Vol. 66, No. 3
JOURNAL OF VIROLOGY, Mar. 1992, p. 1476-1483
0022-538X/92/031476-08$02.00/0 Copyright © 1992, American Society for Microbiology
Internal Ribosome Entry Site within Hepatitis C Virus RNA KYOKO 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. HCV RNA was transcribed from the cDNA that corresponded to nucleotide positions 9 to 1772 of the genome by using phage T7 RNA polymerase. Both capped and uncapped RNAs thus transcribed were active as mRNAs in a cell-free protein synthesis system with lysates prepared from HeLa S3 cells or rabbit reticulocytes, and the translation products were detected by anti-gp35 antibodies. The data indicate that protein synthesis starts at the fourth AUG, which was the initiator AUG at position 333 of the HCV RNA used in this study. Efficiency of 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 capped unmethylated RNA in rabbit reticulocyte lysates when the RNA lacked a nucleotide sequence upstream of position 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 region of HCV RNA plays an important role in efficient translation. Our data suggest that the IRES resides in this region of the RNA. Furthermore, an IRES in the group II HCV RNA was found to be more efficient than that in the group I HCV RNA.
IRES (internal ribosome entry site) (10, 11). IRES function requires a specific segment of the 5' UTR of approximately 400 nucleotides which does not include the extreme 5'proximal nucleotide sequences. Structural similarities of the 5' UTR of HCV RNA to those of picornavirus RNAs provide the possibility that IRES function resides in the 5' UTR of HCV RNA, although the 5' UTR of HCV RNA is shorter than a segment of picornavirus RNAs required for its IRES function, and it is not known whether the 5' end of HCV RNA is capped. Here we demonstrate, by using cell-free translation systems, that the translation initiation on HCV RNAs occurs by entry of ribosomes to the internal sequence within the 5' UTR and that IRES function requires a segment of nucleotide positions 101 to 332 at most, which is the shortest sequence thus far identified as an IRES. We also show that the function of the IRES of group II HCV RNA is more efficient than that of the IRES of group I HCV RNA in a cell-free protein synthesis system from HeLa S3 cells.
The nucleotide sequence of almost the entire genome of hepatitis C virus (HCV), the main causative agent of chronic non-A, non-B hepatitis (NANBH), has been elucidated (3, 15, 23, 28). The genome of HCV is a single-stranded RNA with positive polarity and consists of approximately 10,000 nucleotides. This RNA carries only one long open reading frame for the synthesis of a large single polyprotein of 3,010 amino acids. Analysis of the amino acid sequences deduced from the nucleotide sequences of different HCV isolates (3, 15, 28) revealed that the HCV genomes have a gene organization similar to those of flaviviruses and their related viruses (29). Sequence analysis also revealed the existence of a fairly long 5' untranslated region (UTR) that harbors three (15, 28) or four (6) AUG sequences. This feature of the 5' UTR resembles that of picornaviruses rather than that of flaviviruses. Picornavirus RNAs are uncapped messengers and have unusually long 5' UTRs (ranging from 610 to 1,200 residues, depending on the virus species) which contain many silent AUG sequences. These features seem incompatible with efficient translation by the scanning ribosome mechanism, which is the initiation mechanism of most eukaryotic cellular and viral mRNAs (16, 18). The rule of the scanning model is that ribosomes initially bind to the 5' end of mRNAs and scan the RNAs to reach the authentic initiator AUGs, which are, in many cases, the first AUG sequences. Furthermore, the cap structures at the 5' ends of mRNAs are generally believed to play an important role in the initial ribosome entry or binding step. Thus, the initiation of translation on picornavirus RNAs has been considered to take place by an unusual mechanism. Recently, it was proved that translation initiation on picornavirus RNAs occurs by binding of ribosomes to an internal sequence within the 5' UTR (10, 11, 26). An internal entry site for ribosomes has been called the *
MATERIALS AND METHODS DNA procedure. Restriction endonucleases, DNA polymerase I (Klenow fragment), and T4 DNA ligase were purchased from Takara Shuzo Co. (Kyoto, Japan); calf intestinal alkaline phosphatase was from Boehringer GmbH (Mannheim, Germany); KS vector was from Stratagene (La Jolla, Calif.). These enzymes and compounds were used according to the instructions of the manufacturers. DNA primers were made by a DNA synthesizer (Applied Biosys-
tems). Isolation of HCV cDNAs. HCV cDNA that corresponds to nucleotide positions 9 to 791 (28) was isolated from pooled plasma of NANBH patients by a method involving polymerase chain reaction (PCR) as reported by Tsukiyama-Kohara et al. (31), using a sense primer, 5'-GGCGACACTCCAC CATAGATC-3', and an antisense primer, 5'-ATGCGCC
Corresponding author. 1476
VOL. 66, 1992
INTERNAL RIBOSOME ENTRY SITE IN HCV RNA 9
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AGGGCCCTGGCAGC-3'. The cDNA thus synthesized was inserted into the SmaI site of cloning vector pUC119. Nucleotide sequence analysis of 10 clones by using the 7-deaza Sequenase kit (United States Biochemical) revealed that 7 of the 10 clones had the same nucleotide sequence. A cDNA clone, named clone 2-1, was chosen from these seven clones as an HCV cDNA clone and used for further plasmid construction. A cDNA clone, EN2-3, that corresponds to nucleotide positions 676 to 1772 was isolated by immunoscreening from a Xgtll cDNA library prepared from sera of chronic 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 and inserted into the KS vector that had been digested with BamHI and HindIII. The plasmid thus obtained contains an HCV cDNA that corresponds to nucleotide positions 9 to 1772 downstream of the 410 promoter for phage T7 RNA polymerase and was designated pKIV (Fig. 1). Construction of deletion mutants. Mutants with deletions of the 5' UTR (pkI5, pkI6, pkI7, pkI8, pkI9, pkI10, pkIll, and pkI16) were constructed by the PCR method, using synthetic primers. Nucleotide sequences of sense DNA primers were designed to give an XbaI site to the corresponding 5' end of the PCR products. A XbaI-AatII cDNA fragment of plasmid pKIV was replaced by the corresponding cDNA fragments of the PCR products. In the case of pkI16, a