1 CNRS-UPR 2511, IBL/Institut Pasteur de Lille, 59021 Lille Cedex, France
2 Laboratoire de Virologie, Centre Hospitalier Universitaire-Hôpital Sud, 80054 Amiens Cedex, France
3 CERVI (Virologie), UPRES EA 2387, Hôpital Pitié-Salpêtrière, 75651 Paris Cedex 13, France
Correspondence
Czeslaw Wychowski
czeslaw.wychowski{at}ibl.fr
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ABSTRACT |
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INTRODUCTION |
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HCV is related to the flaviviruses and the pestiviruses (Lindenbach & Rice, 2001; Miller & Purcell, 1990
; Takeuchi et al., 1990
). HCV, a positive-stranded RNA virus with a genomic size of approximately 9·6 kb (Choo et al., 1989
, 1991
), is now classified within the genus Hepacivirus, family Flaviviridae. The viral genome contains a large open reading frame encoding a polyprotein of approximately 3000 aa that is cleaved by a combination of host signal peptidases and two virus-encoded proteases to produce the mature structural and non-structural proteins: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B (Lindenbach & Rice, 2001
). The C protein, also called the core protein, is the putative capsid protein; E1 and E2 are thought to be the membrane-associated envelope glycoproteins; p7, a polypeptide of unknown function, is cleaved inefficiently from E2; NS2 through NS5B are the non-structural proteins, which, except for NS2, are involved in the replication of the viral genome (Lohmann et al., 1999
).
The amino acid sequence of the core protein is well conserved among different HCV isolates. Its N-terminal region is highly basic, while its C-terminal region is hydrophobic (reviewed by McLauchlan, 2000). During its maturation, the core protein undergoes two consecutive membrane-dependent cleavage events: (i) the first generates the p23 protein, the immature core protein of 191 aa, and is mediated by a signal peptidase; and (ii) the second yields the p21 protein and is mediated by a signal peptide peptidase (McLauchlan et al., 2002
). The C terminus of p21 has not been mapped correctly yet and different locations have been reported (Hussy et al., 1996
; Liu et al., 1997
; Lo et al., 1995
; Santolini et al., 1994
; Yasui et al., 1998
). Both p23 and p21 core proteins have been termed, in some early publications, as p21 and p19, respectively. In addition, a 16 kDa protein, called p16, can be expressed also from the HCV capsid protein-encoding sequence (Lo et al., 1994
, 1995
; Yeh et al., 2000
). The identity of this product remained unclear until the recent observation of a novel translation mechanism within the capsid-encoding sequence corresponding to a ribosomal frameshift, a mechanism unique among members of the family Flaviviridae (Varaklioti et al., 2002
; Walewski et al., 2001
; Xu et al., 2001
). This protein has been called F (frameshifted) (Xu et al., 2001
) protein or ARFP (alternative ribosomal frameshift protein) (Walewski et al., 2001
).
In this study, we investigated the expression of the F protein in in vitro and in vivo expression systems in the presence or absence of mutations that have been reported previously to be associated with the expression of the p16 protein (Yeh et al., 2000). In the absence of any mutation, no ribosomal frameshifting was observed. In contrast, a framshifted protein was clearly identified when mutations were introduced at nt 367 and 373 (codons 9 and 11) in the capsid-encoding sequence. Furthermore, the data obtained in time-course experiments revealed that the F protein is a very short-lived protein and that its stability can be maintained by the use of the proteasome inhibitor MG132.
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METHODS |
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Generation of recombinant vaccinia viruses.
Transfection and isolation of recombinant viruses were performed essentially as described (Kieny et al., 1984). The following vaccinia virus recombinant has been described previously: vTF7-3 (expressing the T7 DNA-dependent RNA polymerase) (Fuerst et al., 1986
).
Immunoprecipitation and Western blot assays.
Cells expressing HCV proteins were metabolically labelled with [35S]Protein Labelling mix (3·7x106 Bq ml-1), as described previously (Meunier et al., 1999). Cells were lysed with 0·5 % Igepal CA-630 in TBS (50 mM Tris/HCl, pH 7·5, and 150 mM NaCl). Immunoprecipitations were carried out as described (Dubuisson & Rice, 1996
). Immune complexes were boiled for 5 min in Laemmli's buffer before analysis by SDS-PAGE. Gels were then treated as described previously (Meunier et al., 1999
). Proteins bound to nitrocellulose membranes (PVDF transfer membrane, NEN Life Science Products) were revealed by enhanced chemiluminescence detection (Amersham Pharmacia), as recommended by the manufacturer, with the specific monoclonal antibodies (mAbs) anti-C (diluted 1 : 5000) (Maillard et al., 2001
), anti-myc (diluted 1 : 200) (ATCC CRL-1725) or anti-luc (diluted 1 : 1000) (Promega).
