Internal Ribosome Entry Site within Hepatitis C Virus RNA

Vol. 66, No. 3JOURNAL OF VIROLOGY, Mar. 1992, p. 1476-14830022-538X/92/031476-08$02.00/0Copyright © 1992, American Society for Microbiology

Internal Ribosome Entry Site within Hepatitis C Virus RNAKYOKO TSUKIYAMA KOHARA,l* NARUSHI IIZUKA,l MICHINORI KOHARA,2 AND AKIO NOMOTO'

Department of Microbiology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku,Tokyo 113,1 and Fundamental Research Laboratory, Corporate Research and Development Laboratory,

Tonen Co., Nishi-Tsurugaoka, Ohi-machi, Iruma-gun, Saitama 354,2 Japan

Received 12 August 1991/Accepted 11 November 1991

The mechanism of initiation of translation on hepatitis C virus (HCV) RNA was investigated in vitro. HCVRNA was transcribed from the cDNA that corresponded to nucleotide positions 9 to 1772 of the genome byusing phage T7 RNA polymerase. Both capped and uncapped RNAs thus transcribed were active as mRNAsin a cell-free protein synthesis system with lysates prepared from HeLa S3 cells or rabbit reticulocytes, and thetranslation products were detected by anti-gp35 antibodies. The data indicate that protein synthesis starts atthe fourth AUG, which was the initiator AUG at position 333 of the HCV RNA used in this study. Efficiencyof translation of the capped methylated RNA appeared to be similar to that of the capped unmethylated RNA.However, a capped methylated RNA showed a much higher activity as mRNA than did the cappedunmethylated RNA in rabbit reticulocyte lysates when the RNA lacked a nucleotide sequence upstream ofposition 267. The results strongly suggest that HCV RNA carries an internal ribosome entry site (IRES).Artificial mono- and dicistronic mRNAs were prepared and used to identify the region that carried the IRES.The results indicate that the sequence between nucleotide positions 101 and 332 in the 5' untranslated regionof HCV RNA plays an important role in efficient translation. Our data suggest that the IRES resides in thisregion of the RNA. Furthermore, an IRES in the group II HCV RNA was found to be more efficient than thatin the group I HCV RNA.

The nucleotide sequence of almost the entire genome ofhepatitis C virus (HCV), the main causative agent of chronicnon-A, non-B hepatitis (NANBH), has been elucidated (3,15, 23, 28). The genome of HCV is a single-stranded RNAwith positive polarity and consists of approximately 10,000nucleotides. This RNA carries only one long open readingframe for the synthesis of a large single polyprotein of 3,010amino acids. Analysis of the amino acid sequences deducedfrom the nucleotide sequences of different HCV isolates (3,15, 28) revealed that the HCV genomes have a gene organi-zation similar to those of flaviviruses and their relatedviruses (29). Sequence analysis also revealed the existenceof a fairly long 5' untranslated region (UTR) that harborsthree (15, 28) or four (6) AUG sequences. This feature of the5' UTR resembles that of picornaviruses rather than that offlaviviruses.

Picornavirus RNAs are uncapped messengers and haveunusually long 5' UTRs (ranging from 610 to 1,200 residues,depending on the virus species) which contain many silentAUG sequences. These features seem incompatible withefficient translation by the scanning ribosome mechanism,which is the initiation mechanism of most eukaryotic cellularand viral mRNAs (16, 18). The rule of the scanning model isthat ribosomes initially bind to the 5' end of mRNAs andscan the RNAs to reach the authentic initiator AUGs, whichare, in many cases, the first AUG sequences. Furthermore,the cap structures at the 5' ends of mRNAs are generallybelieved to play an important role in the initial ribosomeentry or binding step. Thus, the initiation of translation onpicornavirus RNAs has been considered to take place by anunusual mechanism. Recently, it was proved that translationinitiation on picornavirus RNAs occurs by binding of ribo-somes to an internal sequence within the 5' UTR (10, 11, 26).An internal entry site for ribosomes has been called the

* Corresponding author.

IRES (internal ribosome entry site) (10, 11). IRES functionrequires a specific segment of the 5' UTR of approximately400 nucleotides which does not include the extreme 5'-proximal nucleotide sequences.

Structural similarities of the 5' UTR ofHCV RNA to thoseof picornavirus RNAs provide the possibility that IRESfunction resides in the 5' UTR ofHCV RNA, although the 5'UTR of HCV RNA is shorter than a segment of picornavirusRNAs required for its IRES function, and it is not knownwhether the 5' end of HCV RNA is capped.Here we demonstrate, by using cell-free translation sys-

tems, that the translation initiation on HCV RNAs occurs byentry of ribosomes to the internal sequence within the 5'UTR and that IRES function requires a segment of nucleo-tide positions 101 to 332 at most, which is the shortestsequence thus far identified as an IRES. We also show thatthe function of the IRES of group II HCV RNA is moreefficient than that of the IRES of group I HCV RNA in acell-free protein synthesis system from HeLa S3 cells.

MATERIALS AND METHODS

DNA procedure. Restriction endonucleases, DNA poly-merase I (Klenow fragment), and T4 DNA ligase werepurchased from Takara Shuzo Co. (Kyoto, Japan); calfintestinal alkaline phosphatase was from Boehringer GmbH(Mannheim, Germany); KS vector was from Stratagene (LaJolla, Calif.). These enzymes and compounds were usedaccording to the instructions of the manufacturers. DNAprimers were made by a DNA synthesizer (Applied Biosys-tems).