segment corresponding to nucleotide positions 333 to 434 was synthesized by the PCR method, digested with AatII, and inserted into pKIV that had been digested with Stul and AatII. To exclude the influence of a sequence existing between the T7 promoter and HCV cDNA, the XbaI and HindIII cDNA fragments of pKIV and its deletion plasmids were filled in with Klenow fragment and subcloned with correct orientation into the StuI site of plasmid pNar3, which had been derived from pUC119 (Fig. 1); the resulting constructs 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 Stul site of pNar3, and the resulting plasmid was designated pNSt (Fig. 1). Construction of cDNA clones that carry CAT cDNA. The chloramphenicol acetyltransferase (CAT) cDNA carrying Sacl and XbaI sites at the corresponding 5' and 3' ends, respectively, was introduced into the Sacl and XbaI sites of pKIV, pkI6, pkI7, pkI8, pkI9, pkI10, pkIll, and pkI16. These cDNA clones encoding dicistronic mRNAs were designated pCV, pC6, pC7, pC8, pC9, pC10, pCll, and pC16, respectively (see Fig. 6A). To construct plasmid 5HcCAT, an XbaI-BspHI fragment of pKIV, which carries a segment of untranslated region of HCV, and the CAT cDNA that had BspHI and Sall sites at the corresponding 5' and 3' ends, respectively, were ligated and inserted into KS vector that had been digested with XbaI and SalI (Fig. 1). A StuI-SalI fragment of 5HcCAT, after treatment with Klenow fragment, was inserted with correct orientation into the Stul site of pNar3. The plasmid thus obtained was designated 5HStCAT (Fig. 1). Construction of cDNA that carries the 5' UTR sequence of group II HCV RNA. A segment of group II HCV cDNA (corresponding to nucleotide positions 49 to 408) was prepared from plasma of a patient infected with group II HCV alone by the PCR method, using 5'-CTGTCTTCACGCA GAAAGCG-3' and 5'-CCGGGAACTTGACGTCCTGT-3' as primers, and subcloned into the SmaI site of pUC19; the nucleotide sequence was then determined as described above. An XbaI-AatII cDNA fragment of pKIV was replaced by the XbaI-AatII cDNA fragment containing the 5' UTR sequence of group II HCV. An XbaI-HindIII cDNA fragment (1,724 bp) of the plasmid thus obtained was subcloned into the StuI site of pNar3, and the resulting plasmid was 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
1478
J. VIROL.
TSUKIYAMA-KOHARA ET AL.
pNSt) and transcribed into RNA in vitro with T7 RNA polymerase (Takara Shuzo Co.). In most cases, cap structure, m7GpppG or GpppG (Pharmacia LKB), was incorporated at the 5' ends of RNA transcripts as described previously (14). In vitro translation. Suspension-cultured HeLa S3 cells were infected with coxsackievirus Bi (CVB1) as previously described (8). Cytoplasmic extracts (S10) of mock- or CVB1infected cells were prepared 3 h after the infection and were treated with micrococcal nuclease as previously described (9). Conditions for the translation reaction (12.5 ,ul) were as described by Rose et al. (27). After incubation at 37°C for 1 h in the presence of [35S]methionine (ICN Radiochemicals), 5 ,ul of each of the translation mixtures was mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer, boiled for 1 min, and analyzed by electrophoresis on a 12.5% polyacrylamide gel in Laemmli's buffer system (21). In some cases, translation products were cotranslationally processed in the presence of canine microsomal membrane (Amersham). The processed translation products were mixed with rabbit anti-gp35 antibodies and then with Affi-Gel protein A (Bio-Rad). The precipitates were analyzed by electrophoresis as described above. Rabbit reticulocyte lysate (RRL; Amersham N150) was also used as in vitro translation system. Translation reactions in RRL were performed at 30°C for 1 h. In some cases, translation reactions were carried out in the presence of 20 FM S-adenosylhomocysteine (SAH) to prevent methylation of GpppG-ended RNA during the reaction (2, 5). Preparation of rabbit anti-gp35 antibodies. A cDNA clone, C10-E12, that corresponds to HCV RNA, including a sequence encoding gp35, was isolated by immunoscreening from the Agtll cDNA library prepared from sera of NANBH patients (31). This cDNA clone was inserted into the hemagglutinin gene downstream of the P7.5 promoter of the Lister strain of vaccinia virus (30). Rabbits were immunized by intradermal inoculation of the recombinant vaccinia virus. The specificity of the rabbit serum was verified by its specific reaction with two kinds of products directed by the HCV genome region encoding gp35, that is, materials produced by using a baculovirus expression vector system and an in vitro translation system (1Sa). Secondary structure of the HCV 5' UTR. A possible secondary structure of HCV RNA was predicted for nucleotide positions 1 to 600 by using the Fold program (Biosequence 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) have demonstrated that capped HCV RNAs with a short 5' UTR can be translated into the correct HCV proteins in the RRL cell-free translation system. The RNA used by Hijikata et al. (7) did not have AUG sequences upstream of the initiator AUG. To determine the effect of the upstream AUG sequences on translation, an RNA with a free 5' end synthesized from linearized pNV was examined for mRNA activity in a cell-free translation system prepared from HeLa S3 cells (Fig. 2). Efficient translation occurred, and a polypeptide with the expected size (approximately 52 kDa) was observed on a gel (Fig. 2, lane 2). A small amount of product that migrated slightly faster than the 52-kDa polypeptide was also observed (lane 2). This material may have resulted from partially degraded RNA template. In any event, the data strongly suggest that the translation of uncapped HCV RNA
1
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3
4
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FIG. 2. In vitro translation of uncapped HCV RNA. HCV RNA with a free 5' end corresponding to nucleotide positions 9 to 1772 was used as mRNA in a cell-free translation system with lysates prepared from HeLa S3 cells, and the products were analyzed as described in Materials and Methods. No RNA (lane 1) and uncapped HCV RNA (lanes 2 to 4) were translated in the presence (lanes 3 and 4) 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 and polyacrylamide gel electrophoresis were performed as described in Materials and Methods. Positions of molecular weight markers are indicated at the left; positions of gp35 and p22 are indicated at the right.
