Internal Initiation of Translation of Bovine Viral Diarrhea Virus RNA

Virology 258, 249–256 (1999) Article ID viro.1999.9741, available online at http://www.idealibrary.com on

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provided by Elsevier - Publisher Connector Internal Initiation of Translation of Bovine Viral Diarrhea Virus RNA

Tatyana V. Pestova*,† and Christopher U. T. Hellen*,1

*Department of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York Health Science Center at Brooklyn, 450 Clarkson Avenue, Box 44, Brooklyn, New York 11203; and †A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia Received October 9, 1998; returned to author for revision December 9, 1998; accepted April 5, 1999

Initiation of translation on the bovine viral diarrhea virus (BVDV) internal ribosomal entry site (IRES) was reconstituted in vitro from purified translation components to the stage of 48S ribosomal initiation complex formation. Ribosomal binding and positioning on this mRNA to form a 48S complex did not require the initiation factors eIF4A, eIF4B, or eIF4F, and translation of this mRNA was resistant to inhibition by a trans-dominant eIF4A mutant that inhibited cap-mediated initiation of translation. The BVDV IRES contains elements that are bound independently by ribosomal 40S subunits and by eukaryotic initiation factor (eIF) 3, as well as determinants that mediate direct attachment of 43S ribosomal complexes to the initiation codon. © 1999 Academic Press Key Words: bovine viral diarrhea virus; IRES; pestivirus; RNA–protein interaction; translation.

INTRODUCTION of domain II, near nucleotide 75, and the 3Ј border of this IRES is downstream of the initiation codon (Chon Bovine viral diarrhea virus (BVDV) is the prototype of et al., 1998). The activity of the BVDV IRES is severely the genus Pestivirus of the Flaviviridae family. The impaired by mutations that affect the integrity of sub- other currently accepted members of this genus are domains between these borders (Poole et al., 1995; classical swine fever virus (CSFV) and border disease Chon et al., 1998). virus of sheep. The positive-sense RNA genomes of The first step in initiation of translation of all eukary- pestiviruses contain one long open reading frame otic mRNAs is formation of a 43S preinitiation complex flanked by 5Ј and 3Ј nontranslated regions (NTRs) (Collett et al., 1988; Meyers et al., 1989; Becher et al., that comprises a 40S subunit, initiator tRNA, GTP, and 1998). The 5Ј NTRs of BVDV, CSFV, and of the related eukaryotic initiation factors (eIFs) 2 and 3 (Merrick, Ј hepatitis C virus (HCV) promote translation of the viral 1992). Attachment of this complex to the 5 end of polyprotein by cap-independent internal ribosomal en- capped mRNAs requires eIF4A, eIF4B, and eIF4F and follows binding of the eIF4E subunit of eIF4F to the 5Ј try (Tsukiyama-Kohara et al., 1992; Poole et al., 1995; 7 Rijnbrand et al., 1997). IRES-mediated initiation of terminal m G cap (Merrick, 1992; Pestova et al.,1998a). translation was first identified in picornaviruses, such Attachment of the 43S complex to an internal position as encephalomyocarditis virus (EMCV), which now on the EMCV IRES also requires eIF4A, eIF4B, and the epitomizes the group of IRES elements that have been eIF4G subunit of eIF4F (Pestova et al., 1996a,b). At- characterized most extensively (Hellen and Wimmer, tachment does not require eIF4E; instead, eIF4G binds 1995; Jackson and Kaminski, 1995). Pestivirus IRES specifically to this IRES upstream of the initiation elements have been characterized less well. The codon and is thought to recruit eIF4A, eIF4B, and a CSFV IRES comprises most of the 5Ј NTR, which con- 43S complex to form the 48S complex at the initiation sists of major structural domains I, II, and III, and a codon (Pestova et al., 1996b; Kolupaeva et al., 1998). pseudoknot upstream of the initiation codon that is By contrast, the mechanism of initiation on HCV and essential for IRES activity (Wang et al., 1995; Lemon CSFV IRES elements differs significantly from all and Honda, 1997; Rijnbrand et al., 1997; Pestova et al., known modes of eukaryotic translation initiation. It is 1998b). The CSFV IRES is ϳ100 nt shorter than the exceptionally simple: 43S complexes bind directly to EMCV IRES and has an unrelated sequence and struc- CSFV and HCV IRES elements independently of eIFs ture. The 5Ј border of the BVDV IRES is at the 5Ј end 4A, 4B, and 4F (Pestova et al., 1998b). These two IRES elements contain determinants in domain III that mediate interaction with eIF3 (Buratti et al., 1998; Pestova 1 To whom correspondence and reprint requests should be ad- et al., 1998b; Sizova et al., 1998). In addition, they dressed. Fax (718) 270-2656. contain multiple, poorly defined determinants whose