Isolation of HCV cDNAs. HCV cDNA that corresponds tonucleotide positions 9 to 791 (28) was isolated from pooledplasma of NANBH patients by a method involving polymer-ase chain reaction (PCR) as reported by Tsukiyama-Koharaet al. (31), using a sense primer, 5'-GGCGACACTCCACCATAGATC-3', and an antisense primer, 5'-ATGCGCC

1476

INTERNAL RIBOSOME ENTRY SITE IN HCV RNA 1477

9 333

~saci A1lioKJ T7pcn w~~ p 2

XbbIeiIIbV'

B~~pHI.Saoj4JSaiIB ta.

SalII

BaIt

S THr.siHI Sil

/ Sbal-sill)Mel Sail

BqII?TT7

906 mu 7n (Kb)I I I a

_ ~ ~ ~~~~~~~~~SoJ

_A.dHa* ndill

Saul -Fblll

sac I' Xbol' Sac aXbuI

F.trcs t o e 22r st inrde b

FIG. 1. Strategy for construction of the expression vector. *, restriction site introduced by the PCR method.

AGGGCCCTGGCAGC-3'. The cDNA thus synthesized wasinserted into the SmaI site of cloning vector pUC119.Nucleotide sequence analysis of 10 clones by using the7-deaza Sequenase kit (United States Biochemical) revealedthat 7 of the 10 clones had the same nucleotide sequence. AcDNA clone, named clone 2-1, was chosen from these sevenclones as an HCV cDNA clone and used for further plasmidconstruction. A cDNA clone, EN2-3, that corresponds tonucleotide positions 676 to 1772 was isolated by immuno-screening from a Xgtll cDNA library prepared from sera ofchronic NANBH patients as previously described (31).A BamHI-Clal cDNA fragment (corresponding positions 9

to 700) of clone 2-1 and a ClaI-HindIII cDNA fragment(positions 700 to 1772) of clone EN2-3 were ligated andinserted into the KS vector that had been digested withBamHI and HindIII. The plasmid thus obtained contains anHCV cDNA that corresponds to nucleotide positions 9 to1772 downstream of the 410 promoter for phage T7 RNApolymerase and was designated pKIV (Fig. 1).

Construction of deletion mutants. Mutants with deletionsof the 5' UTR (pkI5, pkI6, pkI7, pkI8, pkI9, pkI10, pkIll,and pkI16) were constructed by the PCR method, usingsynthetic primers. Nucleotide sequences of sense DNAprimers were designed to give an XbaI site to the corre-sponding 5' end of the PCR products. A XbaI-AatII cDNAfragment of plasmid pKIV was replaced by the correspond-ing cDNA fragments of the PCR products. In the case ofpkI16, a segment corresponding to nucleotide positions 333to 434 was synthesized by the PCR method, digested withAatII, and inserted into pKIV that had been digested withStul and AatII. To exclude the influence of a sequenceexisting between the T7 promoter and HCV cDNA, the XbaIand HindIII cDNA fragments of pKIV and its deletionplasmids were filled in with Klenow fragment and subclonedwith correct orientation into the StuI site of plasmid pNar3,which had been derived from pUC119 (Fig. 1); the resultingconstructs were designated pNV, pN5, pN6, pN7, pN8,pN9, pN10, pNll, and pN16 (see Fig. 6A). The StuI-HindIII

cDNA fragment of pKIV was also subcloned into the Stulsite of pNar3, and the resulting plasmid was designated pNSt(Fig. 1).

Construction of cDNA clones that carry CAT cDNA. Thechloramphenicol acetyltransferase (CAT) cDNA carryingSacl and XbaI sites at the corresponding 5' and 3' ends,respectively, was introduced into the Sacl and XbaI sites ofpKIV, pkI6, pkI7, pkI8, pkI9, pkI10, pkIll, and pkI16.These cDNA clones encoding dicistronic mRNAs weredesignated pCV, pC6, pC7, pC8, pC9, pC10, pCll, andpC16, respectively (see Fig. 6A). To construct plasmid5HcCAT, an XbaI-BspHI fragment of pKIV, which carries asegment of untranslated region of HCV, and the CAT cDNAthat had BspHI and Sall sites at the corresponding 5' and 3'ends, respectively, were ligated and inserted into KS vectorthat had been digested with XbaI and SalI (Fig. 1). AStuI-SalI fragment of 5HcCAT, after treatment with Klenowfragment, was inserted with correct orientation into the Stulsite of pNar3. The plasmid thus obtained was designated5HStCAT (Fig. 1).

