From the Research and Development Center,
BioMedical Laboratories, 1361-1 Matoba, Kawagoe, Saitama 350-1101, Japan, the ¶ Research Institute for Microbial Diseases, Osaka
University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan, and
the
Max-Delbrück Center for Molecular Medicine, D-13125
Berlin-Buch, Germany
Received for publication, April 20, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Translational initiation of hepatitis C virus
(HCV) genome RNA occurs via its highly structured 5' noncoding region
called the internal ribosome entry site (IRES). Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex
that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as
the prime candidate protein for binding of the HCV IRES to the 40 S
subunit. RpS9 has a molecular mass of ~25 kDa in UV cross-linking
experiments. In the present study, we examined the ~25-kDa proteins
of the 40 S ribosome that form complexes with the HCV IRES upon UV
cross-linking. Immunoprecipitation with specific antibodies against two
25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the
protein bound to the IRES. Thus, our results support rpS5 as the
critical element in positioning the HCV RNA on the 40 S ribosomal
subunit during translation initiation.
Translational initiation of most eukaryotic messenger RNAs is
mediated by the binding of elongation initiation factor eIF-4 to the
modified nucleotide cap on the 5' end of the mRNA and the binding
of the 40 S ribosomal subunit and other initiation factors (1).
However, in hepatitis C virus
(HCV)1 as well as in members
of the picornavirus family, the positive-stranded RNA genome is not
capped. In these viral genomes, the 5' noncoding region (NCR) contains
an internal ribosomal entry site (IRES) (2, 3). Trans-acting
cellular proteins regulate viral protein synthesis by binding to
multiple sites within the IRES (2). For example, a polypyrimidine
tract-binding protein (PTB) interacts with several sites in the IRES
elements of picornaviruses (e.g. poliovirus (PV),
encephalomyocarditis virus, foot-and-mouth disease virus, and human
rhinovirus) (4-7). A 52-kDa nuclear factor, La protein, appears to be
essential for PV translation initiation, because its addition to
La-deficient rabbit reticulocyte lysates stimulates and corrects
PV translation (8). In addition to these two proteins, other cellular
factors (e.g. the 97-kDa protein and poly(rC)-binding
protein-2) are likely to stimulate translation initiation directed by
the picornavirus IRES (9, 10).
Identification and characterization of IRES-binding proteins are
important to the understanding of the mechanisms of internal initiation
and, ultimately, to the development of novel therapies for HCV. The
binding PTB and La are required for translation by HCV IRES (11, 12).
However, Kaminski et al. (13) reported that recombinant PTB
did not stimulate HCV IRES function, suggesting that PTB may not be
necessary during HCV translation initiation under certain experimental
conditions. Despite a considerably lower level of La protein in rabbit
reticulocyte lysate, HCV IRES promotes efficient translation activity
(14-16). This clearly differs from the case of translation initiation
on the PV genome. Thus, general models proposed for the process of 40 S
ribosome entry to IRES elements on the picornavirus genome do not seem
to apply to the HCV genome. In this respect, the formation of a stable binary complex between HCV IRES and purified 40 S ribosomal subunit is
of great interest. It differs fundamentally from the initiation process
on picornavirus IRES, which depends absolutely on one or more
initiation factors, and direct ribosome binding to IRES may be an
important step in internal initiation of the HCV genome (17).
Although other ribosomal proteins may interact with HCV IRES, the
binding of ribosomal protein S9 (rpS9) to the IRES has been assumed to
be the initial step in translation initiation (17). RpS9, with a
molecular mass of 25 kDa in UV cross-linking experiments, is a primary
target protein of binary IRES-40 S subunit complex. (17, 18). It
remains unclear whether rpS9 binding is essential for function of HCV
IRES, and unambiguous identification of ribosomal protein cross-linked
to HCV IRES is still missing.
We have previously shown that the binding of a 25-kDa cellular protein
(p25) to the HCV IRES is most likely an important step for efficient
translation of HCV (19). This p25 protein interacts specifically with
the HCV IRES and is cross-linked to the IRES when purified 40 S
subunits are being used as a binding material (20). Binding of p25 to
HCV IRES, therefore, seems to be crucial for the interaction between 40 S subunit and IRES and to be a unique feature in the translation
mechanism of HCV. In this study, we purified p25 from cultured HeLa
cells and characterized it by with specific antibodies. As the main
result we present here the first evidence that the HCV IRES-binding
protein of the 40 S ribosomal subunit is ribosomal protein S5 (rpS5)
and not rpS9 as suggested earlier.