coordinated action promotes direct factor-indepen- of prominent toeprints at UGA403–405 was absolutely de- Met dent binding to 40S subunits such that the initiation pendent on the presence of both Met-tRNAi (Fig. 2A, codon is positioned precisely at or in the immediate lane 5) and eIF2 (data not shown). We conclude that vicinity of the ribosomal P site (Pestova et al., 1998b). these toeprints are caused by assembly of a 48S initia- This activity is perturbed by mutations throughout the tion complex at the BVDV initiation codon. Their position HCV IRES (Wang et al., 1994, 1995; Lemon and Honda, relative to the BVDV initiation codon is identical to that of 1997; Hellen and Pestova, 1999; Pestova et al., 1998b); toeprints caused by 48S complexes assembled at the significantly, the IRES is the most conserved part of CSFV initiation codon that are competent to complete the the HCV genome (Simmonds, 1995). remaining stages in translation initiation up to and in- The BVDV 5Ј NTR differs from the CSFV and HCV 5Ј cluding formation of the first peptide bond (Pestova et al., ϳ NTRs at 35 and at over 50% of nucleotides, respectively 1998b). Prominent new toeprints at G388 and particularly

(Hellen and Pestova, 1999). Much smaller sequence dif- at UGA403–405 (indicative of 48S complex formation on the Met ferences between the IRES elements of poliovirus strains BVDV IRES) appeared when eIF3 and Met-tRNAi were or of EMCV variants are sufficient to cause significant added to assembly reactions that already contained changes in factor requirements for initiation (Svitkin et BVDV mRNA, 40S subunits, GMP-PNP, and eIF2 (Fig. 2A, al., 1988; Kaminski and Jackson, 1998). We report here lanes 4 and 5). The observation that new toeprints were that we have dissected the initiation process on the detected at the leading edge of the 40S subunit and at BVDV IRES using an in vitro approach in order to com- the initiation codon at this stage in initiation suggests pare it with initiation on related IRES elements and to that codon–anticodon basepairing is accompanied by a gain further insights into the nature of the RNA determi- small conformational change in the BVDV mRNA at and nants that mediate the different functions in initiation of downstream of the initiation codon. HCV-like IRESes. Neither the presence nor the intensity of the toeprints

at G388 and UGA403–405 was affected by eIF4A, eIF4B, and RESULTS eIF4F together (compare Figs. 2B, lanes 3 and 4, and 2A, 43S complexes binds directly at the initiation codon lanes 3 and 4). We conclude that 48S complex formation of the BVDV IRES on the BVDV IRES is absolutely dependent on eIF2 and does not require or involve eIF4A, eIF4B, or eIF4F. Ribo- To dissect internal ribosomal entry on the BVDV IRES, somal 48S complexes formed on the BVDV IRES in the we reconstituted this process in vitro to the stage of 48S absence of eIF4A, eIF4B, and eIF4F were competent to ribosomal initiation complex formation using purified complete all remaining stages in initiation, up to and translation components (40S ribosomal subunits, initia- including formation of the first peptide bond. Assembly of tion factors, aminoacylated initiator tRNA and mRNA). active 80S ribosomal complexes was mediated by a Ribosomal complexes were analyzed by toeprinting ribosomal salt wash fraction (the 02.–0.8 M KCl phospho- (primer extension inhibition), which involves cDNA syn- cellulose elution fraction derived from the 50–70% A.S. thesis by reverse transcriptase on a template RNA to cut) that did not contain eIF4A, eIF4B, or eIF4F (data not which a ribosome or protein is bound. cDNA synthesis is shown). Additional data that support the conclusion that arrested either directly by the bound ribonucleoprotein eIF4A, eIF4B, and eIF4F are not required for BVDV IRES- (RNP) complex, yielding a toeprint at its leading edge, or mediated initiation are described below. indirectly, by stabilization of adjacent helices. The result- ing toeprints are located on a sequencing gel, so that the Specific binding of eIF3 and 40S ribosomal subunits position of bound proteins or complexes can be located to the BVDV IRES precisely. The BVDV IRES is highly structured (Fig. 1) and as a We correlated each of the toeprints caused by 48S result, RT arrest occurs at several sites on naked BVDV initiation complexes with binding of a specific translation