Construction of cDNA that carries the 5' UTR sequence ofgroup II HCV RNA. A segment of group II HCV cDNA(corresponding to nucleotide positions 49 to 408) was pre-pared from plasma of a patient infected with group II HCValone by the PCR method, using 5'-CTGTCTTCACGCAGAAAGCG-3' and 5'-CCGGGAACTTGACGTCCTGT-3'as primers, and subcloned into the SmaI site of pUC19; thenucleotide sequence was then determined as describedabove. An XbaI-AatII cDNA fragment of pKIV was re-placed by the XbaI-AatII cDNA fragment containing the 5'UTR sequence of group II HCV. An XbaI-HindIII cDNAfragment (1,724 bp) of the plasmid thus obtained was sub-cloned into the StuI site of pNar3, and the resulting plasmidwas designated pNII5.RNA transcription. Plasmids were linearized by digestion

with Sall (for 5HcCAT, pCV, pC6, pC7, pC8, pC9, pC10,pCll, and pC16), XhoI (for 5HStCAT), or EcoRI (for pNV,pN5, pN6, pN7, pN8, pN9, pNlO, pNll, pN16, pNII5, and

.V,C zB

VOL. 66, 1992

Hbdlll T7pa.wW Ski I xho If-----,% I JIV

.1

1478 TSUKIYAMA-KOHARA ET AL.

pNSt) and transcribed into RNA in vitro with T7 RNApolymerase (Takara Shuzo Co.). In most cases, cap struc-ture, m7GpppG or GpppG (Pharmacia LKB), was incorpo-rated at the 5' ends of RNA transcripts as described previ-ously (14).

In vitro translation. Suspension-cultured HeLa S3 cellswere infected with coxsackievirus Bi (CVB1) as previouslydescribed (8). Cytoplasmic extracts (S10) of mock- or CVB1-infected cells were prepared 3 h after the infection and weretreated with micrococcal nuclease as previously described(9). Conditions for the translation reaction (12.5 ,ul) were asdescribed by Rose et al. (27). After incubation at 37°C for 1h in the presence of [35S]methionine (ICN Radiochemicals),5 ,ul of each of the translation mixtures was mixed with anequal volume of sodium dodecyl sulfate (SDS) samplebuffer, boiled for 1 min, and analyzed by electrophoresis ona 12.5% polyacrylamide gel in Laemmli's buffer system (21).In some cases, translation products were cotranslationallyprocessed in the presence of canine microsomal membrane(Amersham). The processed translation products weremixed with rabbit anti-gp35 antibodies and then with Affi-Gelprotein A (Bio-Rad). The precipitates were analyzed byelectrophoresis as described above. Rabbit reticulocyte ly-sate (RRL; Amersham N150) was also used as in vitrotranslation system. Translation reactions in RRL were per-formed at 30°C for 1 h. In some cases, translation reactionswere carried out in the presence of 20 FM S-adenosylhomo-cysteine (SAH) to prevent methylation of GpppG-endedRNA during the reaction (2, 5).

Preparation of rabbit anti-gp35 antibodies. A cDNA clone,C10-E12, that corresponds to HCV RNA, including a se-quence encoding gp35, was isolated by immunoscreeningfrom the Agtll cDNA library prepared from sera ofNANBHpatients (31). This cDNA clone was inserted into the hem-agglutinin gene downstream of the P7.5 promoter of theLister strain of vaccinia virus (30). Rabbits were immunizedby intradermal inoculation of the recombinant vaccinia vi-rus. The specificity of the rabbit serum was verified by itsspecific reaction with two kinds of products directed by theHCV genome region encoding gp35, that is, materials pro-duced by using a baculovirus expression vector system andan in vitro translation system (1Sa).

Secondary structure of the HCV 5' UTR. A possiblesecondary structure of HCV RNA was predicted for nucle-otide positions 1 to 600 by using the Fold program (Biose-quence Analysis System of the Institute of Medical Science,the University of Tokyo) on a VAX-11/750 computer.

RESULTS

HCV protein produced in vitro. Hijikata et al. (7) havedemonstrated that capped HCV RNAs with a short 5' UTRcan be translated into the correct HCV proteins in the RRLcell-free translation system. The RNA used by Hijikata et al.(7) did not have AUG sequences upstream of the initiatorAUG. To determine the effect of the upstream AUG se-quences on translation, an RNA with a free 5' end synthe-sized from linearized pNV was examined for mRNA activityin a cell-free translation system prepared from HeLa S3 cells(Fig. 2). Efficient translation occurred, and a polypeptidewith the expected size (approximately 52 kDa) was observedon a gel (Fig. 2, lane 2). A small amount of product thatmigrated slightly faster than the 52-kDa polypeptide was alsoobserved (lane 2). This material may have resulted frompartially degraded RNA template. In any event, the datastrongly suggest that the translation of uncapped HCV RNA

kDa

97.4 -

69 -

46 --

1 2 3 4 5

WI,

--o-gp3530-

21.5 -- - p22

14.3

FIG. 2. In vitro translation of uncapped HCV RNA. HCV RNAwith a free 5' end corresponding to nucleotide positions 9 to 1772was used as mRNA in a cell-free translation system with lysatesprepared from HeLa S3 cells, and the products were analyzed asdescribed in Materials and Methods. No RNA (lane 1) and uncappedHCV RNA (lanes 2 to 4) were translated in the presence (lanes 3 and4) or absence (lane 2) of a canine microsomal membrane fraction.The products shown in lane 3 were reacted with normal rabbit serum(lane 5) or rabbit anti-gp35 serum (lane 4). Immunoprecipitation andpolyacrylamide gel electrophoresis were performed as described inMaterials and Methods. Positions of molecular weight markers areindicated at the left; positions of gp35 and p22 are indicated at theright.

