JOURNAL OF VIROLOGY,0022-538X/01/$04.0010 DOI: 10.1128/JVI.75.16.7629–7636.2001

Aug. 2001, p. 7629–7636 Vol. 75, No. 16

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

The Ribosome Binding Site of Hepatitis C Virus mRNAJ. ROBIN LYTLE, LILY WU, AND HUGH D. ROBERTSON*

Department of Biochemistry, Weill Medical College of Cornell University,New York, New York 10021

Received 12 February 2001/Accepted 17 May 2001

Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, the majority of whom developa chronic infection which can lead to severe liver disease, and for which no generally effective treatment yetexists. A promising target for treatment is the internal ribosome entry site (IRES) of HCV, a highly conserveddomain within a highly variable RNA. Never before have the ribosome binding sites of any IRES domains,cellular or viral, been directly characterized. Here, we reveal that the HCV IRES sequences most closelyassociated with 80S ribosomes during protein synthesis initiation are a series of discontinuous domainstogether comprising by far the largest ribosome binding site yet discovered.

Nearly all patients infected with hepatitis C virus (HCV)develop a persistent infection that can progress to cirrhosis,hepatocellular carcinoma, and liver failure. Limited therapy isavailable, and a large number of viral isolates are resistant.HCV is able to avoid host defenses in part due to the highvariability among different strains and isolates. The develop-ment of better therapies has also been hindered by the lack ofan established in vitro cell culture system and a useful labora-tory animal model.

HCV has a positive-strand RNA genome about 9,600 baseslong (12). The most conserved and highly structured portion ofthe HCV genome includes the 59 untranslated region (59 UTR)and about 30 bases of coding sequence (14, 15, 32) which havebeen shown to contain an internal ribosome entry site (IRES)(6, 14). Despite the lack of a 59-terminal cap, protein synthesisstill initiates, although at an AUG codon downstream fromseveral others. IRESs have been found in both viral genomesand cellular mRNAs (15, 24, 26), yet they fail to share signif-icant primary sequence homology. These IRESs range in sizefrom 300 to 1,500 nucleotides and possess both secondary andtertiary structural elements. Since there is no extensive homol-ogy among IRES domains from viral or cellular mRNAs, high-er-order structure along with limited and widely separatedprimary sequence elements may combine to create IRES ribo-some recognition sites (13, 14).

Beginning with work by Steitz (31) and others (9, 28), theisolation and sequence analysis of ribosome binding sites(RBSs) from radioactive polycistronic mRNAs of prokaryoteshave been used to define the mRNA sequences primarily re-sponsible for bringing a ribosome into the correct orientationfor translation (25). An RBS is the ribosome-protected regionof an mRNA under conditions which would otherwise lead tocomplete mRNA solubilization. RBSs remain attached to theribosome and, thus, are separated from the bulk of the mRNAmolecule by sedimentation after exhaustive digestion of initi-ation complexes with RNase (usually pancreatic RNase A) (23,

25). A similar approach was used to isolate RBSs from cappedmonocistronic mRNAs of eukaryotes (18–22).

Studies on HCV IRES sequences and ribosomal subunitshave so far concentrated on 40S preinitiation complexes (17,27). No direct characterization of the 80S RBSs from HCV, orany IRES-containing mRNA, has yet been reported. We re-port our discovery that a series of discontinuous sequencescombine to form a ribosome binding domain many times theconventional size, and we compare these findings to 48S preini-tiation complexes.