RNA. These sites include A320,G327, and U352 (Fig. 2A, lane component to the BVDV IRES by systematically omitting 1). Ribosomal complexes were assembled from 40S sub- components and then toeprinting complexes formed in Met units, eIF2, eIF3, GMP-PNP, and Met-tRNAi on BVDV nt their absence. Ribosomal 40S subunits alone arrested Ј 29–1109 mRNA transcripts. GMP-PNP is a nonhydrolyz- primer extension at U362 at the 3 border of the able GTP analog that causes 48S complexes to accumu- pseudoknot, weakly at G381 and G388, as well as at late. These ribosomal complexes yielded many new or AUC395–397,G404, and strongly at AAA400–402 downstream of enhanced toeprints on BVDV mRNA, including weak to- the initiation codon AUG386–388 (Figs. 2A, lane 2, and 2B, eprints at AC260–261,A320,G381, and G388, stronger toeprints lane 2). The many toeprints attributable to binding of 40S at U362 and AUC395–397, and very prominent toeprints at subunits suggest that 40S subunits make multiple con-

AAA400–402 and particularly at UGA403–405 (Fig. 2A, lane 4). tacts with the BVDV IRES, both at or in the vicinity of the These results are summarized in Fig. 1. The appearance pseudoknot, and on both sides of the initiation codon. BVDV IRES-MEDIATED INITIATION OF TRANSLATION 251

FIG. 1. Model secondary and tertiary RNA structures of BVDV nucleotides 29–406 as suggested by Wang et al. (1995) and Lemon and Honda (1997). The nomenclature of domains is as described by Lemon and Honda (1997). The initiation codon is boxed. Primer extension toeprints on the IRES caused or enhanced by binding of eIF3 or 40S subunits individually and additional stops caused by binding of 48S complexes are indicated by symbols as in the key at upper right.

The presence of eIF3 in reconstituted assembly reac- a second assay to confirm the specificity of this interactions resulted in the appearance of toeprints at AC260–261 tion. Purified 40S subunits were incubated with radiola- and A320 and in weakening of toeprints at AAA400–402 rel- beled BVDV IRES-containing nt 29–1109 mRNA and then ative to those at UGA403–405 (Fig. 2A, lanes 3 and 4). The centrifuged through a 10–30% sucrose density gradient toeprints at AC260–261 and A320 were detected if 40S sub- to resolve free RNA from ribosomal complexes. Together, units were omitted from reactions and even if eIF3 alone 40S subunits and this BVDV RNA formed stable binary was incubated with BVDV RNA (Fig. 2A, lanes 6 and 7). (IRES–40S subunit) complexes (Fig. 3). Binding of 40S These results show that the BVDV IRES binds eIF3 spe- subunits to the BVDV IRES did not require the coding cifically. Analysis of complexes formed by binding of eIF3 region, the initiation codon, or nucleotides downstream to the apical half of CSFV domain III yielded toeprints at of the pseudoknot (Fig. 3). Under identical conditions, almost identical positions to those that we have de- 40S subunits did not bind to the EMCV IRES or to ␤-glo- scribed here for BVDV (Pestova et al., 1998b; Sizova et al., bin mRNA transcripts without initiation factors (Pestova 1998). This observation suggests that eIF3 binds to com- et al., 1998a,b) (Fig. 3). The 40S ribosomal subunits used mon determinants in these two IRES elements. in this experiment were therefore not contaminated by initiation factors or by nonspecific RNA-binding proteins. Assembly of stable binary (BVDV IRES–40S ribosomal The BVDV IRES therefore contains determinants that subunit) complexes enable it to bind directly and specifically to 40S subunits The results of toeprinting show that 40S subunits to form stable binary complexes without any requirement alone bind specifically to the BVDV IRES in vitro. We used for initiation factors, including eIF4A, eIF4B, or eIF4F, 252 PESTOVA AND HELLEN