starts at the fourth AUG, the authentic initiator AUG, fromthe 5' end of the RNA. Thus, the upstream AUG seems notto have a deleterious effect on the translation initiation onHCV RNA in this in vitro translation system.To confirm that the translation products contain the cor-

rect HCV polypeptides, the translation reaction was per-formed in the presence of canine microsomal membrane(Fig. 2, lane 3). Two major proteins with approximatemolecular masses of 22 and 35 kDa were observed inaddition to the unprocessed products. This observation iscompatible with that reported previously (7) and thereforeindicates that these products are p22 and gp35, respectively.A faint band at approximately 11 kDa may be the product ofthe region encoding a part of gp7O. An immunoprecipitationexperiment involving rabbit anti-gp35 serum was performedas described in Materials and Methods. The precipitates withthe rabbit serum were separated by the SDS-polyacrylamidegel electrophoresis (Fig. 2, lane 4). The rabbit serum reactedwith both gp35 and the unprocessed products. These resultsindicate that the in vitro translation products contain thecorrect HCV polyprotein. It is therefore possible that ribo-somes bind to an internal sequence within the 5' UTR likethe translation initiation on picornavirus RNAs.

Effect of cap on translation. Since it is known that capstructure linked to the 5' end of picornavirus RNAs has littleeffect on the efficiency of translation initiation (25), mRNAactivities of HCV RNAs carrying m7GpppG (capped meth-ylated) or GpppG (capped unmethylated) at the 5' end werecompared with each other in the absence (Fig. 3a, lanes 2 to7) or presence (Fig. 3b, lanes 2 to 7) of SAH. mRNA activityof capped methylated HCV RNA derived from pNV appearsto be similar to that of capped unmethylated HCV RNA from

J. VIROL.


pNV pNV pNSt1 2 3 4 5 6 7 8.xtog I*

5HcCAT 5HcCAT 5HStCAT

9 10 11 12 13 14

- HCV

- - A--CAT

GpppG--- + + +

m7GpppG- +

b)

kDa

97.4--

69--

46--

30-_.

pNV pNV pNSt

1 2 3 4 5 6 7 8

5HcCAT 5HcCAT 5HStCAT

9 10 11 12 13 14

MM - w -CAT21.5-

14.3--

GpppG--- +

rn7GpppG- + +

FIG. 3. Effect of cap on in vitro translation. In vitro translationreactions were carried out in the absence (a) or presence (b) of 20,uM SAH. Capped methylated (m7GpppG-linked) (lanes 3, 5, 7, 10,12, and 14) or capped unmethylated (GpppG-linked) (lanes 2, 4, 6, 9,11, and 13) RNAs derived from pNV (lanes 2, 3, 4, and 5), pNSt(lanes 6 and 7), 5HcCAT (lanes 9, 10, 11, and 12), and 5HStCAT(lanes 13 and 14), or no RNA (lanes 1 and 8) were translated in HeLacell lysates (lanes 1, 2, 3, 9, and 10) or in RRL (lanes 4 to 8 and 11to 14), and the translation products were analyzed as described inthe legend to Fig. 2. Positions of products from translated regions ofHCV and CAT mRNAs are indicated at the right; positions ofmolecular weight markers are indicated at the left.

pNV in cell-free translation systems prepared from HeLa S3cells (Fig. 3a and b, lanes 2 and 3) and rabbit reticulocytes(Fig. 3a and b, lanes 4 and 5). In the latter system, however,capped methylated RNA seems to be slightly more efficientthan capped unmethylated RNA. In the case of HCV RNAderived from pNSt, which lacks a nucleotide sequenceupstream of position 270, capped methylated RNA is muchmore effective as mRNA than is capped unmethylated RNAin RRL (Fig. 3a and b, lanes 6 and 7). These data stronglysuggest that a segment within the 5' UTR of HCV RNA hasthe ability to initiate protein synthesis independently of capfunctions. The relative efficiency of the translation was notaffected by the presence of SAH, a competitive inhibitor foran RNA (guanine-7-)-methyltransferase activity existing inHeLa S10 and RRL (2, 5). This finding suggested thatdifferences in translation efficiencies observed betweencapped methylated RNAs and capped unmethylated RNAs(Fig. 3a and b, lanes 4 to 7) were not underestimates.To exclude the possibility that the translated region of

HCV RNA is involved in the cap-independent initiation oftranslation, the translated region of HCV RNAs was re-

FIG. 4. Translation of dicistronic mRNA in RRL and CVB1-infected HeLa cell lysates. No RNA (lanes 1 and 4) and cappedmethylated (m7GpppG-linked) dicistronic RNA derived from pCV(lanes 2 and 3) were used as mRNA in RRL (lanes 1 and 2) or

CVB1-infected HeLa cell lysates (lanes 3 and 4), and the translationproducts were analyzed as described in the legend to Fig. 2.Positions of the products from the first cistron (CAT mRNA) and thesecond cistron (HCV RNA) are indicated at the right; positions ofmolecular weight markers are indicated at the left.

placed by CAT mRNA as described in Materials and Meth-ods. Translation experiments similar to those shown in Fig.3a and b, lanes 2 to 7, were carried out on these recombinantmRNAs (Fig. 3a and b, lanes 9 to 14). The data are verysimilar to those obtained in the experiment involving paren-tal HCV RNAs. These results indicate that a segmentrequired for the cap-independent translation initiation re-sides within the 5' UTR of HCV RNA.