starts at the fourth AUG, the authentic initiator AUG, from the 5' end of the RNA. Thus, the upstream AUG seems not to have a deleterious effect on the translation initiation on HCV RNA in this in vitro translation system. To confirm that the translation products contain the correct HCV polypeptides, the translation reaction was performed in the presence of canine microsomal membrane (Fig. 2, lane 3). Two major proteins with approximate molecular masses of 22 and 35 kDa were observed in addition to the unprocessed products. This observation is compatible with that reported previously (7) and therefore indicates that these products are p22 and gp35, respectively. A faint band at approximately 11 kDa may be the product of the region encoding a part of gp7O. An immunoprecipitation experiment involving rabbit anti-gp35 serum was performed as described in Materials and Methods. The precipitates with the rabbit serum were separated by the SDS-polyacrylamide gel electrophoresis (Fig. 2, lane 4). The rabbit serum reacted with both gp35 and the unprocessed products. These results indicate that the in vitro translation products contain the correct HCV polyprotein. It is therefore possible that ribosomes bind to an internal sequence within the 5' UTR like the translation initiation on picornavirus RNAs. Effect of cap on translation. Since it is known that cap structure linked to the 5' end of picornavirus RNAs has little effect on the efficiency of translation initiation (25), mRNA activities of HCV RNAs carrying m7GpppG (capped methylated) or GpppG (capped unmethylated) at the 5' end were compared with each other in the absence (Fig. 3a, lanes 2 to 7) or presence (Fig. 3b, lanes 2 to 7) of SAH. mRNA activity of capped methylated HCV RNA derived from pNV appears to be similar to that of capped unmethylated HCV RNA from
VOL. 66, 1992 a) kDa
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INTERNAL RIBOSOME ENTRY SITE IN HCV RNA
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FIG. 4. Translation of dicistronic mRNA in RRL and CVB1infected HeLa cell lysates. No RNA (lanes 1 and 4) and capped methylated (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 translation products were analyzed as described in the legend to Fig. 2. Positions of the products from the first cistron (CAT mRNA) and the second cistron (HCV RNA) are indicated at the right; positions of molecular weight markers are indicated at the left.
+
FIG. 3. Effect of cap on in vitro translation. In vitro translation reactions 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 HeLa cell lysates (lanes 1, 2, 3, 9, and 10) or in RRL (lanes 4 to 8 and 11 to 14), and the translation products were analyzed as described in the legend to Fig. 2. Positions of products from translated regions of HCV and CAT mRNAs are indicated at the right; positions of molecular weight markers are indicated at the left.
pNV in cell-free translation systems prepared from HeLa S3 cells (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 efficient than capped unmethylated RNA. In the case of HCV RNA derived from pNSt, which lacks a nucleotide sequence upstream of position 270, capped methylated RNA is much more effective as mRNA than is capped unmethylated RNA in RRL (Fig. 3a and b, lanes 6 and 7). These data strongly suggest that a segment within the 5' UTR of HCV RNA has the ability to initiate protein synthesis independently of cap functions. The relative efficiency of the translation was not affected by the presence of SAH, a competitive inhibitor for an RNA (guanine-7-)-methyltransferase activity existing in HeLa S10 and RRL (2, 5). This finding suggested that differences in translation efficiencies observed between
capped 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 of translation, the translated region of HCV RNAs was re-
placed by CAT mRNA as described in Materials and Methods. Translation experiments similar to those shown in Fig. 3a and b, lanes 2 to 7, were carried out on these recombinant mRNAs (Fig. 3a and b, lanes 9 to 14). The data are very similar to those obtained in the experiment involving parental HCV RNAs. These results indicate that a segment required for the cap-independent translation initiation resides within the 5' UTR of HCV RNA. Existence of IRES within the 5' UTR of HCV RNA. To prove the existence of IRES within the 5' UTR of HCV RNA, a capped methylated (m7GpppG-linked) dicistronic mRNA consisting of CAT mRNA as the first cistron and HCV RNA as the second cistron was synthesized from linearized pCV. The structure of pCV is shown in Fig. 1. The translation experiments were performed on this dicistronic mRNA by using RRL (Fig. 4, lane 2) and lysates of CVB1infected HeLa S3 cells (Fig. 4, lane 3). A protein synthesis system prepared from CVB1-infected HeLa S3 cells has the ability to suppress cap-dependent initiation of translation (unpublished results) as observed in that from poliovirusinfected HeLa cells (4, 22). Both CAT mRNA and HCV RNA appear to be translated in RRL (Fig. 4, lane 2). However, only HCV RNA appears to be translated in lysate of CVB1-infected HeLa S3 cells (Fig. 4, lane 3). Since a dicistronic mRNA on polysomes was shown to be intact in HeLa cell extracts (26), the data strongly suggest that the second cistron is translated even when ribosome entry does not occur at the 5' end of the dicistronic mRNA. Therefore, it is very likely that an IRES exists within the 5' UTR of HCV RNA. Possible secondary structure of the 5' UTR of HCV RNA. A possible secondary structure was predicted as described in Materials and Methods and is shown in Fig. 5. Six possible stem-loop structures observed in the 5' UTR were designated A to F, respectively, from the 5' end of RNA. To confirm the validity of the secondary structure, RNA sequences of the 5' UTR of group I HCV and group II HCV RNAs (31) were compared with each other. Nucleotides of group II HCV that are different from those of group I HCV are indicated by arrows in Fig. 5. Many differenw nucleotides
J. VIROL.
TSUKIYAMA-KOHARA ET AL.
1480
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the translation product of the HCV RNAs derived from dicistronic cDNAs, pC1O and pC1l, was not observed (Fig. 6C, lanes 7 and 8). This observation may support the finding that translated dicistronic mRNA is intact during the translation reaction (26). To identify the 3' end of the IRES, a deletion RNA derived from pN16 was synthesized and tested for mRNA activity. This deletion RNA was not an active mRNA (Fig. 6B, lane 8). A similar observation was made when a dicistronic mRNA derived from pC16 was used as mRNA in RRL (Fig. 6C, lane 9). These results indicate that the IRES of the 5' UTR of HCV RNA resides between nucleotide positions 101 and 332. Efficiency of group II HCV RNA. Since nucleotide differences were observed in IRESs within the 5' UTRs between group I and group II HCV RNAs, IRES functions of group I and group II HCV RNAs were compared in a cell-free protein synthesis system of HeLa S3 cells. Capped unmethylated (GpppG-linked) HCV RNAs (nucleotide positions 49 to 1772) containing the 5' UTRs of group I and group II HCV RNAs were prepared from pN5 and pNII5, respectively, as described in Materials and Methods and used as mRNAs. As shown in Fig. 7, translation initiation from the 5' UTR of group II HCV RNA is more efficient than that of group I HCV RNA. Similar results were obtained with three pairs of RNA preparations (data not shown). Furthermore, amounts of radioactivity incorporated into the product increased proportionally with an RNA dose of up to 2 ,ug per reaction mixture. These results may indicate that regions harboring different nucleotides between group I and group II HCV RNAs play an important role(s) in IRES function.