Plasmid Construction--
Plasmid pUC5END-nc containing a
cDNA of full-length HCV 5' NCR located just downstream of the T7
promoter was described earlier (19). The cDNA for human rpS5 was
amplified from a HeLa cDNA library with polymerase chain
reaction primers HS51
(5'-GGATCCGATGACGATGACAAAATGACCGAGTGGGAGACAGCA-3') and HS52
(5'-GAAGCTTTCAGCGGTTGGACTTGGCCAC-3'), digested with BamHI and HindIII, and inserted into the pQE30 vector (Qiagen) to
yield pQE-RS5. Plasmid pQE-RS9, which contains a cDNA for human
rpS9, was constructed with the primers HS91
(5'-GGATCCGATGACGATGACAAACCAGTGGCCCGGAGCTGGGTT-3') and HS92
(5'-GAAGCTTTTAATCCTCCTCCTCGTCGTC-3') by the same strategy as used
for pQE-RS5.
Purification and Characterization of p25--
Cytoplasmic S10
extracts were prepared from HeLa S3 cells cultured in suspension.
Purification of p25 protein was monitored by UV cross-linking a
[32P]UTP-labeled HCV RNA probe to protein chromatographic
fractions. A HeLa S10 cytoplasmic extract containing ~650 mg of
protein was fractionated by gel filtration on Sephacryl S-300 HR
(Amersham Pharmacia Biotech) pre-equilibrated with buffer A (10 mM HEPES-KOH, pH 7.5, 1.5 mM MgCl2,
1 mM dithiothreitol, 5% glycerol) containing 120 mM KCl.
The binding activity of p25 to the HCV 5' NCR RNA probe was measured in
the void fractions. The void fractions were diluted with buffer A and
loaded onto a HiTrap heparin column (Amersham Pharmacia Biotech)
pre-equilibrated with buffer A. Fractions were collected by stepwise
elutions with 0, 150, 300, and 500 mM KCl in buffer A. Fractions containing binding activity to the HCV 5' NCR RNA were eluted
with 300 and 500 mM KCl in buffer A. The 500 mM
KCl fraction was diluted 5-fold with buffer A and loaded onto a Mono S
HR 5/5 column (Amersham Pharmacia Biotech) pre-equilibrated with buffer
A. Fractions were eluted by a gradient of 100-500 mM KCl
in buffer A. The fractions with the highest HCV 5' NCR RNA binding
activities were eluted by 250-340 mM KCl and pooled. Approximately 38 mg of the partially purified protein was obtained.
Amino Acid Sequence Analysis--
Partially purified protein was
separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
on a 14% gel. After electrophoretic transfer to a polyvinylidene
difluoride membrane, the proteins were visualized with the stain
Ponceau S. Selected peptide fragments were eluted, digested with
Achromobactor proteinase I (Lys-C), and subjected to Edman
degradation chemical sequencing.
In Vitro Transcription and UV Cross-linking--
A
[32P]UTP-labeled probe of high specific activity and
corresponding to nucleotides 1-347 of the HCV 5' NCR was transcribed from ScaI-digested pUC5END-nc with the Riboprobe T7 system
(Promega). Analyses of protein-RNA interactions after UV-induced
cross-linking were performed as described (19).
Antibodies--
Preparation of rabbit antisera
against rat 40 S ribosomal proteins, their purification by
immunosorption, and their monospecificity were as described (21, 22).
Preparation of polyclonal antibodies directed against PTB was as
described (23).
Immunoblotting and Immunoprecipitation--
JM109 cells were
transformed with pQE-RS5 and pQE-RS9, and recombinant rpS5 and rpS9
were isolated by Ni-NTA-agarose chromatography according to the
manufacturer's instructions (Qiagen). Recombinant ribosomal proteins
were separated by gradient SDS-PAGE on a 5-20% gel and used as
reference proteins in Western immunoblotting according to the method of
Towbin et al. (24).
Immunoprecipitation was performed with antibodies against rat ribosomal
proteins and PTB. HeLa S10 extracts cross-linked with [32P]UTP-labeled HCV IRES RNA were heat-denatured in
cross-linking buffer containing 0.5% SDS and centrifuged. The
supernatants were diluted 5-fold in sample buffer (25 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, and 1 mM dithiothreitol). After pre-incubation with uncoated
protein G-Sepharose for 60 min, the samples were centrifuged briefly,
and the supernatants were incubated with antibody-coated protein
G-Sepharose. The precipitates were washed with sample buffer six times
and analyzed by SDS-PAGE and autoradiography.