Luciferase assays.
Luc activity was assayed with the Luciferase Reporter Assay system (Promega) on rabbit reticulocyte lysate expressing the appropriate constructs or on cell lysates provided by cells infected with different recombinant viruses expressing the firefly luciferase activity. HepG2 cells (105 cells) plated in a multiwell plate were infected with the appropriate vaccinia virus recombinant at an m.o.i. of 5 p.f.u. per cell. At 6 h post-infection (p.i.), cells were washed twice with PBS, scraped out with 120 µl 1x Reporter Lysis buffer (Promega), lysed by one freezethaw cycle, vortexed and spun at 25 °C to pellet cell debris. Supernatant (4 µl) in 10 µl water was placed in a luminometer (Lumat, LB9501 Berthold) and the reaction was started by injection of 20 µl Luciferase Assay reagent (Promega). Light emission was recorded for 15 s.
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RESULTS |
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Mutations reported previously G367A (codon 9) and C373
A (codon 11) (Yeh et al., 2000
) were introduced into the N terminus of the capsid-encoding region; this construct will be referred to the mutant m (Fig. 1A
). To detect frameshifting and to analyse the level of expression of the framshifted protein, the F protein reading frame was fused in-frame with the Luc-encoding sequence (pTHC/CF
-Luc+ and pTHC/CF
m-Luc+ constructs). With this type of approach, the fully active luciferase protein can be detected only if a ribosomal frameshift occurs during translation. To be closer to the context of HCV expression, the sequences encoding the HCV C protein and firefly luciferase were placed under the translational control of the HCV 5'UTR. All DNA constructs used for this study are illustrated in Fig. 1(B)
. As expected, a band with an apparent molecular mass of 69 kDa and corresponding to the N terminus of the F protein in fusion with Luc (F
-Luc) was detected with the in vitro transcription/translation assay resulting from the plasmid containing the mutations (pTHC/CF
m-Luc+, Fig. 1C
). However, no band was observed when no mutation was introduced into the N terminus of the capsid-encoding region (pTHC/CF
-Luc+, Fig. 1C
). To determine the efficiency of ribosomal frameshifting, the luciferase activity of the F
-Luc fusion protein was measured and compared to that of a fusion protein in which the luciferase sequence was fused in-frame with the N-terminal sequence of the C protein (C
-Luc). About 30 and 3 % of luciferase activity was observed for the mutated and non-mutated F
-Luc proteins, respectively (data not shown).
To confirm that the mutations introduced into the N terminus of the capsid-encoding region have no effect on the level of expression of the capsid protein, the Luc-encoding sequence was fused in-frame with the sequence encoding the first 82 aa of the capsid protein (pTHC/C-Luc+ and pTHC/C
m-Luc+). The fusion product, corresponding to each construct, yielded a 69 kDa protein in an in vitro transcription/translation assay (Fig. 1C
). Levels of expression were similar and correlated with luciferase activities (data not shown). Additional constructs in which the sequence encoding the first 82 aa of the HCV capsid protein was fused out of frame with the firefly luciferase-encoding sequence (pTHC/C
-Luc- and pTHC/C
m-Luc-) were used as negative controls. These constructs are not supposed to produce a luciferase fusion protein even if a +1 ribosomal frameshift occurs in the capsid-encoding sequence due to the presence of termination codons in the +1 frame of the luciferase-encoding sequence. This was confirmed by SDS-PAGE analysis of [35S]methionine-labelled translation products obtained in an in vitro transcription/translation assay using rabbit reticulocyte lysates (Fig. 1C
). Additionally, no firefly luciferase activity associated with these constructs was observed (data not shown).
These results indicate that a frameshifted fusion protein with an active luciferase protein is produced in vitro when modifications at nt 367 and 373 (codons 9 and 11) are introduced within the nucleotide sequence encoding the N terminus of the HCV capsid protein. However, in the absence of any mutation, no frameshift is detectable above background level.