G U
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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 observation confirms the validity of the possible secondary structure shown in Fig. 5, especially with respect to stem-loop structure 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
into
structures
consideration
as
de-
scribed in Materials and Methods (Fig. 6A). Capped unmethylated (GpppG-linked) pN7, pN8, pN9, pNlO,
RNAs
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derived
from pNV,
pN6,
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
derived from pCV, pC6, pC7, and pC8 (Fig.
mRNAs
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 tendency to recognize and bind to the end of RNA (17). Indeed,
DISCUSSION In this study, we demonstrated that initiation of HCV protein synthesis occurs in a cap-independent manner like that of picornavirus-specific protein synthesis and that an IRES within the 5' UTR of HCV RNA resides between nucleotide positions 101 and 332. Previous studies of picornavirus RNA have shown that IRES function requires a specific segment of approximately 400 nucleotides (11, 26). Thus, the IRES of HCV RNA is the shortest among IRESs so far discovered. Mutational analysis of picornavirus RNAs indicated that 21- and 7-base sequences conserved in the 5' UTR of human enterovirus and rhinovirus RNAs are important for translation initiation (8, 9, 19, 25). These sequences were not observed in the 5' UTR of HCV RNA. Therefore, HCV and picornaviruses may have different mechanisms for the entry of ribosomes to the IRES. An oligopyrimidine tract before the AUG at position 206, however, was observed within the 5' UTR of HCV RNA. An oligopyrimidine tract before an AUG is conserved in picornavirus RNA and is considered to be important for translation initiation on the viral RNAs (1, 19, 24). This AUG is the initiator AUG (encephalomyocarditis virus and foot-and-mouth disease virus) or most 3'-proximal AUG (rhinovirus and poliovirus) in the 5' UTR (12, 13, 20). The existence of this RNA sequence in both HCV and picornavirus RNAs suggests that the same initiation factor(s) is involved in the translation initiation on both viral RNAs. Like the IRES of encephalomyocarditis virus, the IRES of HCV is very effective in both HeLa cell lysates and RRL. Indeed, the HCV IRES is much more effective than the CVB1 IRES both in mock- and CVB1-infected HeLa cell lysates (unpublished result). Capped methylated and capped unmethylated HCV RNAs did not show any difference in the
INTERNAL RIBOSOME ENTRY SITE IN HCV RNA
VOL. 66, 1992
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-
CAT
FIG. 6. Structures of mutant cDNAs and translation products of their transcripts in RRL. (A) Structures of mutant cDNAs. Positions of stem-loop structures (A to F) (boxes) and nucleotide numbers and positions of AUG sequences are shown at the top. Regions missing in plasmids are indicated by broken lines. Numbers on cDNAs represent nucleotide positions of the terminal nucleotides. Open triangles represent 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 legend to Fig. 2. As a control, a sample with no RNA (lane 1) was also analyzed. Positions of the translation products (HCV and CAT) are indicated at the right.
efficiency of translation in HeLa S3 lysates (Fig. 3a and b, lanes 2 and 3). However, capped methylated HCV RNA is more efficient than capped unmethylated HCV RNA in RRL (Fig. 3a and b, lanes 4 and 5). It is therefore possible that HCV RNA requires a cap structure to be a fully active mRNA in RRL. This phenomenon is difficult to explain at present. It is of interest that the IRES of group II HCV is more effective than the IRES of group I HCV in HeLa cell lysates (Fig. 7), although group I HCV appears to be the major group of HCV (31). In any event, different nucleotides observed in the IRESs of group I and group II HCV RNAs
must influence IRES function, which may provide some insight into the mechanism of the IRES function of HCV. Mutational analysis of HCV RNA with respect to IRES function is currently under way. Although the nucleotide sequence of almost the entire genome 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 indicates that HCV has a mechanism for translation initiation totally different from that of a member of the family Flaviridiae;
1482
TSUKIYAMA-KOHARA ET AL. pNII5
kDa
1
2
pN5 3
974_ 6946-.
30-a.
21.5-
14.3-
FIG. 7. Translation efficiency from the 5' UTRs of group I and 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 a cell-free protein synthesis system prepared from HeLa S3 cells. Both HCV RNAs correspond to nucleotide positions 49 to 1772, as described in Materials and Methods. The translation products were analyzed as described in the legend to Fig. 2. Positions of molecular weight markers are indicated at the left.
group
therefore, creation of new virus family may be desirable for classification of HCV. ACKNOWLEDGMENTS
We thank F. Ochikubo for help in computer analysis, and we acknowledge the significant support of S. Tanaka and N. Hattori in the collection of patient plasma. We also thank K. Mizumoto for helpful discussions. This work was supported by research grants from The Ministry of Education, Science and Culture of Japan and The Ministry of Health and Welfare of Japan.
REFERENCES 1.
Bienkowska-Szewczyk, K., and E. Ehrenfeld. 1988. An internal 5'-noncoding region required for translation of poliovirus RNA
in vitro. J. Virol. 62:3068-3072. Both, G. W., A. K. Barnerjee, and A. J. Shatkin. 1975. Methylation 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 protein synthesis, p. 177-221. In H. Fraenkel-Conrat and R. R. Wagner 2.
(ed.), Comprehensive virology, vol. 19. Plenum Press, New York.
Ensinger, M. J., and B. Moss. 1976. Modification of the 5' terminus of mRNA by an RNA (guanine-7-)-methyltransferase from 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, and M. Houghton. 1991. Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 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 of the 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. Komatsu, K. Tago, M. Arita, and A. Nomoto. 1989. Construction of less neurovirulent polioviruses by introducing deletions into the 5' noncoding sequence of the genome. J. Virol. 63:5354-5363. 5.