FIG. 2. (A and B) Toeprint analysis of 48S ribosomal initiation complex formation on the BVDV IRES. Ribosomal preinitiation complexes were assembled on BVDV nucleotides 29 to 1109 RNA under standard reaction conditions in the presence of translation components as indicated and were then analyzed by primer extension. Full-length cDNA is marked E, and other cDNA products terminated at the sites indicated on the right. Reference lanes C, T, A, and G depict BVDV sequences; BVDV nucleotides are indicated on the right. which mediate attachment of ribosomal complexes to tion complexes bind to the BVDV IRES without eIF4A, capped mRNAs (Merrick, 1992; Pestova et al., 1998a). eIF4B, or eIF4F. BVDV mRNA translation would therefore This result is consistent with the results of toeprinting, be expected to be unaffected by eIF4A [R362Q]. which showed that 40S subunits make multiple contacts Preincubation of RRL with eIF4A [R362Q] prior to it with the BVDV IRES, both at or in the vicinity of the being programmed with EMCV-GUS mRNA alone or pseudoknot and flanking the initiation codon. together with BVDV mRNA abolished EMCV IRES-mediated translation of the GUS cistron (Fig. 4, lanes 1–3 BVDV translation is resistant to inhibition by trans- and 6–9). In parallel experiments, this mutant form of dominant eIF4A (R362Q) eIF4A inhibited translation of capped luciferase mRNA Ribosomal attachment to capped mRNAs and to the (data not shown). However, BVDV IRES-mediated EMCV IRES requires eIF4A, probably as a subunit of translation of the p20 cistron was unaffected by eIF4A eIF4F (Merrick, 1992; Pause et al., 1994; Pestova et al., [R362Q] (Fig. 4, lanes 4–9). Inclusion of wt recombi- 1998a). Free eIF4A and the eIF4A subunit of eIF4F are in nant eIF4A in translation reactions had no effect on equilibrium (Yoder-Hill et al., 1993), so that a translation of EMCV-GUS, luciferase, or BVDV mRNAs [R362Q]trans-dominant mutant form of eIF4A also rapidly (data not shown). These results indicate that eIF4A impairs the function of eIF4F (Pause et al., 1994). Trans- (and probably eIF4F) are not required for BVDV IRES- lation of mRNAs that require eIF4A, eIF4F, or both is mediated initiation and strongly support the results therefore inhibited by this eIF4A mutant (Pause et al., obtained by in vitro reconstitution of 48S complex 1994). Data presented above indicate that 43S preinitia- formation on this mRNA. BVDV IRES-MEDIATED INITIATION OF TRANSLATION 253