MATERIALS AND METHODS

In vitro transcription. The plasmid pN(1–4728) containing the first 4.7 kb ofHCV sequence adjacent to the phage T7 promoter was a gift from StanleyLemon, University of Texas, Galveston. The plasmid was cleaved by three dif-ferent restriction enzymes: AatII, SacII, and BamHI. When these templates weretranscribed in vitro, three 32P-labeled RNAs spanning bases 1 to 402, 1 to 645,and 1 to 1349 were produced. The plasmid pbHb containing rabbit b-globincDNA was a gift from Karen Browning, University of Texas, Austin. This plasmidwas cleaved with HindIII to produce the proper template for transcription. Thetranscription reactions are based on earlier published work from this laboratory(2, 7), and specific activities of 4.13 3 106 or 8.25 3 107 dpm/mg were generallyused. [a-32P]GTP (NEN Life Science Products, Boston, Mass.) was used in alllabeled transcriptions unless noted otherwise.

Ribosome binding. Ribosome binding reactions followed the standard trans-lation protocol of Promega (Madison, Wis.). Each sample contained 35 ml ofnuclease-treated rabbit reticulocyte lysate (RRL) (Promega), 1 ml of amino acidmixture (Promega), and enough water so that the final reaction volume equaled50 ml. If necessary, 10 mM EDTA was added to control reactions at this time(34). Each reaction mixture was incubated at 30°C, and 100 mM anisomycin orsparsomycin was added after 1 min. Edeine (1 mM) or aurin tricarboxylic acid(100 mM) was added after 4.5 min of incubation. Edeine was a gift from Z.Kurylo-Borowska. After 5 min at 30°C, 1 mg of radiolabeled mRNA was added.The samples were incubated for another 10 min (15-min total incubation) andthen immediately placed on ice. Pancreatic RNase A was added to each sample(25.6 mg/ml [final concentration] for HCV RNAs and 9.6 mg/ml for b-globinRNA) followed by incubation on ice for 15 min.

Sucrose density gradient analysis. After 15 min on ice, 250 ml of gradientbuffer (25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl [pH 7.5], and1 mM dithiothreitol) was added to each reaction and the samples were loadedonto 5-ml 15 to 30% sucrose gradients using the same gradient buffer. Gradientbuffer similar to that described above, but without MgCl2 and containing 10 mMEDTA, was used for both dilution of samples and formation of 5-ml 15 to 30%sucrose gradients containing EDTA. All samples were then centrifuged for 2 h at2°C and 45,000 rpm (189,378 3 g average). Two-drop fractions were collected asdescribed before (28, 31), and those corresponding to the appropriate peakswere pooled. The total volume containing the 80S or 48S peaks was approxi-mately 1 ml. Protected RNA was recovered from pooled fractions as described

* Corresponding author. Mailing address: Room E-013, Depart-ment of Biochemistry, Weill Medical College of Cornell University,1300 York Ave., New York, NY 10021. Phone: (212) 746-6400. Fax:(212) 746-8144. E-mail: hdrober@mail.med.cornell.edu.

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FIG. 1. HCV RNAs of 645 bases and b-globin RNAs of 602 bases bind to 80S ribosomal initiation complexes. (A and B) Sucrose gradientsedimentation profiles of radiolabeled 645-base HCV or 602-base b-globin RNAs incubated in RRL containing anisomycin, treated with RNase,and centrifuged in sucrose density gradients containing 1 mM MgCl2. (C and D) Sedimentation profiles of radiolabeled HCV or b-globin RNAs

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before (20–22). Pooled 80S or 48S peaks were added to 1 ml of phenol containing0.5 g of urea, 10 ml of mercaptoethanol, 10 ml of 10% sodium dodecyl sulfate, and10 ml of 200 mM EDTA at ambient temperature. The mixture was vortexed, 1 mlof chloroform was added, and after more vortexing, the phases were separated bycentrifugation. The aqueous layer was collected and ethanol precipitated.

RNA fingerprinting. RNA fingerprinting, a technique in which RNA mole-cules are digested with RNase T1 (Calbiochem, San Diego, Calif.) and thedigestion products are separated in two dimensions by charge and size, has beendescribed previously (3).

