.2 mM each], and 125 mM HEPES-KOH [pH 7.3]) (68), 2 µl of L-methionine (0.4 mM), 4 µl of a salt solution (0.8 M KCl, 5 mM MgCl2, 2.5 mM spermidine), and HCV IRES (
106 cpm) or poliovirus (105 cpm) RNA. Following incubation, samples were chilled on ice, diluted fourfold with ice-cold high-salt buffer, and analyzed on 5-ml 15 to 30% linear sucrose gradients as described previously (66).
Assembly of 48S ribosome initiation complexes on HCV IRES was measured in a similar way, except that the GTP analogue ß,
-imido-GTP (GMP-PNP) (1 mM) was substituted for GTP in the reaction mixture and a low-salt buffer (23) was used to prepare 10 to 30% sucrose gradients. Gradients were centrifuged for 180 min at 54,000 rpm using a Beckman SW55 rotor. Ribosome binding was calculated by two methods. The total area under the 80S or 48S ribosome initiation complex curve was measured using the histogram function of Adobe Photoshop. Alternatively, the graphs were printed out, and the areas under the curves were cut out and weighed. No significant differences between the two methods were noted.
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RESULTS |
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La85-siRNA inhibits poliovirus IRES-driven protein synthesis. To determine whether the effect of La siRNA on viral protein synthesis in vivo is a consequence of inhibition of translation, the effect of La85-siRNA on in vivo expression of a bicistronic reporter mRNA, RLuc-PolioIRES-FLuc (56), was examined. The bicistronic mRNA contains the poliovirus IRES inserted between the RLuc and FLuc open reading frames (ORFs) (Fig. 2). Thus, translation of RLuc is cap dependent, whereas translation of the second cistron (FLuc) is IRES dependent. Cotransfection of HeLa cells with pcDNA3-RLuc-PolioIRES-FLuc and La-siRNA significantly reduced the FLuc/RLuc ratio (more than threefold) (Fig. 2). In striking contrast, the nonspecific siRNA (Ctlr-siRNA) failed to inhibit FLuc synthesis, indicating a specific reduction of IRES-dependent translation by La85-siRNA. Importantly, RLuc synthesis, which is cap dependent, did not vary significantly among cells transfected with La85-siRNA, cells transfected with a nonspecific siRNA (Ctlr-siRNA), or mock-transfected cells (Fig. 2), indicating that the decrease in IRES-dependent translation does not reflect a general decrease in translation. Taken together, these results bolster the conclusion that La protein enhances IRES activity in vivo.
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15%) was still detected (Fig. 3A, lane 5). Thus, the magnitude of inhibition at the level of protein synthesis is dramatically amplified at the downstream step of replication. The specific effect of La(226-348) was confirmed in an in vitro translation extract from HeLa cells using the bicistronic construct RLuc-poliovirus-Fluc mRNA in which only poliovirus IRES-driven translation was reduced (data not shown). Taken together, our data show that inactivation of La renders HeLa cell extract defective in supporting poliovirus translation and in turn virus replication.
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HCV IRES-driven translation is impaired in La85-siRNA-treated cells. Since HCV does not grow in cultured cells (3), we used a poliovirus-HCV(IRES-core) chimeric model (44, 77) to assess the requirement of La for HCV IRES activity in vivo. The poliovirus-HCV chimera (P/H 701-2A) contains the 5' cloverleaf structure of poliovirus, followed by the HCV IRES (nucleotides 9 to 332), the first 369 nucleotides of the HCV core region, the entire poliovirus ORF, 3'UTR, and the poly(A) tail (77). In HeLa cells that were depleted of La by siRNA, the P/H 701-2A chimera grew to a titer that was approximately sevenfold lower than that for control cells (see Fig. S1C and D in the supplemental material). In addition, LaDN drastically inhibited translation of the poliovirus-HCV chimera mRNA in HeLa cell extracts (data not shown).
La protein promotes the initiation of formation of 48S and 80S ribosome complexes on poliovirus and HCV IRESs.