We used a HeLa S10 cytoplasmic extract as the starting material in
our efforts to purify and characterize p25 cross-linked to HCV IRES.
Purification of p25 was monitored by UV-induced cross-linking of the
protein fractions to a HCV probe. During subsequent gel filtration on a
Sephacryl S-300 column, p25 bound to the HCV probe and migrated close
to the void fractions, suggesting that p25 exists as a component of a
macromolecular complex rather than as a single cytoplasmic molecule
with an affinity for HCV (20).
The initial void fraction was subjected to heparin-Sepharose and cation
exchange chromatography. Maximum activities of HCV 5' NCR RNA binding
were eluted from cation exchange column by 250-340 mM KCl
(Fig. 1A). The proteins for
this fraction were separated by SDS-PAGE, and silver staining revealed
two closely migrating proteins of ~25 kDa and a peptide corresponding
to p25 just between them (Fig. 1B). The gel sections
containing each of the two proteins were cut out (Fig. 1C),
and the Lys-C-digested fragments were subjected to amino acid
sequencing. Nine of 10 amino acids of the slower migrating band
(a) matched amino acids 155-163 of human rpS9 (Fig.
1D). Ten amino acids of the faster migrating band
(b) were identical to amino acids 23-32 of human rpS5.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (52K):
[in a new window]
Fig. 1.
Isolation of p25 cross-linked to HCV
IRES. HeLa S10 extracts were applied to a Sephacryl S-300 column.
Void fractions from gel filtration were loaded onto heparin affinity
columns, and the samples eluted with 500 mM KCl were
fractionated on cation exchange columns. A, the result of UV
cross-linking of fractions 8-30 eluted with 200-430 mM
KCl are shown. An arrow indicates p25. B, silver
staining of the fractions described in A are also shown. The
molecular masses of the protein standards for SDS-PAGE are indicated on
the left. C, the position of the p25 fractions
from each purification step was determined by SDS-PAGE on 14% gels and
silver staining. Crude S10 extracts (lane 1), void
fractions from gel filtration (lane 2), 500 mM
KCl fraction from heparin affinity column (lane 3), and the
pool of 250-340 mM KCl fraction from cation-exchange
column (lane 4) are shown. Two protein bands (a
and b) migrated with an apparent molecular mass of 25 kDa.
Protein standards are indicated on the left. D,
amino acid sequence of a Lys-C fragment of the p25. Isolated peptide
corresponding to peptides a and b were subjected
to Edman degradation chemical sequencing. The resulting amino acid
sequences were used to search the SwissProt data base and were aligned
with the sequences of human rpS9 and rpS5.
To identify which protein corresponded to p25, we analyzed
the fractions by immunoprecipitation with rabbit anti-rpS5 and anti-rpS9 antibodies. The monospecificities of these antibodies had
been established previously (21), and we reconfirmed those by
immunoblot analysis. The anti-rpS5 and anti-rpS9 antibodies specifically recognized recombinant human rpS5 and rpS9, respectively (Fig. 2A). These antibodies
were mixed with HeLa cytoplasmic extracts that had been UV cross-linked
to [32P]UTP-labeled HCV IRES. Immunoprecipitation was
carried out under highly stringent conditions to avoid
nonspecific precipitation of aggregated protein during incubation
with antibodies (see "Experimental Procedures"). Analysis by
SDS-PAGE and autoradiography demonstrated that the cross-linked p25
was immunoprecipitated by anti-rpS5 antibodies but not by
anti-rpS9 antibodies (Fig. 2B). Furthermore, p25 was not
precipitated with a different rabbit anti-rpS9 preparation raised
against recombinant human rpS9 (data not shown). Among the control
antibodies, anti-PTB precipitated a single protein of 58 kDa, a size
corresponding to that of PTB. Anti-ribosomal protein S26 did not
precipitate any protein, indicating that nonspecific precipitation did
not exist under our experimental conditions. Thus, we conclude that the
p25 protein cross-linked to HCV IRES was rpS5.
|
In contrast, a previous report (17) indicated that rpS9 was the 25-kDa protein cross-linked to HCV RNA. We cannot explain the discrepancy between their results and ours. However, it is obviously difficult to clearly identify two ribosomal proteins of similar molecular mass and electrophoretic mobility, particularly when they are cross-linked to the HCV IRES. Although the radioactive signal of p25 shown on the autoradiogram is closer to rpS9 than to rpS5, special caution is required when identifying cross-linked proteins. Proteins covalently cross-linked with nucleotides can migrate more slowly than the native molecules. For these reasons, we believe our immunoprecipitation experiments were of particular value.