initiation by a common mechanism even though their sequences differ from one another by 35–50%. In general terms, translation initiation mediated by BVDV, CSFV, and HCV IRES elements is described by a model in which unpaired loops in the IRES constitute specific binding sites for 40S ribosomal subunits and translation initiation factors and in which helices play a primarily structural role, orienting these binding sites in such a way that their interaction with components of the translation apparatus leads to assembly of a 48S complex (Hellen and Pestova 1998; Pestova et al., 1998b; FIG. 3. Formation of binary ribosomal complexes on the BVDV IRES. Sizova et al., 1998). Pestivirus IRES elements contain Ribosomal complexes were assembled by incubating rabbit 40S sub- insertions relative to the HCV IRES (Lemon and Honda, units with either [32P]UTP-labeled BVDV or ␤-globin mRNAs and were analyzed by sucrose density gradent centrifugation. Sedimentation was 1997; Hellen and Pestova, 1999) but the majority of se- from right to left. The position of binary (40S subunit–BVDV IRES) quence differences between these IRES elements are complexes is indicated by an arrow. covariant substitutions that map to basepaired structures, so that sequence identity between these IRES elements is concentrated in internal and apical loops DISCUSSION (Hellen and Pestova, 1999). Observations that several We have used purified translation components to re- complementary sequences in HCV and CSFV IRES ele- constitute internal ribosomal entry on the BVDV IRES in ments can be replaced by alternative basepairing se- vitro to the stage of 48S initiation complex formation. This quences without loss of IRES activity are consistent with analysis simplifies the process of initiation on this mRNA the hypothesis that the role of conserved helices in these because it occurs in the absence of competing capped RNAs is primarily structural (Wang et al., 1994, 1995; mRNAs and RNA-binding proteins that are normal con- Reynolds et al., 1996; Lemon and Honda, 1997; Hellen stituents of the cytoplasm. However, this approach has and Pestova, 1999). enabled us to identify the minimal set of factors required The BVDV IRES contains determinants that enable it to for BVDV IRES-mediated initiation and to characterize bind directly to 40S subunits without any requirement for their role and that of the IRES in it. Ribosomal 43S initiation factors, including eIF4F, which mediates ribo- preinitiation complexes bound precisely at the BVDV somal attachment to capped mRNAs (Pestova et al., initiation codon without any requirement for eIF4A, 1998a) and to the EMCV IRES (Pestova et al., 1996a,b). eIF4B, or eIF4F. This conclusion was strongly supported Toeprints caused by 40S subunits bound to the BVDV by the observation that BVDV IRES-mediated translation IRES are located both within the pseudoknot and on was resistant to inhibition by an [R362Q] mutant form of either side of the initiation codon. However, binding of eIF4A that is a trans-dominant inhibitor of cap-mediated 40S subunits to this IRES does not require the coding translation as well as of translation mediated by poliovirus and EMCV IRES elements (Pause et al., 1994; Pestova et al., 1998b; Fig. 4). The mechanism of ribosomal attachment to the BVDV IRES therefore differs significantly both from the canonical mechanism of cap- mediated initiation and from initiation mediated by two well-characterized picornavirus IRES elements. How- ever, it is similar in many respects to the mechanism of initiation on HCV and CSFV IRES elements (Hellen and Pestova 1999; Pestova et al., 1998b). These similarities include the presence of independent binding sites for eIF3 and for 40S subunits on each of these three IRES elements, as well as of determinants that direct binding of 43S ribosomal complexes precisely to the initiation codon of the viral polyprotein (Buratti et al., 1998; Pestova et al., 1998b; Sizova et al., 1998; and this work). The 48S FIG. 4. Resistance of BVDV IRES-mediated translation to inhibition by complex formation on all three of these IRES elements trans-dominant [R362Q] mutant eIF4A. RRL was preincubated alone (lanes 1, 4, and 7), with [R362Q] mutant eIF4A (1.2 ␮g) (lanes 2, 5, and has no requirement for any noncanonical initiation fac- 8), or with [R362Q] mutant eIF4A (2.4 ␮g) (lanes 3, 6, and 9) and was tors nor for the canonical factors eIF4A, eIF4B, and eIF4F. then incubated with EMCV-GUS mRNA (lanes 1–3), BVDV-p20 mRNA We conclude that these three IRES elements mediate (lanes 4–6), or both mRNAs (lanes 7–9). 