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9. Iizuka, N., H. Yonekawa, and A. Nomoto. 1991. Nucleotide sequences important for translation initiation of enterovirus RNA. 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 ribosomes into the 5' nontranslated region of encephalomyocarditis virus 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 directs 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. In R. E. Thach (ed.), Translationally regulated genes in higher eukaryotes, in press. Karger, Basel. 13. Jang, S. K., and E. Wimmer. 1990. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kDa RNA binding protein. Genes Dev. 4:1560-1572. 14. Kaminski, A., M. T. Howell, and R. J. Jackson. 1990. Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism. EMBO J. 9:3753-3759. 15. Kato, N., M. Hijikata, Y. Ootsuyama, M. Nakagawa, S. Ohkoshi, T. Sugiyama, and K. Shimotohno. 1990. Molecular cloning of the human hepatitis C virus genome from Japanese patients with 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 deletion and 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 the internal translation initiation site of foot-and-mouth disease virus. J. Virol. 64:4625-4631. 21. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 22. Lee, K. A. W., and N. Sonenberg. 1982. Inactivation of capbinding proteins accompanies the shut-off of host protein synthesis 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 codons of 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 conferred by sequence elements within the 5' noncoding region. Mol. Cell. Biol. 8:1103-1112. 26. Pelletier, J., and N. Sonenberg. 1988. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from 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 specific initiation 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 genome isolated from human carriers. J. Virol. 65:1105-1113. 29. Takeuchi, K., Y. Kubo, S. Boonmar, Y. Watanabe, T.
VOL. 66, 1992 Katayama, Q.-L. Choo, G. Kuo, M. Houghton, I. Saito, and T. Miyamura. 1990. The putative nucleotide and envelope protein genes of hepatitis C virus determined by comparison of the nucleotide sequences of two isolates derived from an experimentally infected chimpanzee and healthy human carrier. J. Gen. Virol. 71:3021-3033. 30. Tsukiyama, K., Y. Yoshikawa, H. Kamata, K. Imaoka, K.
INTERNAL RIBOSOME ENTRY SITE IN HCV RNA
1483
Asano, S. Funahashi, T. Maruyama, H. Shida, M. Sugimoto, and K. Yamanouchi. 1989. Development of heat-stable recombinant rinderpest 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 5:243254.
JOURNAL OF VIROLOGY, Mar. 1992, p. 1476-1483
0022-538X/92/031476-08$02.00/0 Copyright © 1992, American Society for Microbiology
Internal Ribosome Entry Site within Hepatitis C Virus RNA KYOKO 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. HCV RNA was transcribed from the cDNA that corresponded to nucleotide positions 9 to 1772 of the genome by using phage T7 RNA polymerase. Both capped and uncapped RNAs thus transcribed were active as mRNAs in a cell-free protein synthesis system with lysates prepared from HeLa S3 cells or rabbit reticulocytes, and the translation products were detected by anti-gp35 antibodies. The data indicate that protein synthesis starts at the fourth AUG, which was the initiator AUG at position 333 of the HCV RNA used in this study. Efficiency of 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 capped unmethylated RNA in rabbit reticulocyte lysates when the RNA lacked a nucleotide sequence upstream of position 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 region of HCV RNA plays an important role in efficient translation. Our data suggest that the IRES resides in this region of the RNA. Furthermore, an IRES in the group II HCV RNA was found to be more efficient than that in the group I HCV RNA.
IRES (internal ribosome entry site) (10, 11). IRES function requires a specific segment of the 5' UTR of approximately 400 nucleotides which does not include the extreme 5'proximal nucleotide sequences. Structural similarities of the 5' UTR of HCV RNA to those of picornavirus RNAs provide the possibility that IRES function resides in the 5' UTR of HCV RNA, although the 5' UTR of HCV RNA is shorter than a segment of picornavirus RNAs required for its IRES function, and it is not known whether the 5' end of HCV RNA is capped. Here we demonstrate, by using cell-free translation systems, that the translation initiation on HCV RNAs occurs by entry of ribosomes to the internal sequence within the 5' UTR and that IRES function requires a segment of nucleotide positions 101 to 332 at most, which is the shortest sequence thus far identified as an IRES. We also show that the function of the IRES of group II HCV RNA is more efficient than that of the IRES of group I HCV RNA in a cell-free protein synthesis system from HeLa S3 cells.
The nucleotide sequence of almost the entire genome of hepatitis C virus (HCV), the main causative agent of chronic non-A, non-B hepatitis (NANBH), has been elucidated (3, 15, 23, 28). The genome of HCV is a single-stranded RNA with positive polarity and consists of approximately 10,000 nucleotides. This RNA carries only one long open reading frame for the synthesis of a large single polyprotein of 3,010 amino acids. Analysis of the amino acid sequences deduced from the nucleotide sequences of different HCV isolates (3, 15, 28) revealed that the HCV genomes have a gene organization similar to those of flaviviruses and their related viruses (29). Sequence analysis also revealed the existence of a fairly long 5' untranslated region (UTR) that harbors three (15, 28) or four (6) AUG sequences. This feature of the 5' UTR resembles that of picornaviruses rather than that of flaviviruses. Picornavirus RNAs are uncapped messengers and have unusually long 5' UTRs (ranging from 610 to 1,200 residues, depending on the virus species) which contain many silent AUG sequences. These features seem incompatible with efficient translation by the scanning ribosome mechanism, which is the initiation mechanism of most eukaryotic cellular and viral mRNAs (16, 18). The rule of the scanning model is that ribosomes initially bind to the 5' end of mRNAs and scan the RNAs to reach the authentic initiator AUGs, which are, in many cases, the first AUG sequences. Furthermore, the cap structures at the 5' ends of mRNAs are generally believed to play an important role in the initial ribosome entry or binding step. Thus, the initiation of translation on picornavirus RNAs has been considered to take place by an unusual mechanism. Recently, it was proved that translation initiation on picornavirus RNAs occurs by binding of ribosomes to an internal sequence within the 5' UTR (10, 11, 26). An internal entry site for ribosomes has been called the *
MATERIALS AND METHODS DNA procedure. Restriction endonucleases, DNA polymerase I (Klenow fragment), and T4 DNA ligase were purchased from Takara Shuzo Co. (Kyoto, Japan); calf intestinal alkaline phosphatase was from Boehringer GmbH (Mannheim, Germany); KS vector was from Stratagene (La Jolla, Calif.). These enzymes and compounds were used according to the instructions of the manufacturers. DNA primers were made by a DNA synthesizer (Applied Biosys-
tems). Isolation of HCV cDNAs. HCV cDNA that corresponds to nucleotide positions 9 to 791 (28) was isolated from pooled plasma of NANBH patients by a method involving polymerase chain reaction (PCR) as reported by Tsukiyama-Kohara et al. (31), using a sense primer, 5'-GGCGACACTCCAC CATAGATC-3', and an antisense primer, 5'-ATGCGCC
Corresponding author. 1476
VOL. 66, 1992
INTERNAL RIBOSOME ENTRY SITE IN HCV RNA 9