Existence of IRES within the 5' UTR of HCV RNA. Toprove the existence of IRES within the 5' UTR of HCVRNA, a capped methylated (m7GpppG-linked) dicistronicmRNA consisting of CAT mRNA as the first cistron andHCV RNA as the second cistron was synthesized fromlinearized pCV. The structure of pCV is shown in Fig. 1. Thetranslation experiments were performed on this dicistronicmRNA by using RRL (Fig. 4, lane 2) and lysates of CVB1-infected HeLa S3 cells (Fig. 4, lane 3). A protein synthesissystem prepared from CVB1-infected HeLa S3 cells has theability to suppress cap-dependent initiation of translation(unpublished results) as observed in that from poliovirus-infected HeLa cells (4, 22). Both CAT mRNA and HCVRNA appear to be translated in RRL (Fig. 4, lane 2).However, only HCV RNA appears to be translated in lysateof CVB1-infected HeLa S3 cells (Fig. 4, lane 3). Since a

dicistronic mRNA on polysomes was shown to be intact inHeLa cell extracts (26), the data strongly suggest that thesecond cistron is translated even when ribosome entry doesnot occur at the 5' end of the dicistronic mRNA. Therefore,it is very likely that an IRES exists within the 5' UTR ofHCV RNA.

Possible secondary structure of the 5' UTR of HCV RNA. Apossible secondary structure was predicted as described inMaterials and Methods and is shown in Fig. 5. Six possiblestem-loop structures observed in the 5' UTR were desig-nated A to F, respectively, from the 5' end of RNA. Toconfirm the validity of the secondary structure, RNA se-

quences of the 5' UTR of group I HCV and group II HCVRNAs (31) were compared with each other. Nucleotides ofgroup II HCV that are different from those of group I HCVare indicated by arrows in Fig. 5. Many differenw nucleotides

a)

kDa

97.4-.-69--

46_-.

30--

kDa

21.5--

1 2 3 4

14.3-_

97.4 -

69-

46 -

30-

OW v.-" _ HCV

21.5_--t

14.3-_

-CAT

VOL. 66, 1992


AACU

u cA CG C

A UU GA UCC G

A A

C G

C G

U AACC U

A UCAC G 50 GAA U C A A UAG C UCA GC AGCGUC GN

*, 1 C G-AGU UG UUGCGG CB5- CGAUUGGGGG U A A UAC

AACCCUU G

A UG C

CG A CC3' AG GC-CUCCG4f CC

taCCG GAGG CClr CCU A A GC CC

ucA uA A

CGU GG UC GC GA UG CA U

U GG C

FAGCC GUAUGCUG UAG-GGUr'UGCG I$WC-CCCG GGCCAC UDFCAGGGCGU A3C0G0 C GUA

AA505CA

AAUGC A G A+ GQJG CC-U

G U

U U

UCU

E

FIG. 5. Possible secondary structure of the 5' UTR of group I

HCV RNA predicted by computer analysis. Different nucleotides

within the 5' UTR of group II HCV RNA are indicated by arrows

and bold letters. Major stem-loop structures are designated A to F.

The initiator AUG is indicated by bold letters.

were observed in stem-loop structure E. However, most of

the nucleotide differences in stem-loop structure E occurred

without destruction of possible base pairing. This observa-

tion confirms the validity of the possible secondary structure

shown in Fig. 5, especially with respect to stem-loop struc-

ture E.

Identification of an IRES. To identify the IRES of HCV

RNA, artificial mono- and dicistronic mRNAs were con-

structed, taking positions of upstream AUG sequences and

possible stem-loop structures into consideration as de-

scribed in Materials and Methods (Fig. 6A). Capped unme-

thylated (GpppG-linked) RNAs derived from pNV, pN6,

pN7, pN8, pN9, pNlO, pN11, and pNl6 and capped meth-

ylated (m7GpppG-linked) RNAs from pCV, pC6, pC7, pC8,

pC9, pCl0, pCll, and pCl6 were used as mRNAs in RRL

(Fig. 6B and C). RNAs derived from pNV, pN6, and pN7

were active mRNAs in RRL, and an RNA derived from pN8

was a very inefficient mRNA (Fig. 6B). Similar results were

obtained from experiments involving dicistronic mRNAs

derived from pCV, pC6, pC7, and pC8 (Fig. 6C). These

results indicate that an IRES appears to reside downstream

of nucleotide position 101. RNAs from pNl0 and pNl1, in

which the first AUG is the initiator AUG, restored mRNA

activities (Fig. 6B, lanes 6 and 7). This phenomenon may

result from the fact that eukaryotic ribosomes have a ten-

dency to recognize and bind to the end of RNA (17). Indeed,

the translation product of the HCV RNAs derived fromdicistronic cDNAs, pC1O and pC1l, was not observed (Fig.6C, lanes 7 and 8). This observation may support the findingthat translated dicistronic mRNA is intact during the trans-lation reaction (26). To identify the 3' end of the IRES, adeletion RNA derived from pN16 was synthesized andtested for mRNA activity. This deletion RNA was not anactive mRNA (Fig. 6B, lane 8). A similar observation wasmade when a dicistronic mRNA derived from pC16 was usedas mRNA in RRL (Fig. 6C, lane 9). These results indicatethat the IRES of the 5' UTR of HCV RNA resides betweennucleotide positions 101 and 332.

