1 Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, QLD 4029, Australia
2 Clinical Medical Virology Research Centre, University of Queensland, St Lucia, QLD 4067, Australia
Correspondence
Eric Gowans (at Macfarlane Burnet Institute)
gowans{at}burnet.edu.au
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ABSTRACT |
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Present address: Macfarlane Burnet Institute for Medical Research and Public Health, Cnr Punt & Commercial Roads, Prahran, VIC 3181, Australia.
Present address: Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA.
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INTRODUCTION |
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HCV is a member of the Flaviviridae, along with the pestiviruses and flaviviruses (Robertson et al., 1998). The genome is a single-strand positive-sense RNA molecule of approximately 9600 nucleotides that contains a single long open reading frame (ORF) which is flanked by untranslated regions (UTR) at the 5' and 3' ends. The 5' UTR is a highly conserved region that contains an internal ribosome entry site (IRES), which initiates translation by a cap-independent mechanism (Rijnbrand & Lemon, 2000
). Translation yields a polyprotein, which is cleaved into three structural (core, E1, E2/p7) and six non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B).
The core protein, which is thought to form the viral capsid, is located in the most N-terminal portion of the polyprotein. Evidence has accumulated to suggest that the core protein may inhibit the host response to virus infection through multiple mechanisms (Lai & Ware, 1999; McLauchlan, 2000
). In addition, the core protein has been shown to bind heterogeneous nuclear ribonucleoprotein K (hnRNP K), which is involved in cellular pre-mRNA splicing and nuclear RNA transport, and modulate cellular RNA transcription (Ray et al., 1995
, 1997
; Shrivastava et al., 1998
).
It has also been suggested that HCV has a self-modulating mechanism to maintain a low level of replication and expression that may promote virus persistence. To account for this, it was speculated that stemloop IV of the HCV IRES might be stabilized by interaction with a viral protein, to result in inhibition of translation (Honda et al., 1996). The core protein was later shown to bind positive- but not negative-strand HCV RNA, an interaction that resulted in suppression of translation (Shimoike et al., 1999
). Amino acids (aa) 175 of the core protein were previously reported to be responsible for the interaction with the viral RNA (Santolini et al., 1994
). In addition to binding the viral RNA, the core protein can interact with itself and with the E1 and E2 proteins (Lo et al., 1996
). It was also reported that the HCV core protein reduced the efficiency of HCV translation by binding to the IRES (Shimoike et al., 1999
) and aa 3344 were recently shown to interact with the IRES and contribute to the inhibition of translation (Zhang et al., 2002
). In contrast, a previous study suggested that the core protein did not appear to have any specific effect on HCV IRES-directed translation, and instead, it was reported that suppression of IRES-directed translation resulted from an RNARNA interaction (Wang et al., 2000
).
The potential role of the HCV core protein in the efficiency of HCV IRES-directed translation is a key issue in understanding the replication and expression of HCV. Consequently, the aim of this study was to clarify the nature of the HCV coreIRES interaction. We examined the effect of the wild-type and mutated core protein on the expression of reporter genes in an in vitro system and the effect of the protein expressed from a recombinant vaccinia virus on HCV IRES-directed translation in different cell lines.
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METHODS |
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Plasmids.
The plasmids pCore and p20, which encode aa 1167 and aa 21167 of the HCV core protein respectively, were synthesized by insertion of the appropriate PCR-generated fragment from p5'UTR-A2 (Trowbridge & Gowans, 1998
) into pcDNA3 (Invitrogen). p
20 was engineered to encode a methionine at the start of the ORF and a stop codon at the end. Plasmid p
1, in which the start codon of the core protein was deleted from pCore, was made using the Quikchange Site-Directed Mutagenesis Kit (Stratagene), following the manufacturer's instructions. A series of C-terminal truncated core proteins was generated by appropriate restriction enzyme digestion of pCore. The reporter plasmid pIRES-CAT (Lott et al., 2001
), which contains the HCV 5' UTR sequence and 27 nt of the downstream core protein-coding sequence ligated in-frame with the chloramphenicol acetyltransferase (CAT) gene, was constructed in pGEM-T (Promega). pCAP-LUC was constructed by inserting the firefly luciferase gene into pGEM-T. Transcription from these plasmids is controlled by the T7 promoter and expression controlled by the HCV IRES and a cap-dependent mechanism respectively.
A bicistronic reporter pcCAT, from which expression of firefly luciferase and CAT were controlled by cap- and IRES-dependent mechanisms respectively, was constructed by ligating pCAP-LUC and pIRES-CAT in pcDNA3. Three additional bicistronic constructs, which contained the IRES elements from HCV, encephalomyocarditis virus (EMCV) and classical swine fever virus (CSFV) (referred to as HIRES, EIRES and CIRES) inserted between the Renilla luciferase (R-LUC) and CAT genes (Lott et al., 2001), were also used as reporter molecules. Thus, the expression of R-LUC and CAT was directed by cap- and IRES-dependent mechanisms respectively.
In vitro transcription of RNA.
The plasmids were linearized by the appropriate restriction enzyme and then purified by phenol/chloroform extraction followed by isopropanol precipitation or by the BRESAclean DNA purification system (Bresatec). RNA was synthesized from each plasmid by T7 RNA polymerase with or without the addition of RNA capping analogue (Gibco-BRL) as appropriate. The RNAs were purified by phenol/chloroform extraction and isopropanol precipitation followed by two washes with 70 % ethanol. The RNA pellets were dissolved in RNase-free water. The quality and quantity of the RNAs were checked by agarose gel electrophoresis and the concentration was determined by optical density measurement.