RESULTS

80S ribosomal complex formation on HCV and b-globinRNAs. Internally labeled transcripts containing the completeHCV 59 UTR and various lengths of HCV mRNA codingsequence were used to form 80S initiation complexes in RRL.Eukaryotic b-globin mRNA was used as a control since its RBShas already been characterized (21, 22). To interrupt the trans-lation process at the point of first peptide bond formation,translation inhibitors, anisomycin and sparsomycin, wereadded to the reactions (33). At the concentrations used here,these inhibitors allow the 80S ribosomal complex to bind to thestart codon but prevent translation elongation on cappedmRNAs beyond the dipeptide state, thus permitting a stable80S ribosomal-mRNA complex to form (20–22). The radiola-beled RNAs were incubated in the lysate in the presence ofeither anisomycin or sparsomycin, treated with RNase A tosolubilize all parts of the mRNA not protected by ribosomes,and loaded on 15 to 30% sucrose gradients for centrifugation.

RNA sedimentation profiles were obtained for HCV andb-globin RNAs after collecting fractions and determining theirradioactivity (Fig. 1). When the RNAs are centrifuged alone,without any RNase treatment, they both are found in the topof the gradients (Fig. 1K and L). In the presence of anisomycinand after treatment with RNase, peaks corresponding to the80S ribosomal initiation complex and protected radiolabeledRNA form and can be seen in the profiles of both HCV andb-globin RNAs (Fig. 1A and B). Similar results were seenwhen sparsomycin was added to the reaction (data not shown).These 80S ribosomal complexes cannot form when the RRL ispretreated with 10 mM EDTA (Fig. 1C and D). Similarly,these complexes dissociate when centrifuged in sucrose densitygradients containing 10 mM EDTA (Fig. 1E and F). Whenfractions from 80S regions obtained with and without EDTAwere quantified from a scaled-up preparation, the EDTA-treated samples had only 4 to 5% of the radioactivity found inthose obtained with the complete system. Thus, recovery of thevast majority of the 80S-protected RNA fragments requiresprotein synthesis initiation.

Interestingly, the HCV profile of the RNA incubated inRRL containing anisomycin (Fig. 1A) or sparsomycin (datanot shown) also contains a peak corresponding to a 40S sub-

unit-mRNA complex (fractions 18 to 22) not observed withb-globin mRNA (Fig. 1B) (21, 22). Furthermore, addition ofthe translation inhibitor aurin tricarboxylic acid, which hasbeen shown to block all binding of globin mRNA to rabbitreticulocyte ribosomes (20–22, 33), abolishes both globin andHCV 80S ribosome-protected peaks but allows the HCV 40Speak to persist (Fig. 1G and H). In the presence of edeine,which allows 48S complexes to accumulate but prevents 60Ssubunit association, similar 48S peaks are observed for bothHCV and b-globin mRNAs (Fig. 1I and J).

Analysis of HCV and b-globin protected RNA fragments.The fractions containing the 80S ribosome peaks were pooled,and the radioactive, protected RNA comprising the RBSs wasisolated (20–22). These RNAs were then electrophoresed on adenaturing polyacrylamide gel (Fig. 2). The b-globin RNAprotected by the 80S ribosomal initiation complex (Fig. 1B) isfour bands approximately 40 to 35 bases in length (Fig. 2, leftlane). In contrast, the protected HCV RNA is up to 30 bandsvarying in length from more than 100 bases to approximately 9bases. It is evident that many more bases of sequence areprotected in the HCV IRES than in the b-globin mRNA. Byidentifying these IRES sequences, we will have the first directindication of how the IRES specifies ribosome binding.