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AGGGCCCTGGCAGC-3'. The cDNA thus synthesized was inserted into the SmaI site of cloning vector pUC119. Nucleotide sequence analysis of 10 clones by using the 7-deaza Sequenase kit (United States Biochemical) revealed that 7 of the 10 clones had the same nucleotide sequence. A cDNA clone, named clone 2-1, was chosen from these seven clones as an HCV cDNA clone and used for further plasmid construction. A cDNA clone, EN2-3, that corresponds to nucleotide positions 676 to 1772 was isolated by immunoscreening from a Xgtll cDNA library prepared from sera of chronic 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 and inserted into the KS vector that had been digested with BamHI and HindIII. The plasmid thus obtained contains an HCV cDNA that corresponds to nucleotide positions 9 to 1772 downstream of the 410 promoter for phage T7 RNA polymerase and was designated pKIV (Fig. 1). Construction of deletion mutants. Mutants with deletions of the 5' UTR (pkI5, pkI6, pkI7, pkI8, pkI9, pkI10, pkIll, and pkI16) were constructed by the PCR method, using synthetic primers. Nucleotide sequences of sense DNA primers were designed to give an XbaI site to the corresponding 5' end of the PCR products. A XbaI-AatII cDNA fragment of plasmid pKIV was replaced by the corresponding cDNA fragments of the PCR products. In the case of pkI16, a segment corresponding to nucleotide positions 333 to 434 was synthesized by the PCR method, digested with AatII, and inserted into pKIV that had been digested with Stul and AatII. To exclude the influence of a sequence existing between the T7 promoter and HCV cDNA, the XbaI and HindIII cDNA fragments of pKIV and its deletion plasmids were filled in with Klenow fragment and subcloned with correct orientation into the StuI site of plasmid pNar3, which had been derived from pUC119 (Fig. 1); the resulting constructs 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 Stul site of pNar3, and the resulting plasmid was designated pNSt (Fig. 1). Construction of cDNA clones that carry CAT cDNA. The chloramphenicol acetyltransferase (CAT) cDNA carrying Sacl and XbaI sites at the corresponding 5' and 3' ends, respectively, was introduced into the Sacl and XbaI sites of pKIV, pkI6, pkI7, pkI8, pkI9, pkI10, pkIll, and pkI16. These cDNA clones encoding dicistronic mRNAs were designated pCV, pC6, pC7, pC8, pC9, pC10, pCll, and pC16, respectively (see Fig. 6A). To construct plasmid 5HcCAT, an XbaI-BspHI fragment of pKIV, which carries a segment of untranslated region of HCV, and the CAT cDNA that had BspHI and Sall sites at the corresponding 5' and 3' ends, respectively, were ligated and inserted into KS vector that had been digested with XbaI and SalI (Fig. 1). A StuI-SalI fragment of 5HcCAT, after treatment with Klenow fragment, was inserted with correct orientation into the Stul site of pNar3. The plasmid thus obtained was designated 5HStCAT (Fig. 1). Construction of cDNA that carries the 5' UTR sequence of group II HCV RNA. A segment of group II HCV cDNA (corresponding to nucleotide positions 49 to 408) was prepared from plasma of a patient infected with group II HCV alone by the PCR method, using 5'-CTGTCTTCACGCA GAAAGCG-3' and 5'-CCGGGAACTTGACGTCCTGT-3' as primers, and subcloned into the SmaI site of pUC19; the nucleotide sequence was then determined as described above. An XbaI-AatII cDNA fragment of pKIV was replaced by the XbaI-AatII cDNA fragment containing the 5' UTR sequence of group II HCV. An XbaI-HindIII cDNA fragment (1,724 bp) of the plasmid thus obtained was subcloned into the StuI site of pNar3, and the resulting plasmid was 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
1478
J. VIROL.
TSUKIYAMA-KOHARA ET AL.
pNSt) and transcribed into RNA in vitro with T7 RNA polymerase (Takara Shuzo Co.). In most cases, cap structure, m7GpppG or GpppG (Pharmacia LKB), was incorporated at the 5' ends of RNA transcripts as described previously (14). In vitro translation. Suspension-cultured HeLa S3 cells were infected with coxsackievirus Bi (CVB1) as previously described (8). Cytoplasmic extracts (S10) of mock- or CVB1infected cells were prepared 3 h after the infection and were treated with micrococcal nuclease as previously described (9). Conditions for the translation reaction (12.5 ,ul) were as described by Rose et al. (27). After incubation at 37°C for 1 h in the presence of [35S]methionine (ICN Radiochemicals), 5 ,ul of each of the translation mixtures was mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer, boiled for 1 min, and analyzed by electrophoresis on a 12.5% polyacrylamide gel in Laemmli's buffer system (21). In some cases, translation products were cotranslationally processed in the presence of canine microsomal membrane (Amersham). The processed translation products were mixed with rabbit anti-gp35 antibodies and then with Affi-Gel protein A (Bio-Rad). The precipitates were analyzed by electrophoresis as described above. Rabbit reticulocyte lysate (RRL; Amersham N150) was also used as in vitro translation system. Translation reactions in RRL were performed at 30°C for 1 h. In some cases, translation reactions were carried out in the presence of 20 FM S-adenosylhomocysteine (SAH) to prevent methylation of GpppG-ended RNA during the reaction (2, 5). Preparation of rabbit anti-gp35 antibodies. A cDNA clone, C10-E12, that corresponds to HCV RNA, including a sequence encoding gp35, was isolated by immunoscreening from the Agtll cDNA library prepared from sera of NANBH patients (31). This cDNA clone was inserted into the hemagglutinin gene downstream of the P7.5 promoter of the Lister strain of vaccinia virus (30). Rabbits were immunized by intradermal inoculation of the recombinant vaccinia virus. The specificity of the rabbit serum was verified by its specific reaction with two kinds of products directed by the HCV genome region encoding gp35, that is, materials produced by using a baculovirus expression vector system and an in vitro translation system (1Sa). Secondary structure of the HCV 5' UTR. A possible secondary structure of HCV RNA was predicted for nucleotide positions 1 to 600 by using the Fold program (Biosequence 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) have demonstrated that capped HCV RNAs with a short 5' UTR can be translated into the correct HCV proteins in the RRL cell-free translation system. The RNA used by Hijikata et al. (7) did not have AUG sequences upstream of the initiator AUG. To determine the effect of the upstream AUG sequences on translation, an RNA with a free 5' end synthesized from linearized pNV was examined for mRNA activity in a cell-free translation system prepared from HeLa S3 cells (Fig. 2). Efficient translation occurred, and a polypeptide with the expected size (approximately 52 kDa) was observed on a gel (Fig. 2, lane 2). A small amount of product that migrated slightly faster than the 52-kDa polypeptide was also observed (lane 2). This material may have resulted from partially degraded RNA template. In any event, the data strongly suggest that the translation of uncapped HCV RNA
1
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FIG. 2. In vitro translation of uncapped HCV RNA. HCV RNA with a free 5' end corresponding to nucleotide positions 9 to 1772 was used as mRNA in a cell-free translation system with lysates prepared from HeLa S3 cells, and the products were analyzed as described in Materials and Methods. No RNA (lane 1) and uncapped HCV RNA (lanes 2 to 4) were translated in the presence (lanes 3 and 4) 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 and polyacrylamide gel electrophoresis were performed as described in Materials and Methods. Positions of molecular weight markers are indicated at the left; positions of gp35 and p22 are indicated at the right.