Efficiency of group II HCV RNA. Since nucleotide differ-ences were observed in IRESs within the 5' UTRs betweengroup I and group II HCV RNAs, IRES functions of group Iand group II HCV RNAs were compared in a cell-freeprotein synthesis system of HeLa S3 cells. Capped unmeth-ylated (GpppG-linked) HCV RNAs (nucleotide positions 49to 1772) containing the 5' UTRs of group I and group II HCVRNAs were prepared from pN5 and pNII5, respectively, asdescribed in Materials and Methods and used as mRNAs. Asshown in Fig. 7, translation initiation from the 5' UTR ofgroup II HCV RNA is more efficient than that of group IHCV RNA. Similar results were obtained with three pairs ofRNA preparations (data not shown). Furthermore, amountsof radioactivity incorporated into the product increasedproportionally with an RNA dose of up to 2 ,ug per reactionmixture. These results may indicate that regions harboringdifferent nucleotides between group I and group II HCVRNAs play an important role(s) in IRES function.

DISCUSSION

In this study, we demonstrated that initiation of HCVprotein synthesis occurs in a cap-independent manner likethat of picornavirus-specific protein synthesis and that anIRES within the 5' UTR of HCV RNA resides betweennucleotide positions 101 and 332. Previous studies of picor-navirus RNA have shown that IRES function requires aspecific segment of approximately 400 nucleotides (11, 26).Thus, the IRES of HCV RNA is the shortest among IRESsso far discovered. Mutational analysis of picornavirus RNAsindicated that 21- and 7-base sequences conserved in the 5'UTR of human enterovirus and rhinovirus RNAs are impor-tant for translation initiation (8, 9, 19, 25). These sequenceswere not observed in the 5' UTR of HCV RNA. Therefore,HCV and picornaviruses may have different mechanisms forthe entry of ribosomes to the IRES. An oligopyrimidine tractbefore the AUG at position 206, however, was observedwithin the 5' UTR of HCV RNA. An oligopyrimidine tractbefore an AUG is conserved in picornavirus RNA and isconsidered to be important for translation initiation on theviral RNAs (1, 19, 24). This AUG is the initiator AUG(encephalomyocarditis virus and foot-and-mouth disease vi-rus) or most 3'-proximal AUG (rhinovirus and poliovirus) inthe 5' UTR (12, 13, 20). The existence of this RNA sequencein both HCV and picornavirus RNAs suggests that the sameinitiation factor(s) is involved in the translation initiation onboth viral RNAs.

Like the IRES of encephalomyocarditis virus, the IRES ofHCV is very effective in both HeLa cell lysates and RRL.Indeed, the HCV IRES is much more effective than theCVB1 IRES both in mock- and CVB1-infected HeLa celllysates (unpublished result). Capped methylated and cappedunmethylated HCV RNAs did not show any difference in the

J. VIROL.


A _E I37AUiG AUG

1c1C i;%5u. C 101 128 1 162IAi..Ll_ ; 1 i_

A -3 C D!i

206ALIG

I333AUG309I2216 267

bI

pNV DpN6 -> --------

pN7>7D- --------- -- C-L:7pN8 >--

pN9 --

pN10 _________________

pNl6 >

pN16T CAT

pC6 ><//v-pC8 D{tM 147

pC0 --------- -^ ---dpCR - -163pC11 >14Iz---____pCl6 - -- - - - ----- --- - --

IRES

B

1 2 3 4 5 6 7 8 9

a_

" -'I..

C

_ HCV

q qe

qe

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- HCV

%---m - VW - CAT

FIG. 6. Structures of mutant cDNAs and translation products of their transcripts in RRL. (A) Structures of mutant cDNAs. Positions ofstem-loop structures (A to F) (boxes) and nucleotide numbers and positions of AUG sequences are shown at the top. Regions missing inplasmids are indicated by broken lines. Numbers on cDNAs represent nucleotide positions of the terminal nucleotides. Open trianglesrepresent T7 f10 promoters. The region including IRES is indicated by the arrowed line at the bottom. (B) Capped unmethylated(GpppG-linked) monocistronic HCV RNAs derived from pNV (lane 1), pN6 (lane 2), pN7 (lane 3), pN8 (lane 4), pN9 (lane 5), pNlO (lane 6),pNll (lane 7), and pN16 (lane 8) were used as mRNAs in RRL, and the translation products were analyzed as described in the legend to Fig.2. A translation reaction was also carried out with no RNA (lane 9). The position of the translation product (HCV) is indicated at the right.(C) Capped methylated (m7GpppG-linked) dicistronic mRNAs from pCV (lane 2) pC6 (lane 3), pC7 (lane 4), pC8 (lane 5), pC9 (lane 6), pClO(lane 7), pCll (lane 8), and pC16 (lane 9) were used as mRNAs in RRL, and the translation products were analyzed as described in the legendto Fig. 2. As a control, a sample with no RNA (lane 1) was also analyzed. Positions of the translation products (HCV and CAT) are indicatedat the right.