In vivo analysis of the frameshifting
Due to the presence of some factors that may influence ribosomemRNA interactions (Parkin et al., 1992; Reil et al., 1993
), it is well known that in vivo frameshifting results are generally different from those observed in vitro. To confirm the above-mentioned conclusions by in vivo experiments, recombinant vaccinia viruses were generated by homologous recombination and used to infect HepG2 cells. The proteins expressed by the different recombinant vaccinia viruses were analysed by SDS-PAGE followed by Western blot analysis using anti-luc or anti-C mAbs (Fig. 2
). All constructs analysed in vitro were examined in vivo. A product fused in-frame with the fully active firefly protein was detected in HepG2 cells infected with the recombinant vaccinia virus vvpTHC/CF
m-Luc+ (Fig. 2B
, lane 7), indicating that frameshifting was observed in vivo. In addition, 16 % luciferase activity has been observed for this construct (Fig. 2D
, lane 7). In contrast, the firefly luciferase activity was only slightly above background in the absence of any mutation in the F
-Luc fusion protein (Fig. 2D
, lane 6), which correlates with the absence of detection of the corresponding product by Western blotting (Fig. 2B
, lane 6). These data indicate that the mutations introduced in the capsid-encoding sequence are necessary to improve the in vivo expression of a frameshifted protein. To confirm that all constructs could be expressed in vivo, the presence of capsid-derived proteins was analysed by Western blotting with an anti-C mAb. A truncated form of the capsid protein of the expected size (10 kDa) was detected from cell extracts of HepG2 cells infected with the recombinant vaccinia viruses vvpTHC/C
-Luc-, vvpTHC/C
m-Luc-, vvpTHC/CF
-Luc+ and vvpTHC/CF
m-Luc+ as expected (Fig. 2C
, lanes 2, 3, 6 and 7). The small size of these proteins is due to the presence of termination codons in the Luc sequence, which are not in the same frame as the capsid protein. The fused proteins resulting from the capsid protein with the luciferase were only detected when the HepG2 cells were infected with the recombinant viruses vvpTHC/C
-Luc+ and vvpTHC/C
m-Luc+ (Fig. 2A
, lanes 4 and 5). The corresponding fused proteins were also detected by Western blotting with anti-luc mAb (Fig. 2B
, lanes 4 and 5). These constructs gave the highest reproducible levels of luciferase activity (Fig. 2D
, lanes 4 and 5).
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Analysis of F protein stability
We also wanted to determine whether the F protein is stable during its translation in mammalian cells. The expression of the F protein was analysed in pulsechase experiments by infecting HepG2 cells with the recombinant vaccinia virus vvpTHC/CFm-myc. As shown in Fig. 4(A), the level of expression of the F protein decreased very quickly. After 15 min of chase, the intensity of the band was already very low. The estimated half-life of the F protein was approximately 10 min (Fig. 4A
). A decrease in the amount of the F protein was also observed when the HepG2 cells were infected with the recombinant virus vvmyc-F1161 (Fig. 4B
). However, after 2 h of chase, 20 % of the F protein was still detected, suggesting that overexpression of the F protein might reduce its degradation. The capsid protein tagged with the Myc epitope at its N terminus (myc-C1173) was used as a control. As shown in Fig. 4(C)
, this protein was very stable during the same pulsechase conditions.
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DISCUSSION |
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Reporter enzymes, such as luciferase, CAT and -galactosidase, are used commonly in vivo and in vitro (Naylor, 1999
). Such enzymes are not expressed naturally in mammalian cells, making them useful to study gene expression in cells. Luciferase-tagging experiments used in this study have revealed that the ribosomal frameshift is more efficient when mutations at nt 367 (codon 9) and 373 (codon 11) are introduced in the capsid-encoding sequence compared to the results obtained with the wild-type sequence. The luciferase activity determined in vivo was about 16 % relative to the control construct and was less than that observed in vitro (30 %). This lower efficiency observed in mammalian cells is not unusual and can depend on the genes and the expression systems used (Ivanov et al., 2000
). In our study, tagging the C terminus of the F protein has made its detection easier in mammalian cells by immunoblot or immunoprecipitation analyses. Our results have shown that, besides its expression in an in vitro system, the F protein can be expressed also in mammalian cells. These data are reinforced by the fact that antibodies directed against the F protein are present in some patient sera, indicating that the F protein is produced during a natural HCV infection in patients (Varaklioti et al., 2002
; Xu et al., 2001
).