254 PESTOVA AND HELLEN region, the initiation codon, or nucleotides downstream recruit the factors eIF1 and eIF5 to the 48S complex of the pseudoknot (Fig. 3). Attachment of the BVDV IRES (Asano et al., 1998; Phan et al., 1998; Bandyopadhyay and to 40S subunits may therefore occur in two phases: initial Maitra, 1999); these two factors are involved in initiation binding of 40S subunits to primary determinants in the codon selection and in the release of factors from the pseudoknot or its vicinity is sufficient for formation of 48S complex, leaving initiator tRNA in the ribosomal P stable binary complexes and is followed by “accommo- site prior to subunit joining (Huang et al., 1997; Pestova et dation” or rearrangement of nucleotides flanking the ini- al., 1998b). tiation codon in the mRNA binding cleft of the 40S subunit so that the initiation codon is placed precisely in the METHODS ribosomal P site. The binding site for eIF3 has been mapped to the Transcription of mRNAs upper cloverleaf structure of domain III of BVDV, CSFV, Plasmids pTE3-GUS (Borovjagin et al., 1991), pBSϪ(␤- and HCV IRES elements by toeprinting (e.g., Fig. 2A). globin) (Hellen et al., 1992), and pT75Јp20 (Wiskirchen et Several nucleotides in this domain are absolutely con- al., 1991) were linearized at appropriate sites and then served in all pestivirus and HCV isolates; those centered used to transcribe EMCV-GUS, ␤-globin, and BVDV mR- on a bulge in the apical IIIb domain coincide exactly with NAs in vitro using T7 RNA polymerase and either with or nucleotides that are protected from chemical modifica- without [32P]UTP (ϳ3000 Ci/mmol; ICN Radiochemicals, tion and enzymatic cleavage by eIF3 (Hellen and Irvine, CA). RNA was purified using Nuc-Trap columns Pestova, 1999; Sizova et al., 1998). The binding site for (Stratagene, La Jolla, CA) as described (Pestova et al., eIF3 on these IRES elements is therefore likely to consist 1991). Radiolabeled RNAs had specific activities of of a conserved structural “scaffold” formed by the helices ϳ400,000 cpm/␮g. IIIa, IIIb, and IIIc and of these conserved unpaired nucleotides. Mutations in the HCV IRES that impair binding Purification of factors and 40S ribosomal subunits of eIF3 are localized to this irregular “cloverleaf” and all The 40S ribosomal subunits and eIF2, eIF3, and eIF4F reduce its activity (Buratti et al., 1998; Pestova et al., were purified from rabbit reticulocyte lysate (RRL; Green 1998b; Sizova et al., 1998). Deletion of subdomain IIIa Hectares, Oregon, WI) as described (Pestova et al., and most of subdomain IIIb strongly reduced the activity 1996a,b, 1998a,b). No cross-contamination of these of the BVDV IRES (Poole et al., 1995; Chon et al., 1998). translation components was detected by Western blot- eIF3 is involved in different stages in initiation on ting using specific antibodies and functional assays de- pestivirus IRES elements, including formation of 48S pendent on each of these factors (Pestova et al., 1996a,b, complexes and their subsequent joining to 60S ribo- 1998a,b). Recombinant eIF4A, [R362Q] mutant eIF4A, and somal subunits to form active 80S ribosomes. eIF3 en- eIF4B proteins were expressed in Escherichia coli and hances 48S complex formation on the BVDV IRES in vitro purified as described (Pause et al., 1994). (Fig. 2) and probably also does so in vivo. In the cytoplasm, eIF3 is stoichiometrically associated with free Assembly and analysis of ribosomal complexes 40S ribosomal subunits and is therefore effectively a constitutive component of native 40S subunits (Sundkvist Ribosomal complexes were assembled on 1 ␮gof and Staehelin, 1975). The affinity of 40S subunits and of BVDV mRNA in reaction volumes of 40 ␮l that contained Met eIF3 for distinct structural elements in pestivirus IRES 0.4 mM GMP-PNP and Met-tRNAi (6 pmol), 40S sub- elements may therefore enhance binding of native 40S units (6 pmol), eIF2 (3 ␮g), and eIF3 (7 ␮g), as indicated 35 Met subunits to these viral RNAs in the cytoplasm, where in the text. [ S]Methionine-labeled Met-tRNAi was pre- they compete with cellular mRNAs for the translation pared using rabbit tRNA (Novagen, Madison, WI) and apparatus. The presence of binding sites for two different aminoacyl tRNA synthetase purified from E. coli MRE600 components of native 40S subunits on these IRES ele- (American Type Culture Collection, Manassas, VA), as ments is also likely to promote ribosomal attachment to described (Pestova et al., 1996a). These ribosomal and them in an orientation that promotes entry of the initia- RNP complexes were analyzed by primer extension, tion codon into the ribosomal P site. This binding mech- done using the primer 5Ј GCTTTAGCGTCGATTG-3Ј (com- anism would explain the unusual mode of initiation on plementary to BVDV nt 524–509) and avian myeloblasto- these IRES elements by direct assembly of 80S ribo- sis virus reverse transcriptase (Promega, Madison, WI) in somes at the initiation codon without prior scanning from the presence of [␣-32P]dATP (ϳ6000 Ci/mmol) as de- an upstream position (Reynolds et al., 1996; Rijnbrand et scribed (Pestova et al., 1996a, b). cDNA products were al., 1996; Pestova et al., 1998b). eIF3 enhances 48S com- ethanol-precipitated, resuspended and analyzed by elec- plex formation, but is absolutely required for subsequent trophoresis through 6% polyacrylamide sequencing gels. subunit joining to form active 80S ribosomes (Pestova et cDNA products were compared with appropriate al., 1998b). A likely function of eIF3 in this process is to dideoxynucleotide sequence ladders. BVDV IRES-MEDIATED INITIATION OF TRANSLATION 255

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