The protected RNA fragments were then subjected to RNAfingerprinting analysis (3). These b-globin mRNA fragmentsand the control intact b-globin mRNA were fingerprinted (Fig.3A and B). The complex fingerprint pattern of the intact con-trol (Fig. 3A) is greatly simplified for the ribosome-protectedRNA (Fig. 3B), signifying that much of the 600-base RNA wasnot protected and is missing from the pattern. The spots fromthe 80S-protected b-globin mRNA fingerprint were eluted,and secondary RNase digestions were performed to determinethe sequence of each RNase T1-resistant oligonucleotide. Thepresence of the previously reported 80S ribosome-protectedsequence (21, 22) was thus confirmed and found to containbases 43 to 82 of rabbit b-globin mRNA, a sequence 40 basesin length (see Fig. 5A). The shorter bands seen in the left laneof Fig. 2 contain this same sequence minus a few bases fromeither end, a “frayed” end. The shortest protected band isdefined as the “core” sequence. The intact HCV mRNA (Fig.3C) and the 80S ribosome-protected fragments (Fig. 3D) werealso fingerprinted. A simpler pattern is seen in the 80S ribo-some-protected HCV fingerprint.

The HCV RBS contains a series of discontinuous fragments.The gel pattern for the HCV RBS (Fig. 2, right lane) is repro-ducible, and each band was analyzed in detail. On two occa-sions, separate HCV or b-globin mRNA transcripts labeledwith [a-32P]GTP or [a-32P]CTP were analyzed. In the case ofthe HCV RBS, at least 12 gel band sequences (Fig. 2, rightlane) were characterized in each of the four preparations. Each

incubated in RRL containing anisomycin and 10 mM EDTA, treated with RNase, and centrifuged in sucrose density gradients containing 1 mMMgCl2. (E and F) Sedimentation profiles of radiolabeled HCV or b-globin RNAs incubated in RRL containing anisomycin, treated with RNase,and centrifuged in sucrose density gradients containing 10 mM EDTA. (G and H) Sedimentation profiles of radiolabeled HCV or b-globin RNAsincubated in RRL containing anisomycin and ATA, treated with RNase, and centrifuged in sucrose density gradients containing 1 mM MgCl2. (Iand J) Sedimentation profiles of radiolabeled HCV or b-globin RNAs incubated in RRL containing edeine, treated with RNase, and centrifugedin sucrose density gradients containing 1 mM MgCl2. (K and L) Sedimentation profiles of radiolabeled HCV or b-globin RNAs incubated in RRLand centrifuged in sucrose density gradients containing 1 mM MgCl2. Both the HCV and b-globin RNAs are of approximately equal lengths, about600 bases. All profiles show the counts per minute plotted against the fraction number of the gradient. The first fraction is taken from the bottomof the gradient, and the last is taken from the top. The 80S and 48S ribosomal peaks are indicated.

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of the individual gel bands of HCV protected RNA was elutedand fingerprinted (data not shown). Using secondary RNasedigestions of the RNase T1-resistant oligonucleotides of eachfingerprint (1, 3), the sequence of each purified 80S ribosome-protected fragment was determined. The termini of these se-quences identified points of RNase cleavage within, or at theborders of, HCV RBS elements. The longest protected se-quence contains 128 bases, and the shortest contains 9 bases.All of the protected fragments fall between bases 124 and 392of the HCV map (Fig. 4). The largest fragment covers bases204 to 331, which contain the right side of stem-loop III andmuch of the pseudoknot region. Another large fragment con-tains the left side of stem-loop III from bases 124 to 191.Another fragment contains the start codon and 36 bases ofdownstream coding sequence. The sequences recovered fourtimes or more from the HCV IRES, either alone or as part ofa larger fragment, are defined here as core sequences. Theseprotected fragments must interact to form a specific ribosomebinding domain.