The C-terminal amino acid sequence of rat rpS5 protein is also highly conserved in the ribosomal protein S7 (rpS7) from other species, including eubacteria, archaebacteria, and chloroplasts (25). The stringent conservation of this region in ribosomal proteins from evolutionarily diverse species strongly implies a common function. RpS7 controls translation of the str operon by binding to an intercistronic region of the mRNA (26). Affinity labeling experiments with mRNA analogues in bacterial ribosome systems revealed rpS7 as a frequent binding target (27, 28). Cross-linking and immunological analysis of a 30 S ribosomal protein indicate that rpS7 is a primary target for binding to an upstream region of mRNA (29, 30). A binary complex consisting of mRNA and the small ribosomal subunit has been identified in prokaryotes and suggests a parallel binding mechanism via specific ribosomal proteins (31). These observations support our findings that the analogous eukaryotic rpS5 functions as a primary target at the 40 S ribosomal subunit when binding to HCV IRES.
The affinity of p25 for the HCV IRES correlates well with the efficiency of translation initiation of HCV RNA, indicating a critical role for this protein in HCV translation (19). Furthermore, the interaction between p25 and the IRES is specific for HCV (20). This protein may also be important in HCV translation by favoring preferential complex formation with the 40 S subunit. In this way, it may differ from the mechanism of ribosome recruitment during translation of normal cellular mRNAs in infected cells.
Other studies support our finding that rpS5 interacts with the IRES. Mapping by cryo-electron microscopy revealed that domain II of HCV IRES interacts with the head region of the 40 S ribosomal subunit (32). This location corresponds to that of rpS5 on the surface of the 40 S subunit as demonstrated by immune electron microscopy (22). Deletion or alteration of domain II RNA sequences abolished the cross-linking between HCV IRES and the 25-kDa cellular protein (18, 19), suggesting that the binding of rpS5 is dependent on domain II. In this way, rpS5 may be involved in positioning the IRES in the ribosomal decoding center, a process accompanied by pronounced conformational changes in the structure of the 40 S subunit (32).
In summary, we have shown that the rpS5 protein interacts specifically
with the IRES. Additional studies of this interaction may reveal
insights into potential target for the specific inhibition of HCV
translation in the infected cells. Further understanding of these
mechanisms may provide new strategies for development of novel
antiviral drugs.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81-492-32-0440; Fax: 81-492-32--5480; E-mail: sfukushi@alk.co.jp.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.C100206200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HCV, hepatitis C virus; IRES, internal ribosomal entry site; NCR, noncoding region; PTB, polypyrimidine tract-binding protein; PV, poliovirus; PAGE, polyacrylamide gel electrophoresis; rp, ribosomal protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Merrick, W. C. (1992) Microbiol. Rev. 56, 291-315[Abstract] |
2. | Jackson, R. J., and Kaminski, A. (1995) RNA 1, 985-1000[Medline] [Order article via Infotrieve] |
3. | Wang, C., and Siddiqui, A. (1995) Curr. Top. Microbiol. Immunol. 203, 99-115[Medline] [Order article via Infotrieve] |
4. | Hellen, C. U., Pestova, T. V., Litterst, M., and Wimmer, E. (1994) J. Virol. 68, 941-950[Abstract] |
5. |
Hellen, C. U.,
Witherell, G. W.,
Schmid, M.,
Shin, S. H.,
Pestova, T. V.,
Gil, A.,
and Wimmer, E.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7642-7646 |
6. | Rojas-Eisenring, I. A., Cajero-Juarez, M., and del Angel, R. M. (1995) J. Virol. 69, 6819-6824[Abstract] |
7. | Niepmann, M., Petersen, A., Meyer, K., and Beck, E. (1997) J. Virol. 71, 8330-8339[Abstract] |
8. | Meerovitch, K., Svitkin, Y. V., Lee, H. S., Lejbkowicz, F., Kenan, D. J., Chan, E. K., Agol, V. I., Keene, J. D., and Sonenberg, N. (1993) J. Virol. 67, 3798-3807[Abstract] |
9. | Blyn, L. B., Towner, J. S., Semler, B. L., and Ehrenfeld, E. (1997) J. Virol. 71, 6243-6246[Abstract] |
10. | Borman, A., Howell, M. T., Patton, J. G., and Jackson, R. J. (1993) J. Gen. Virol. 74, 1775-1788[Abstract] |
11. |
Ali, N.,
Pruijn, G. J.,
Kenan, D. J.,
Keene, J. D.,
and Siddiqui, A.