starts at the fourth AUG, the authentic initiator AUG, from the 5' end of the RNA. Thus, the upstream AUG seems not to have a deleterious effect on the translation initiation on HCV RNA in this in vitro translation system. To confirm that the translation products contain the correct HCV polypeptides, the translation reaction was performed in the presence of canine microsomal membrane (Fig. 2, lane 3). Two major proteins with approximate molecular masses of 22 and 35 kDa were observed in addition to the unprocessed products. This observation is compatible with that reported previously (7) and therefore indicates that these products are p22 and gp35, respectively. A faint band at approximately 11 kDa may be the product of the region encoding a part of gp7O. An immunoprecipitation experiment involving rabbit anti-gp35 serum was performed as described in Materials and Methods. The precipitates with the rabbit serum were separated by the SDS-polyacrylamide gel electrophoresis (Fig. 2, lane 4). The rabbit serum reacted with both gp35 and the unprocessed products. These results indicate that the in vitro translation products contain the correct HCV polyprotein. It is therefore possible that ribosomes bind to an internal sequence within the 5' UTR like the translation initiation on picornavirus RNAs. Effect of cap on translation. Since it is known that cap structure linked to the 5' end of picornavirus RNAs has little effect on the efficiency of translation initiation (25), mRNA activities of HCV RNAs carrying m7GpppG (capped methylated) or GpppG (capped unmethylated) at the 5' end were compared with each other in the absence (Fig. 3a, lanes 2 to 7) or presence (Fig. 3b, lanes 2 to 7) of SAH. mRNA activity of capped methylated HCV RNA derived from pNV appears to be similar to that of capped unmethylated HCV RNA from
VOL. 66, 1992 a) kDa
1
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INTERNAL RIBOSOME ENTRY SITE IN HCV RNA
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FIG. 4. Translation of dicistronic mRNA in RRL and CVB1infected HeLa cell lysates. No RNA (lanes 1 and 4) and capped methylated (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 translation products were analyzed as described in the legend to Fig. 2. Positions of the products from the first cistron (CAT mRNA) and the second cistron (HCV RNA) are indicated at the right; positions of molecular weight markers are indicated at the left.
+
FIG. 3. Effect of cap on in vitro translation. In vitro translation reactions 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 HeLa cell lysates (lanes 1, 2, 3, 9, and 10) or in RRL (lanes 4 to 8 and 11 to 14), and the translation products were analyzed as described in the legend to Fig. 2. Positions of products from translated regions of HCV and CAT mRNAs are indicated at the right; positions of molecular weight markers are indicated at the left.
pNV in cell-free translation systems prepared from HeLa S3 cells (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 efficient than capped unmethylated RNA. In the case of HCV RNA derived from pNSt, which lacks a nucleotide sequence upstream of position 270, capped methylated RNA is much more effective as mRNA than is capped unmethylated RNA in RRL (Fig. 3a and b, lanes 6 and 7). These data strongly suggest that a segment within the 5' UTR of HCV RNA has the ability to initiate protein synthesis independently of cap functions. The relative efficiency of the translation was not affected by the presence of SAH, a competitive inhibitor for an RNA (guanine-7-)-methyltransferase activity existing in HeLa S10 and RRL (2, 5). This finding suggested that differences in translation efficiencies observed between
capped 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 of translation, the translated region of HCV RNAs was re-
placed by CAT mRNA as described in Materials and Methods. Translation experiments similar to those shown in Fig. 3a and b, lanes 2 to 7, were carried out on these recombinant mRNAs (Fig. 3a and b, lanes 9 to 14). The data are very similar to those obtained in the experiment involving parental HCV RNAs. These results indicate that a segment required for the cap-independent translation initiation resides within the 5' UTR of HCV RNA. Existence of IRES within the 5' UTR of HCV RNA. To prove the existence of IRES within the 5' UTR of HCV RNA, a capped methylated (m7GpppG-linked) dicistronic mRNA consisting of CAT mRNA as the first cistron and HCV RNA as the second cistron was synthesized from linearized pCV. The structure of pCV is shown in Fig. 1. The translation experiments were performed on this dicistronic mRNA by using RRL (Fig. 4, lane 2) and lysates of CVB1infected HeLa S3 cells (Fig. 4, lane 3). A protein synthesis system prepared from CVB1-infected HeLa S3 cells has the ability to suppress cap-dependent initiation of translation (unpublished results) as observed in that from poliovirusinfected HeLa cells (4, 22). Both CAT mRNA and HCV RNA appear to be translated in RRL (Fig. 4, lane 2). However, only HCV RNA appears to be translated in lysate of CVB1-infected HeLa S3 cells (Fig. 4, lane 3). Since a dicistronic mRNA on polysomes was shown to be intact in HeLa cell extracts (26), the data strongly suggest that the second cistron is translated even when ribosome entry does not occur at the 5' end of the dicistronic mRNA. Therefore, it is very likely that an IRES exists within the 5' UTR of HCV RNA. Possible secondary structure of the 5' UTR of HCV RNA. A possible secondary structure was predicted as described in Materials and Methods and is shown in Fig. 5. Six possible stem-loop structures observed in the 5' UTR were designated A to F, respectively, from the 5' end of RNA. To confirm the validity of the secondary structure, RNA sequences of the 5' UTR of group I HCV and group II HCV RNAs (31) were compared with each other. Nucleotides of group II HCV that are different from those of group I HCV are indicated by arrows in Fig. 5. Many differenw nucleotides
J. VIROL.
TSUKIYAMA-KOHARA ET AL.