The RBS of the approximately 600-base b-globin mRNA is40 bases in length (blue box) with the start codon located at

bases 56 to 58 (Fig. 5A). The core sequence (orange box), thesmallest protected fragment, is 35 bases in length. In contrast,the RBS of the HCV genomic mRNA, drawn to a similar scale,comprises two segments 68 and 189 bases long, respectively(blue boxes), separated by a 12-base region of unprotectedRNA. The start codon is located at bases 342 to 344. The coreelements of the HCV RBS, the most frequently protectedsequences, consist of five noncontiguous RNA segments (orange

FIG. 2. The 80S ribosomal initiation complex protects many frag-ments of HCV RNA of various lengths. The protected RNA fragmentsfrom the 80S peaks of both b-globin (left lane) and HCV (1 to 1349)(right lane) mRNAs were isolated, electrophoresed on a 15% dena-turing polyacrylamide gel, and subjected to autoradiography. The 80Sribosomal complexes accumulated after treatment with sparsomycin.O, origin; XC, xylene cyanol; Bf, bromophenol.

FIG. 3. The RNA fingerprints of the 80S ribosomal complex-pro-tected b-globin and HCV mRNAs are much simpler. (A) Intact b-glo-bin mRNA was subjected to RNA fingerprinting. The radiolabeledRNA was subjected to exhaustive RNase T1 digestion. The resultingoligonucleotides then were separated in the first dimension by charge(from right to left) and in the second dimension by size (from bottomto top). (B) The protected b-globin RNA fragments were isolated fromthe 80S ribosomal initiation complex peak (like that of Fig. 1B) andsubjected to RNA fingerprinting. The 80S ribosome-protected mRNAproduces a simpler pattern than does the intact control mRNA. (C)Control HCV mRNA containing bases 1 to 1349 was fingerprinted.(D) HCV mRNA (bases 1 to 1349) from the 80S ribosomal peak (likethat of Fig. 1A) was fingerprinted. This fingerprint pattern is muchsimpler than the control shown in panel C.

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boxes) which contain a total of 108 bases, as follows: 128 to 138(Fig. 5A, domain 1), 145 to 156 (Fig. 5A, domain 2), 237 to 249(Fig. 5A, domain 3), 286 to 324 (Fig. 5A, domain 4), and 342to 373 (Fig. 5A, domain 5). Each core is substantially or com-pletely protected from RNase digestion and was recoveredboth alone and covalently linked to additional RNA segments(Fig. 4). Two kinds of noncore elements are recovered only aspart of larger segments containing at least one core element.Noncore elements are sometimes cleaved by RNase A duringRBS isolation, although much less frequently than the rest ofthe HCV mRNA. One subset of HCV noncore elements hasnot been observed to undergo cleavage. The three segmentscomprising this group are indicated by pairs of vertical arrowsconnected by horizontal dashed lines (Fig. 5A).

The protected regions of the HCV IRES are shown in blue,and the core sequences are shown in orange (Fig. 5B). Coredomain 114 contains the pseudoknot of the HCV IRES, coredomain 213 is located in the base-paired region below stem-loops IIIa and IIIc, and core domain 5 contains the initiatorAUG codon and is the most similar to the globin mRNA coredomain. Arrows indicate termini of noncore elements lackingcleavage sites (Fig. 5).

As previously described in the literature (21), the edeine-induced 48S ribosomal complex protected a larger region ofb-globin mRNA of approximately 60 bases in length (Fig. 6,lanes 1 and 3). The 48S-protected HCV RNA fragments, likethe 80S-protected RNA fragments, range in length from 10bases to over 100 bases (Fig. 6, lane 4). These protected frag-ments were isolated and characterized in a manner similar tothat described for the 80S-protected fragments (data notshown). The largest protected fragment lies between bases 259and 355. Another large fragment covers bases 125 to 192. The48S-protected region is very similar to that protected by theentire 80S ribosomal complex. However, in contrast to theRBS, the most 39 protected base is nucleotide 355, whichcorresponds with the end of stem-loop IV. No other down-stream nucleotides are protected. Otherwise, the 48S-pro-tected region, like the RBS, contains stem-loop III minus theapical loop, the pseudoknot region, and stem-loop IV.