The molecular mechanism of frameshifting leading to the translation of the HCV F protein remains to be determined. However, given the data presented in this study, it could be concluded that the modifications at nt 367 and 373 do not simply increase efficiency but are necessary for the production of the F protein. Interestingly, a similar observation was made using the sequence of a HCV genotype 1b (data not shown). Modifications of codons ArgLys (codon 9) and Thr
Asn (codon 11) lead to the modification of a G
A (nt 367) and a C
A (nt 373), generating an A-rich region (10 A residues) between nt 363 and 374 of the HCV sequence. In this context, a slippery sequence can emerge from this region. Indeed, defined initially by in vitro translation assays, two structural motifs in mRNA have been characterized as important for an efficient -1 ribosomal frameshift (Brierley, 1995
; Dinman, 1995
; Gesteland & Atkins, 1996
). One is the slippery sequence, the heptanucleotide XXXYYYZ (X is any base, Y is A or U and Z is not G) (Jacks et al., 1988
), and the other component is a downstream RNA structural element, either a simple hairpin structure or, more frequently, a pseudoknot (Chamorro et al., 1992
; Jacks et al., 1988
; ten Dam et al., 1990
). In contrast, the +1 ribosomal frameshift in the yeast retrotransposon Ty requires only a short slippery sequence with a rare codon in the original reading frame (Belcourt & Farabaugh, 1990
). Frameshifting in human immunodeficiency virus requires only the short slippery sequence but not the 3' sequence with its predicted stemloop structure (Wilson et al., 1988
). This unusual process occurs also among other viruses, including coronaviruses (Brierley, 1995
; Herold & Siddell, 1993
) and human astrovirus serotype 1 (Marczinke et al., 1994
). In HCV, a single mutation of the codon AGA (Arg) to AAA (Lys) is also sufficient to generate in vitro the expression of the F protein (Lo et al., 1994
). This mutation creates a heptanucleotide sequence between nt 364 and 372 or 368 and 374. As mutations introduced in this region may recreate a functional slippery sequence, as described for other viruses, it can be suggested that this sequence constitutes the first control element for the translation of the F protein. Comparative studies of the sequence of the codons 814 reveal that these codons are also very conserved among the HCV sequences, reinforcing the importance of this region (Rijnbrand & Lemon, 2000
). However, if the slippery sequence can be suggested, no available data allow us to determine whether an RNA structural element is also necessary for the ribosomal frameshift.
Interestingly, the F protein is very unstable when expressed in mammalian cells. The degradation of most proteins in mammalian cells occurs via the ubiquitin-proteasome pathway (Lee & Goldberg, 1998). Under these circumstances, ubiquitin is linked covalently to the proteins and is then targeted to a large multiproteinase complex (700 kDa) that constitutes the catalytic core of the 26S proteasome used as an intracellular protein-degrading machine of eukaryotic organisms. The proteasome is essential for the normal turnover of regulatory proteins that controls cell growth and metabolism or is necessary for the removal of damaged or mutated proteins (Molinari et al., 1999
). Since the discovery that lactacystin is a potent inhibitor of the 26S proteasome (Fenteany et al., 1995
), different novel proteasome inhibitors, like MG132 or proteasome inhibitor I, were developed. In our study, pulsechase assays indicate that the decreased protein level of the HCV F protein in mammalian cells is due to proteasome degradation. A specific inhibitor of 26S proteasome activity blocks degradation of the F protein during its translation and stabilizes the level of its expression. It is possible that the F protein expressed under our experimental conditions is not folded properly and is, therefore, targeted directly for degradation, as observed for some damaged or mutated proteins (Molinari et al., 1999
). In this way, it would be very interesting also to determine whether HCV proteins could be involved in the stabilization of the F protein. Alternatively, it is not the F protein itself but some of its degradation product(s) that might play a functional role in the HCV life cycle.