efficiency of translation in HeLa S3 lysates (Fig. 3a and b,lanes 2 and 3). However, capped methylated HCV RNA ismore efficient than capped unmethylated HCV RNA in RRL(Fig. 3a and b, lanes 4 and 5). It is therefore possible thatHCV RNA requires a cap structure to be a fully activemRNA in RRL. This phenomenon is difficult to explain atpresent. It is of interest that the IRES of group II HCV ismore effective than the IRES of group I HCV in HeLa celllysates (Fig. 7), although group I HCV appears to be themajor group ofHCV (31). In any event, different nucleotidesobserved in the IRESs of group I and group II HCV RNAs

must influence IRES function, which may provide someinsight into the mechanism of the IRES function of HCV.Mutational analysis of HCV RNA with respect to IRESfunction is currently under way.

Although the nucleotide sequence of almost the entiregenome of HCV has been elucidated (3, 15, 28), the 5' and 3'structures of the genome are not clear at present. Therefore,it is not known whether HCV RNA is capped. However,elucidation of cap-independent translation of HCV indicatesthat HCV has a mechanism for translation initiation totallydifferent from that of a member of the family Flaviridiae;

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L . I .


pNII5 pN5kDa 1 2 3

974_

69-

46-.

30-a.

21.5-

14.3-

FIG. 7. Translation efficiency from the 5' UTRs of group I andgroup II HCV RNAs. No RNA (lane 1) and capped unmethylated(GpppG-linked) HCV RNAs that carried the 5' UTRs of group II

HCV (lane 2) and group I HCV (lane 3) were used as mRNAs in acell-free protein synthesis system prepared from HeLa S3 cells.Both HCV RNAs correspond to nucleotide positions 49 to 1772, asdescribed in Materials and Methods. The translation products were

analyzed as described in the legend to Fig. 2. Positions of molecularweight markers are indicated at the left.

therefore, creation of new virus family may be desirable forclassification of HCV.

ACKNOWLEDGMENTS

We thank F. Ochikubo for help in computer analysis, and we

acknowledge the significant support of S. Tanaka and N. Hattori inthe collection of patient plasma. We also thank K. Mizumoto forhelpful discussions.

This work was supported by research grants from The Ministry ofEducation, Science and Culture of Japan and The Ministry of Healthand Welfare of Japan.

REFERENCES1. Bienkowska-Szewczyk, K., and E. Ehrenfeld. 1988. An internal

5'-noncoding region required for translation of poliovirus RNAin vitro. J. Virol. 62:3068-3072.

2. Both, G. W., A. K. Barnerjee, and A. J. Shatkin. 1975. Meth-ylation dependent translation of viral messenger RNA in vitro.Proc. Natl. Acad. Sci. USA 72:1189-1193.

3. Choo, Q.-L., K. H. Richman, J. H. Han, K. Berger, C. Lee, C.Dong, C. Gallegos, D. Coit, A. Medina-Selby, P. J. Barr, A. J.Weiner, D. W. Bradley, G. Kuo, and M. Houghton. 1991.Genetic organization and diversity of the hepatitis C virus. Proc.Natl. Acad. Sci. USA 88:2451-2455.

4. Ehrenfeld, E. 1984. Picornavirus inhibition of host cell proteinsynthesis, p. 177-221. In H. Fraenkel-Conrat and R. R. Wagner(ed.), Comprehensive virology, vol. 19. Plenum Press, NewYork.

5. Ensinger, M. J., and B. Moss. 1976. Modification of the 5'terminus of mRNA by an RNA (guanine-7-)-methyltransferasefrom HeLa cells. J. Biol. Chem. 251:5283-5291.

6. Han, J. H., V. Shyamala, K. H. Richman, M. J. Brauer, B.Irvine, M. S. Urdea, P. Tekamp-Olson, G. Kuo, Q.-L. Choo, andM. Houghton. 1991. Characterization of the terminal regions ofhepatitis C viral RNA: identification of conserved sequences inthe 5' untranslated region and poly (A) tails at 3' end. Proc.Natl. Acad. Sci. USA 88:1711-1715.

7. Hijikata, M., N. Kato, Y. Ootsuyama, M. Nakagawa, and K.Shimotohno. 1991. Gene mapping of putative structural region ofthe hepatitis C virus genome by in vitro processing analysis.Proc. Natl. Acad. Sci. USA 88:5547-5551.

8. lizuka, N., M. Kohara, K. Hagino-Yamagishi, S. Abe, T. Ko-matsu, K. Tago, M. Arita, and A. Nomoto. 1989. Construction ofless neurovirulent polioviruses by introducing deletions into the5' noncoding sequence of the genome. J. Virol. 63:5354-5363.

9. Iizuka, N., H. Yonekawa, and A. Nomoto. 1991. Nucleotidesequences important for translation initiation of enterovirusRNA. J. Virol. 65:4867-4873.

10. Jang, S. K., M. V. Davies, R. J. Kaufman, and E. Wimmer.1989. Initiation of protein synthesis by internal entry of ribo-somes into the 5' nontranslated region of encephalomyocarditisvirus RNA in vivo. J. Virol. 63:1651-1660.