1480
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the translation product of the HCV RNAs derived from dicistronic cDNAs, pC1O and pC1l, was not observed (Fig. 6C, lanes 7 and 8). This observation may support the finding that translated dicistronic mRNA is intact during the translation reaction (26). To identify the 3' end of the IRES, a deletion RNA derived from pN16 was synthesized and tested for mRNA activity. This deletion RNA was not an active mRNA (Fig. 6B, lane 8). A similar observation was made when a dicistronic mRNA derived from pC16 was used as mRNA in RRL (Fig. 6C, lane 9). These results indicate that the IRES of the 5' UTR of HCV RNA resides between nucleotide positions 101 and 332. Efficiency of group II HCV RNA. Since nucleotide differences were observed in IRESs within the 5' UTRs between group I and group II HCV RNAs, IRES functions of group I and group II HCV RNAs were compared in a cell-free protein synthesis system of HeLa S3 cells. Capped unmethylated (GpppG-linked) HCV RNAs (nucleotide positions 49 to 1772) containing the 5' UTRs of group I and group II HCV RNAs were prepared from pN5 and pNII5, respectively, as described in Materials and Methods and used as mRNAs. As shown in Fig. 7, translation initiation from the 5' UTR of group II HCV RNA is more efficient than that of group I HCV RNA. Similar results were obtained with three pairs of RNA preparations (data not shown). Furthermore, amounts of radioactivity incorporated into the product increased proportionally with an RNA dose of up to 2 ,ug per reaction mixture. These results may indicate that regions harboring different nucleotides between group I and group II HCV RNAs play an important role(s) in IRES function.
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 observation confirms the validity of the possible secondary structure shown in Fig. 5, especially with respect to stem-loop structure 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
into
structures
consideration
as
de-
scribed in Materials and Methods (Fig. 6A). Capped unmethylated (GpppG-linked) pN7, pN8, pN9, pNlO,
RNAs
pN11,
derived
from pNV,
pN6,
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
derived from pCV, pC6, pC7, and pC8 (Fig.
mRNAs
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 tendency to recognize and bind to the end of RNA (17). Indeed,
DISCUSSION In this study, we demonstrated that initiation of HCV protein synthesis occurs in a cap-independent manner like that of picornavirus-specific protein synthesis and that an IRES within the 5' UTR of HCV RNA resides between nucleotide positions 101 and 332. Previous studies of picornavirus RNA have shown that IRES function requires a specific segment of approximately 400 nucleotides (11, 26). Thus, the IRES of HCV RNA is the shortest among IRESs so far discovered. Mutational analysis of picornavirus RNAs indicated that 21- and 7-base sequences conserved in the 5' UTR of human enterovirus and rhinovirus RNAs are important for translation initiation (8, 9, 19, 25). These sequences were not observed in the 5' UTR of HCV RNA. Therefore, HCV and picornaviruses may have different mechanisms for the entry of ribosomes to the IRES. An oligopyrimidine tract before the AUG at position 206, however, was observed within the 5' UTR of HCV RNA. An oligopyrimidine tract before an AUG is conserved in picornavirus RNA and is considered to be important for translation initiation on the viral RNAs (1, 19, 24). This AUG is the initiator AUG (encephalomyocarditis virus and foot-and-mouth disease virus) or most 3'-proximal AUG (rhinovirus and poliovirus) in the 5' UTR (12, 13, 20). The existence of this RNA sequence in both HCV and picornavirus RNAs suggests that the same initiation factor(s) is involved in the translation initiation on both viral RNAs. Like the IRES of encephalomyocarditis virus, the IRES of HCV is very effective in both HeLa cell lysates and RRL. Indeed, the HCV IRES is much more effective than the CVB1 IRES both in mock- and CVB1-infected HeLa cell lysates (unpublished result). Capped methylated and capped unmethylated HCV RNAs did not show any difference in the
INTERNAL RIBOSOME ENTRY SITE IN HCV RNA
VOL. 66, 1992
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FIG. 6. Structures of mutant cDNAs and translation products of their transcripts in RRL. (A) Structures of mutant cDNAs. Positions of stem-loop structures (A to F) (boxes) and nucleotide numbers and positions of AUG sequences are shown at the top. Regions missing in plasmids are indicated by broken lines. Numbers on cDNAs represent nucleotide positions of the terminal nucleotides. Open triangles represent 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 legend to Fig. 2. As a control, a sample with no RNA (lane 1) was also analyzed. Positions of the translation products (HCV and CAT) are indicated at the right.
efficiency of translation in HeLa S3 lysates (Fig. 3a and b, lanes 2 and 3). However, capped methylated HCV RNA is more efficient than capped unmethylated HCV RNA in RRL (Fig. 3a and b, lanes 4 and 5). It is therefore possible that HCV RNA requires a cap structure to be a fully active mRNA in RRL. This phenomenon is difficult to explain at present. It is of interest that the IRES of group II HCV is more effective than the IRES of group I HCV in HeLa cell lysates (Fig. 7), although group I HCV appears to be the major group of HCV (31). In any event, different nucleotides observed in the IRESs of group I and group II HCV RNAs
must influence IRES function, which may provide some insight into the mechanism of the IRES function of HCV. Mutational analysis of HCV RNA with respect to IRES function is currently under way. Although the nucleotide sequence of almost the entire genome 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 indicates that HCV has a mechanism for translation initiation totally different from that of a member of the family Flaviridiae;
1482
TSUKIYAMA-KOHARA ET AL. pNII5
kDa
1
2
pN5 3
974_ 6946-.
30-a.
21.5-
14.3-
FIG. 7. Translation efficiency from the 5' UTRs of group I and 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 a cell-free protein synthesis system prepared from HeLa S3 cells. Both HCV RNAs correspond to nucleotide positions 49 to 1772, as described in Materials and Methods. The translation products were analyzed as described in the legend to Fig. 2. Positions of molecular weight markers are indicated at the left.
group
therefore, creation of new virus family may be desirable for classification of HCV. ACKNOWLEDGMENTS
We thank F. Ochikubo for help in computer analysis, and we acknowledge the significant support of S. Tanaka and N. Hattori in the collection of patient plasma. We also thank K. Mizumoto for helpful discussions. This work was supported by research grants from The Ministry of Education, Science and Culture of Japan and The Ministry of Health and Welfare of Japan.
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