(2000)
J. Biol. Chem.
275,
27531-27540 |
12. |
Anwar, A.,
Ali, N.,
Tanveer, R.,
and Siddiqui, A.
(2000)
J. Biol. Chem.
275,
34231-34235 |
13. | Kaminski, A., Hunt, S. L., Patton, J. G., and Jackson, R. J. (1995) RNA 1, 924-938[Abstract] |
14. | Fukushi, S., Katayama, K., Kurihara, C., Ishiyama, N., Hoshino, F. B., Ando, T., and Oya, A. (1994) Biochem. Biophys. Res. Commun. 199, 425-432[CrossRef][Medline] [Order article via Infotrieve] |
15. | Rijnbrand, R., Bredenbeek, P., van der Straaten, T., Whetter, L., Inchauspe, G., Lemon, S., and Spaan, W. (1995) FEBS Lett. 365, 115-119[CrossRef][Medline] [Order article via Infotrieve] |
16. | Kamoshita, N., Tsukiyama-Kohara, K., Kohara, M., and Nomoto, A. (1997) Virology 233, 9-18[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Pestova, T. V.,
Shatsky, I. N.,
Fletcher, S. P.,
Jackson, R. J.,
and Hellen, C. U.
(1998)
Genes Dev.
12,
67-83 |
18. |
Kolupaeva, V. G.,
Pestova, T. V.,
and Hellen, C. U.
(2000)
J. Virol.
74,
6242-6250 |
19. | Fukushi, S., Kurihara, C., Ishiyama, N., Hoshino, F. B., Oya, A., and Katayama, K. (1997) J. Virol. 71, 1662-1666[Abstract] |
20. | Fukushi, S., Okada, M., Kageyama, T., Hoshino, F. B., and Katayama, K. (1999) Virus Genes 19, 153-161[CrossRef][Medline] [Order article via Infotrieve] |
21. | Theise, H., Noll, F., and Bielka, H. (1978) Acta Biol. Med. Ger. 37, 1353-1362[Medline] [Order article via Infotrieve] |
22. | Lutsch, G., Bielka, H., Enzmann, G., and Noll, F. (1983) Biomed. Biochim. Acta 42, 705-723[Medline] [Order article via Infotrieve] |
23. | Fukushi, S., Okada, M., Kageyama, T., Hoshino, F. B., Nagai, K., and Katayama, K. (2001) Virus Res. 73, 67-79[CrossRef][Medline] [Order article via Infotrieve] |
24. | Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
25. |
Kuwano, Y.,
Olvera, J.,
and Wool, I. G.
(1992)
J. Biol. Chem.
267,
25304-25308 |
26. | Spiridonova, V. A., Rozhdestvensky, T. S., and Kopylov, A. M. (1999) FEBS Lett. 460, 353-356[CrossRef][Medline] [Order article via Infotrieve] |
27. | Gimautdinova, O. I., Karpova, G. G., Knorre, D. G., and Kobetz, N. D. (1981) Nucleic Acids Res. 9, 3465-3481[Abstract] |
28. | Vladimirov, S. N., Babkina, G. T., Venijaminova, A. G., Gimautdinova, O. I., Zenkova, M. A., and Karpova, G. G. (1990) Biochim. Biophys. Acta 1048, 245-256[Medline] [Order article via Infotrieve] |
29. | Stade, K., Rinke-Appel, J., and Brimacombe, R. (1989) Nucleic Acids Res. 17, 9889-9908[Medline] [Order article via Infotrieve] |
30. | Dontsova, O., Kopylov, A., and Brimacombe, R. (1991) EMBO J. 10, 2613-2620[Abstract] |
31. | Hartz, D., McPheeters, D. S., Green, L., and Gold, L. (1991) J. Mol. Biol. 218, 99-105[Medline] [Order article via Infotrieve] |
32. |
Spahn, C. M.,
Kieft, J. S.,
Grassucci, R. A.,
Penczek, P. A.,
Zhou, K.,
Doudna, J. A.,
and Frank, J.
(2001)
Science
291,
1959-1962 |