11. Jang, S. K., H.-G. Krausslich, M. J. H. Nicklin, G. M. Duke,A. C. Palmenberg, and E. Wimmer. 1988. A segment of the 5'nontranslated region of encephalomyocarditis virus RNA di-rects internal entry of ribosomes during in vitro translation. J.Virol. 62:2636-2643.

12. Jang, S. K., T. Pestova, C. U. T. Hellen, G. W. Witherell, and E.Wimmer. Cap-independent translation of picornavirus RNAs:structure and function of the internal ribosomal entry site. InR. E. Thach (ed.), Translationally regulated genes in highereukaryotes, in press. Karger, Basel.

13. Jang, S. K., and E. Wimmer. 1990. Cap-independent translationof encephalomyocarditis virus RNA: structural elements of theinternal ribosomal entry site and involvement of a cellular57-kDa RNA binding protein. Genes Dev. 4:1560-1572.

14. Kaminski, A., M. T. Howell, and R. J. Jackson. 1990. Initiationof encephalomyocarditis virus RNA translation: the authenticinitiation site is not selected by a scanning mechanism. EMBOJ. 9:3753-3759.

15. Kato, N., M. Hijikata, Y. Ootsuyama, M. Nakagawa, S. Ohko-shi, T. Sugiyama, and K. Shimotohno. 1990. Molecular cloningof the human hepatitis C virus genome from Japanese patientswith non-A non-B hepatitis. Proc. Natl. Acad. Sci. USA 87:9524-9528.

15a.Kohara, M., et al. Unpublished data.16. Kozak, M. 1978. How do eukaryotic ribosomes select initiation

regions in messenger RNA? Cell 15:1109-1123.17. Kozak, M. 1979. Inability of circular mRNA to attach to

eukaryotic ribosomes. Nature (London) 280:82-85.18. Kozak, M. 1989. Context effects and inefficient initiation at

non-AUG codons in eukaryotic cell-free translation systems.Mol. Cell. Biol. 9:5073-5080.

19. Kuge, S., and A. Nomoto. 1987. Construction of viable deletionand insertion mutants of the Sabin strain of type 1 poliovirus:function of the 5' noncoding sequence in viral replication. J.Virol. 61:1478-1487.

20. Kuhn, R., N. Luz, and E. Beck. 1990. Functional analysis of theinternal translation initiation site of foot-and-mouth diseasevirus. J. Virol. 64:4625-4631.

21. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

22. Lee, K. A. W., and N. Sonenberg. 1982. Inactivation of cap-binding proteins accompanies the shut-off of host protein syn-thesis by poliovirus. Proc. Natl. Acad. Sci. USA 79:3447-3451.

23. Okamoto, H., S. Okada, Y. Sugiyama, S. Yotsumoto, T. Tanaka,H. Yoshizawa, F. Tsuda, Y. Miyakawa, and M. Mayumi. 1990.The 5'-terminal sequence of the hepatitis C virus genome. Jpn.J. Exp. Med. 60:167-177.

24. Pelletier, J., M. E. Flynn, G. Kaplan, R. Racaniello, and N.Sonenberg. 1988. Mutational analysis of upstream AUG codonsof poliovirus RNA. J. Virol. 62:4486-4492.

25. Pelletier, J., G. Kaplan, V. R. Racaniello, and N. Sonenberg.1988. Cap-independent translation of poliovirus mRNA is con-ferred by sequence elements within the 5' noncoding region.Mol. Cell. Biol. 8:1103-1112.

26. Pelletier, J., and N. Sonenberg. 1988. Internal initiation oftranslation of eukaryotic mRNA directed by a sequence derivedfrom poliovirus RNA. Nature (London) 334:320-325.

27. Rose, J. K., H. Trachsel, K. Leong, and D. Baltimore. 1978.Inhibition of translation by poliovirus: inactivation of a specificinitiation factor. Proc. Natl. Acad. Sci. USA 75:2732-2736.

28. Takamizawa, A., C. Mori, I. Fuke, S. Manabe, S. Murakami, J.Fujita, E. Onishi, T. Andoh, I. Yoshida, and H. Okayama.Structure and organization of the hepatitis C virus genomeisolated from human carriers. J. Virol. 65:1105-1113.

29. Takeuchi, K., Y. Kubo, S. Boonmar, Y. Watanabe, T.

J. VIROL.


Katayama, Q.-L. Choo, G. Kuo, M. Houghton, I. Saito, and T.Miyamura. 1990. The putative nucleotide and envelope proteingenes of hepatitis C virus determined by comparison of thenucleotide sequences of two isolates derived from an experi-mentally infected chimpanzee and healthy human carrier. J.Gen. Virol. 71:3021-3033.

30. Tsukiyama, K., Y. Yoshikawa, H. Kamata, K. Imaoka, K.

Asano, S. Funahashi, T. Maruyama, H. Shida, M. Sugimoto, andK. Yamanouchi. 1989. Development of heat-stable recombinantrinderpest vaccine. Arch. Virol. 107:225-235.

31. Tsukiyama-Kohara, K., M. Kohara, K. Yamaguchi, N. Maki, A.Toyoshima, K. Miki, S. Tanaka, N. Hattori, and A. Nomoto.1991. A second group of hepatitis C virus. Virus Genes 5:243-254.

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Internal Ribosome Entry Site within Hepatitis C Virus RNA