No function has been attributed to the F protein yet. Its localization in the cytosol suggests that this protein plays a functional role in this compartment. However, we cannot exclude that it moves to another compartment, e.g. the nucleus, after interacting with a cellular component. Its very low level of expression in the absence of any mutation suggests that the F protein might also be expressed at very low levels in HCV-infected cells, which is not in favour for a role of this protein in virus assembly. Interestingly, mutations leading to higher levels of expression of the F protein ex vivo can be observed in HCV isolated from chronically infected patients with HCV-related hepatocellular carcinoma, leading to the hypothesis that higher levels of F protein expression might be linked to virus pathogenesis. Further studies on the F protein should lead to a better understanding of the role of this protein in the HCV life cycle and in pathogenesis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Brierley, I. (1995). Ribosomal frameshifting viral RNAs. J Gen Virol 76, 18851892.[Medline]
Chamorro, M., Parkin, N. & Varmus, H. E. (1992). An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA. Proc Natl Acad Sci U S A 89, 713717.[Abstract]
Choo, Q.-L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359362.[Medline]
Choo, Q.-L., Richman, K. H., Han, J. H. & 11 other authors (1991). Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A 88, 24512455.[Abstract]
Dinman, J. D. (1995). Ribosomal frameshifting in yeast viruses. Yeast 11, 11151127.[Medline]
Dubuisson, J. & Rice, C. M. (1996). Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin. J Virol 70, 778786.[Abstract]
Feinstone, S. M., Alter, H. J., Dienes, H. P., Shimizu, Y., Popper, H., Blackmore, D., Sly, D., London, W. T. & Purcell, R. H. (1981). Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis 144, 588598.[Medline]
Fenteany, G., Standaert, R. F., Lane, W. S., Choi, S., Corey, E. J. & Schreiber, S. L. (1995). Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 268, 726731.[Medline]
Fournillier-Jacob, A., Cahour, A., Escriou, N., Girard, M. & Wychowski, C. (1996). Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells. J Gen Virol 77, 10551064.[Abstract]
Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. (1986). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83, 81228126.[Abstract]
Gesteland, R. F. & Atkins, J. F. (1996). Recoding: dynamic reprogramming of translation. Annu Rev Biochem 65, 741768.[CrossRef][Medline]
Herold, J. & Siddell, S. G. (1993). An elaborated pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA. Nucleic Acids Res 21, 58385842.[Abstract]
Hussy, P., Langen, H., Mous, J. & Jacobsen, H. (1996). Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase. Virology 224, 93104.[CrossRef][Medline]
Ivanov, I. P., Matsufuji, S., Murakami, Y., Gesteland, R. F. & Atkins, J. F. (2000). Conservation of polyamine regulation by translational frameshifting from yeast to mammals. EMBO J 19, 19071917.
Jacks, T., Madhani, H. D., Masiarz, F. R. & Varmus, H. E. (1988). Signals for ribosomal frameshifting in the Rous sarcoma virus gagpol region. Cell 55, 447458.[Medline]
Kieny, M.-P., Lathe, R., Drillien, R., Spehner, D., Skory, S., Schmitt, D., Wiktor, T., Koprowski, H. & Lecocq, J.-P. (1984). Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature 312, 163166.[Medline]
Lee, D. H. & Goldberg, A. L. (1998). Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol 8, 397403.[CrossRef][Medline]
Lindenbach, B. D. & Rice, C. M. (2001). Flaviviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 9911042. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Liu, Q., Tackney, C., Bhat, R. A., Prince, A. M. & Zhang, P. (1997). Regulated processing of hepatitis C virus core protein is linked to subcellular localization. J Virol 71, 657662.[Abstract]
Lo, S. Y., Selby, M., Tong, M. & Ou, J. H. (1994). Comparative studies of the core gene products of two different hepatitis C virus isolates: two alternative forms determined by a single amino acid substitution. Virology 199, 124131.[CrossRef][Medline]
Lo, S. Y., Masiarz, F., Hwang, S. B., Lai, M. M. & Ou, J. H. (1995). Differential subcellular localization of hepatitis C virus core gene products. Virology 213, 455461.[CrossRef][Medline]
Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L. & Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110113.
Maillard, P., Krawczynski, K., Nitkiewicz, J. & 7 other authors (2001). Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients. J Virol 75, 82408250.