DISCUSSION

We report here the initial results of our studies involvingdirect analysis of the interaction between mammalian ribo-somes and the HCV IRES using the conventional cappedb-globin mRNA for comparison. Under conditions of pancre-

atic RNase digestion which lead to complete solubilization ofboth HCV mRNA and the control globin mRNA species, wefind, in both cases, specific protection of RBS domains byinitiating 80S ribosomes. The globin mRNA RBS, under thetranslation-blocked conditions used here, is typical of a con-ventional capped mRNA (18–22), comprising a 40-base regionwith a 35-base core domain containing the AUG start codon(Fig. 5A). In contrast, the RBS sequences of the HCV IREScontain substantially more bases (257) than those of the globinmRNA RBS, including five core elements totaling 108 bases.Unlike globin RNA, in which the start codon is present in allRNase-protected fragments, many of the HCV IRES-pro-tected fragments do not contain the initiator AUG codon.

It is clear that, even when we adopt the conventional sec-ondary-structure folding pattern for the HCV IRES (Fig. 5B)(4, 10, 11), we see three separate core domains: 114, contain-ing the pseudoknot structure; 213, a duplex stem betweenstem-loop IIId and stem-loops IIIb and IIIc; and 5, whichcontains the AUG start codon and the first 30 bases of codingsequence. These results suggest to us a minimum of threedifferent sites on the 80S ribosome which protect substantialIRES core sequences while ensuring accurate initiation. In thisregard, it is likely that core sequence 5 occupies the ribosomalmRNA binding groove with its AUG triplet precisely alignedwith initiator tRNA in the P site. HCV IRES core domain 5would thus occupy the position analogous to that of the RBS ofa capped mRNA.

Which factors or ribosomal proteins are responsible for pro-tection of such a large amount of the HCV IRES RNA? TheHCV IRES has a distinctive mechanism for ribosomal complexformation (27) and, therefore, may require novel factorsand/or a heretofore unknown order of assembly; in this sense,the HCV-80S ribosomal initiation complex may be unlike typ-ical 80S ribosomal complexes with capped mRNAs and maycontain unknown factors or factors normally not present afterribosomal complex initiation. Since our 80S ribosomal com-plexes do not include purified components and are formed inRRL, all cellular factors or proteins are available for the per-haps unique HCV-80S ribosomal initiation complex formation.In this light, perhaps the ribosomal entity responsible for pro-tecting core domain 213 could be eukaryotic initiation factor3 (eIF3). While this initiation factor is thought to exit theribosome upon 60S subunit addition in the binding of conven-tional capped mRNAs (8), the HCV IRES shows a strong anddirect binding reaction to purified eIF3 (5, 17, 27, 29). There-fore, an HCV IRES domain remaining bound to eIF3 could

FIG. 4. The 80S-protected fragments of HCV mRNA are discontinuous and noncontiguous. The HCV RNA genome is depicted here as asingle thick black line. The recovered, protected fragments are depicted above the genome as thin black lines. All protected fragments lie withinbases 124 to 392. No protected fragments were found between bases 191 and 202.

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lead to a delayed exit of this factor from 80S ribosomes, re-sulting in the protection of core domain 213 observed here.

The protection from RNase of our core domain 114, whichcontains the HCV IRES pseudoknot region, leads us to pos-tulate the involvement of a third ribosomal domain. We havefound in collaboration with J. Gomez and colleagues of Bar-celona, Spain, that the host pre-tRNA processing enzymeRNase P can cleave HCV RNA at two sites, one of which islocated in the HCV IRES near the initiator AUG codon (A.Nadal, M. Martell, J. R. Lytle, H. D. Robertson, B. Cabot, J. I.Esteban, R. Esteban, J. Guardia, and J. Gomez, submitted forpublication). Since RNase P, in the absence of external guidesequences, cleaves only tRNA-like structures, this result sug-gests that a tRNA-like structure exists within the HCV IRES ata site near our pseudoknot-containing core domain 114. Ri-bosomes bind tRNA at three sites: the A site, the P site, andthe E site. We suggest that this tRNA-like domain of the HCVIRES, and perhaps analogous domains in other IRESs, couldoccupy one of the tRNA binding sites of the initiating 80Sribosome.