To further characterize La function in translation initiation of poliovirus mRNA, 80S ribosome binding assays were performed with poliovirus mRNA, and the effect of the La(226-348) dominant-negative mutant was examined. Initiation of formation of poliovirus 80S complexes, albeit inefficient, in agreement with earlier data (22), was inhibited by the addition of the La(226-348) dominant-negative mutant (Fig. 5A) (the position of the 80S ribosome initiation complex was determined by sedimenting a pure preparation of 80S ribosomes in a parallel tube). Thus, La plays a critical role in the initiation of formation of 80S ribosome complexes. Next, to determine the importance of La in the initiation of formation of 80S ribosome complexes on HCV IRES, the effect of the LaDN mutant on ribosome binding was examined. The addition of the GST-La(226-348) mutant resulted in a significant decrease (60%, three replicate samples) in viral RNA binding to ribosomes (Fig. 5B). In sharp contrast, binding of globin mRNA to ribosomes, which is not known to be dependent on La, was not affected by the LaDN mutant (Fig. 5C) (the second sedimenting complex [fractions 18 to 25] in Fig. 5C most likely represents disomes [24], but this complex has not been characterized further). To rule out the possibility that the ribosome binding inhibition seen by GST-LaDN is due to the GST tag, we cleaved the tag from the protein, and after purification, the untagged LaDN was tested for inhibiting the initiation of formation of 80S ribosome complexes. Untagged LaDN also inhibits HCV 80S ribosome binding in a dose-dependent manner (see Fig. S2 in the supplemental material). Thus, La protein is a specific ITAF for poliovirus and HCV IRESs but is not required for globin mRNA translation.
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45%, two replicate samples) the binding of the 40S ribosomes to the HCV IRES (Fig. 6). Thus, the LaDN mutant inhibits the initiation of formation of complexes by preventing the recruitment of the 40S ribosomal subunit. In contrast, GST had no effect on 48S ribosome complex formation (Fig. 6). Similar results were obtained with the 48S ribosome initiation complex for poliovirus mRNA (data not shown). These results indicate that the La protein is directly involved in the initiation of formation of 48S ribosome complexes on poliovirus and HCV mRNAs.
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DISCUSSION |
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The physiological significance of earlier results demonstrating the importance of La in vitro was questioned because of the large amounts of La used in the earlier studies (35-37, 72). There are several possible explanations for this unusual requirement. First, the recombinant protein may have been rendered partially inactive during the purification process. Second, the effector domain (C-terminal region) of La is known to undergo phosphorylation (18), and this modification (which does not occur in the recombinant protein) could be important for optimal activity of the protein. Indeed, different RNAs are preferentially associated with the phosphorylated form of La compared to the nonphosphorylated form, and a fraction of the phosphorylated form of La is cytoplasmic (33). Finally, poliovirus causes the cleavage of La (62), which is then redistributed to the cytoplasm (34, 48), raising the possibility that cleaved La is more potent than wild-type La in translation.
What is the molecular mechanism by which La stimulates 40S ribosome recruitment? Our data demonstrate that a LaDN mutant protein inhibits 80S (Fig. 5) and 48S initiation complex formation (Fig. 6) on HCV and poliovirus mRNAs. The use of a highly fractionated system in combination with a toe-printing assay would allow further characterization of molecular mechanisms underlying this inhibition. Originally, it was suggested that La functions as an RNA chaperone to fold the IRES into a structure that can recruit initiation factors, such as eIF4G (6, 49, 65). Indeed, studies in yeast show that the yeast homologue of La protein (LHP1) functions as a molecular chaperone as it promotes the refolding of a mutant Met-tRNA (74). Alternatively, it is also possible that La stimulates translation indirectly by displacing an inhibitory protein from the IRES. In addition, La might aid in the recruitment of ribosomes in a more direct fashion, since human La protein has been reported to sediment with the 40S ribosomal subunit and to bind the 18S rRNA (52).
In summary, we determined that La functions to recruit the 40S ribosome subunit to the viral mRNA. In addition, we provided direct evidence based on in vivo studies that La protein is a bona fide ITAF for poliovirus and HCV. These results have important implications for studies on inhibition of virus replication in cells. Inhibition of poliovirus and HCV replication has been reported for siRNAs directly targeting poliovirus (21) or HCV RNA (40, 59, 71, 73). However, viruses are likely to evade siRNA by adaptive mutations of the target sequences (20). To circumvent this problem, it would be important to design siRNAs against cellular factors, such as La protein, which are required for optimal virus replication. We expect that the knowledge gained from this study will aid in the understanding of how cellular proteins may regulate IRES activity and might also be important in the design of new IRES-targeted antiviral strategies.
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ADDENDUM IN PROOF |
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Canadian Institute of Health Research (CIHR) to N.S., who is recipient of a CIHR Distinguished Scientific Award and a Howard Hughes Medical Institute International Scholarship. M.C.-M is a postdoctoral fellow supported by a CIHR fellowship.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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