Marczinke, B., Bloys, A. J., Brown, T. D., Willcocks, M. M., Carter, M. J. & Brierley, I. (1994). The human astrovirus RNA-dependent RNA polymerase coding region is expressed by ribosomal frameshifting. J Virol 68, 55885595.[Abstract]
McHutchison, J. G. & Poynard, T. (1999). Combination therapy with interferon plus ribavirin for the initial treatment of chronic hepatitis C. Semin Liver Dis 19, 5765.[Medline]
McHutchison, J. G., Gordon, S. C., Schiff, E. R. & 7 other authors (1998). Interferon 2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med 339, 14851492.
McLauchlan, J. (2000). Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes. J Viral Hep 7, 214.[CrossRef]
McLauchlan, J., Lemberg, M. K., Hope, G. & Martoglio, B. (2002). Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J 21, 39803988.
Meunier, J. C., Fournillier, A., Choukhi, A., Cahour, A., Cocquerel, L., Dubuisson, J. & Wychowski, C. (1999). Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex. J Gen Virol 80, 887896.[Abstract]
Miller, R. H. & Purcell, R. H. (1990). Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci U S A 87, 20572061.[Abstract]
Molinari, E., Gilman, M. & Natesan, S. (1999). Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo. EMBO J 18, 64396447.
Naylor, L. H. (1999). Reporter gene technology: the future looks bright. Biochem Pharmacol 58, 749757.[CrossRef][Medline]
Parkin, N. T., Chamorro, M. & Varmus, H. E. (1992). Human immunodeficiency virus type 1 gagpol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo. J Virol 66, 51475151.[Abstract]
Pietschmann, T., Lohmann, V., Rutter, G., Kurpanek, K. & Bartenschlager, R. (2001). Characterization of cell lines carrying self-replicating hepatitis C virus RNAs. J Virol 75, 12521264.
Reil, H., Kollmus, H., Weidle, U. H. & Hauser, H. (1993). A heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells. J Virol 67, 55795584.[Abstract]
Rijnbrand, R. C. & Lemon, S. M. (2000). Internal ribosome entry site-mediated translation in hepatitis C virus replication. Curr Top Microbiol Immunol 242, 85116.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Santolini, E., Migliaccio, G. & La Monica, N. (1994). Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol 68, 36313641.[Abstract]
Schalm, S. W., Weiland, O., Hansen, B. E. & 9 other authors (1999). Interferon-ribavirin for chronic hepatitis C with and without cirrhosis: analysis of individual patient data of six controlled trials. Eurohep Study Group for Viral Hepatitis. Gastroenterology 117, 408413.[Medline]
Stemmer, W. P. C. & Morris, S. K. (1992). Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site directed mutagenesis. Biotechniques 13, 214220.[Medline]
Takeuchi, K., Kubo, Y., Boonmar, S. & 7 other authors (1990). The putative nucleocapsid 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 carriers. J Gen Virol 71, 30273033.[Abstract]
ten Dam, E. B., Pleij, C. W. & Bosch, L. (1990). RNA pseudoknots: translational frameshifting and readthrough on viral RNAs. Virus Genes 4, 121136.[Medline]
Varaklioti, A., Vassilaki, N., Georgopoulou, U. & Mavromara, P. (2002). Alternate translation occurs within the core coding region of the hepatitis C viral genome. J Biol Chem 277, 1771317721.
Walewski, J. L., Keller, T. R., Stump, D. D. & Branch, A. D. (2001). Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame. RNA 7, 710721.
Wilson, W., Braddock, M., Adams, S. E., Rathjen, P. D., Kingsman, S. M. & Kingsman, A. J. (1988). HIV expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems. Cell 55, 11591169.[Medline]
Xu, Z., Choi, J., Yen, T. S., Lu, W., Strohecker, A., Govindarajan, S., Chien, D., Selby, M. J. & Ou, J. (2001). Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO J 20, 38403848.
Yasui, K., Wakita, T., Tsukiyama-Kohara, K., Funahashi, S. I., Ichikawa, M., Kajita, T., Moradpour, D., Wands, J. R. & Kohara, M. (1998). The native form and maturation process of hepatitis C virus core protein. J Virol 72, 60486055.
Yeh, C. T., Lo, S. Y., Dai, D. I., Tang, J. H., Chu, C. M. & Liaw, Y. F. (2000). Amino acid substitutions in codons 911 of hepatitis C virus core protein lead to the synthesis of a short core protein product. J Gastroenterol Hepatol 15, 182191.[CrossRef][Medline]
Received 24 December 2002;
accepted 27 March 2003.