Our work here focuses on the HCV RNA protected by 80Sribosomal initiation complexes, while work done by others hasdetermined locations of HCV RNA involved in 40S preinitia-tion complexes (with or without additional factors) (17, 27).Significantly, several of our domains have already been shownto be important regions for binding. Core domain 3 has beenshown to be involved in eIF3 binding to the HCV IRES (27).Similarly, bases from our core domains 4 and 5 have beenshown to interact with the 40S subunit alone using the indirecttoeprinting assay to measure 40S subunits’ ability to blockprimed DNA synthesis (27). Several laboratories have deter-mined stem-loop IIId to be important for translation (16, 17),and we find these bases in a protected, but not core, domain.This region may be more important for an earlier initiationstep in protein synthesis and not as strongly protected at thepeptide formation stage.

The HCV RNA sequences protected by the 48S preinitia-tion complex (Fig. 1I and J) are very similar to those protectedby the 80S ribosomal complex. This result is similar to thatdescribed previously for b-globin mRNA: the 48S-protectedregion of b-globin mRNA contains the entire RBS sequencebut also additional 59 sequence from the 59 UTR (21). Initially,the large amount of 48S-protected HCV RNA may seem sur-prising. However, the recent cryoelectron microscopy mapmade by Spahn et al. (30) of the HCV IRES complexed withthe 40S ribosomal subunit shows that almost all of the HCVRNA binds to the 40S subunit on the side opposite where the60S subunit associates. Only stem-loop II wraps around the40S subunit to the 60S side, and this domain has so far notbeen shown to be protected from digestion by either the 48S orthe 80S complex. This finding implies that the 60S subunitwould not afford any additional protection from RNase andthat the protection of the HCV IRES by the 48S preinitiation

complex could indeed be very similar to that provided by theentire 80S ribosomal complex.

The HCV RBS is the largest RBS found to date. The gen-erality of this HCV RBS paradigm for other cellular and viralIRESs remains to be seen. However, the discontinuous natureand considerable size of the HCV RBS are likely to be sharedby related, viral IRESs. Confirmation of a pattern of commonstructural features will help explain the action and specificity ofIRESs.

ACKNOWLEDGMENTS

This work was supported in part by grant U35-8010 from the NewYork State Science and Technology Foundation’s Centers for Ad-

FIG. 6. The 48S ribosomal initiation complex also protects manyfragments of HCV RNA of various lengths. The protected fragmentsfrom the anisomycin-induced 80S peaks of both b-globin (lane 1) andHCV (bases 1 to 645) (lane 2) mRNAs were electrophoresed on a 15%denaturing polyacrylamide gel and subjected to autoradiography. Theedeine-induced 48S ribosomal complexes of b-globin (lane 3) andHCV (bases 1 to 645) (lane 4) mRNAs are also shown. XC, xylenecyanol; Bf, bromophenol.

FIG. 5. The RBS of HCV mRNA is large and discontinuous. (A) The RBSs of b-globin and HCV mRNAs are shown as blue boxes. The coreregions are represented by orange boxes. HCV mRNA has five core regions which are discontinuous. The three segments comprising noncoreelements which have not been observed to undergo cleavage are indicated by vertical arrows connected by horizontal dashed lines. (B) The HCVRBS domains are mapped on the secondary structure of the HCV IRES. The protected regions are shown in blue, and the core sequences areshown in orange.

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vanced Technology Program and by Public Health Service grant DK-56424 from NIH.

We thank S. Genus and P. Mercado for excellent technical assis-tance, E. Kanner for help in growing DNA plasmids, K. Browning andS. Lemon for gifts of plasmid DNA, Z. Kurylo-Borowska for the gift ofedeine, and J. Gomez for helpful discussions.

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