In vitro translation.
Wild-type core protein and the p1 and p
20 protein products were expressed in a 25 µl rabbit reticulocyte lysate translation reaction (RRL; Promega) loaded with 500 ng of the respective RNA. This reaction will be referred to as RRL1. The products were analysed by SDS-PAGE and immunoblot as previously described (Wang et al., 1997
). Radiolabelled products were visualized by SDS-PAGE followed by PhosphorImager analysis (Molecular Dynamics) and by immunoblot to confirm the authenticity of the synthesized protein. To examine the effect of the expressed protein on IRES- or cap-dependent translation, an aliquot of the above RRL1 was then added to a second RRL containing 500 ng of pIRES-CAT RNA and/or 100 ng of pCAP-LUC RNA. This reaction will be referred to as the reporter translation reaction (RRL2). All translation reactions were carried out at 30 °C for 90 min unless noted otherwise. The products of RRL2 were analysed as described above and all experiments were carried out in triplicate.
In some experiments, synthetic peptides were added to the RRL2. The peptides were synthesized by Mimotopes (Australia) to >90 % purity. The sequences of the peptides are: (1) HCV core aa 1-20-MSTNPKPQRKTKRNTNRRPQ; (2) HCV E2 HVR1 aa 384-419-DTHTTGGVAGRDTLRFTGFFSFGPKQK; (3) HBV core aa141-160-STLPETTVVRRRGRSPRRRT; (4) HBsAg aa 202-213-IPQSLDSWWTSL.
RNAprotein binding assay.
The RNAprotein binding assay was carried out essentially as described (Furuya & Lai, 1993) with slight modifications. Briefly, 32P-labelled RNA was mixed with peptide in 10 µl of binding buffer (10 mM HEPES, 2·5 mM MgCl2, 40 mM HCl, 5 % glycerol, 2·5mM DTT, 20 U RNase inhibitor). The binding reaction was incubated at 30 °C for 10 min and then mixed with 5x loading buffer (50 % glycerol, 0·05 % bromophenol blue, 0·05 % xylene cyanol, 2x TBE). The samples were analysed by electrophoresis on a 4 % native polyacrylamide gel. The gel was pre-electrophoresed at 80 V for 1 h prior to loading the samples in 0·5x TBE and electrophoresis was performed at 80 V for 1·5 h. Labelled RNA was detected by PhosphorImager analysis of the dried gel.
Recombinant vaccinia viruses.
Two recombinant vaccinia viruses (RecVV), RecVV-HCC and RecVV-HBC, were used to express either the full-length HCV core protein (aa 1191) or the full-length HBV core protein (aa 1183), respectively. These were synthesized and supplied by J. Hammond and B. Couper (Australian Animal Health Laboratory, Geelong, Australia). Expression of the core proteins from the recombinant viruses was controlled by the VVp7.5 promoter in the thymidine kinase (TK) gene locus of Western Reserve (WR). The virus stocks were prepared and titrated as previously described (Boyle et al., 1985).
To construct the N-terminal truncated version of RecVV-HCC (RecVV-20), the PCR product, encoding aa 21191 of the HCV core protein, was inserted into the pBCB06 transfer vector (Boyle et al., 1985
). The recombinant plasmid was transfected into CV1 cells, previously infected with WR, then followed by two cycles of TK phenotype selection in 143B tk- cells. The DNA sequence of the recombinant transfer vector was confirmed and the authenticity of the recombinant virus was further confirmed by PCR amplification of the inserted fragment.
Infection and transfection.
Confluent monolayers of CV-1, HuH7 and HepG2 cells were prepared in 6-well plates (TPP) and infected with trypsinized VV at an m.o.i. of 10 in 0·5 ml of DMEM + 1% FCS). The inoculum was replaced with fresh culture medium after incubation for 1 h in a humidified CO2 incubator at 37 °C and the cells were subsequently incubated overnight (1824 h). Before transfection, the cells were washed twice with 1 ml OptiMEM (Gibco-BRL). 1 µg each of pIRES-CAT and pCAP-LUC RNA, or 2 µg of each bicistronic reporter RNA, were prepared with Lipofectin reagent (Gibco-BRL) per well according to the manufacturer's protocol.
CAT and luciferase assays.
The cells were washed once with ice-cold PBS, and then lysed with 0·3 ml of buffer provided with the dual luciferase assay (DLA; Promega) for 10 min. The cell debris was removed by centrifugation and the supernatant transferred to a fresh tube. CAT activity was determined by ELISA (Roche) and LUC and R-LUC activities were measured by the DLA, following the instructions of the manufacturer.
Relative translational efficiency (RTE).
The RTE was calculated by normalizing the activities of the reporter molecules in the RRL2 containing the RNA(-)Control and in RecVV-HBC-infected cells to 100 %.
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RESULTS |
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HCV core protein inhibits HCV IRES-directed translation in vivo in a cell-specific manner
To examine the effect of the HCV core protein on HCV IRES-directed translation in vivo, the HCV core protein was expressed from a recombinant vaccinia virus (RecVV-HCC), and an HBV core recombinant vaccinia virus (RecVV-HBC) represented a control. We have shown that the level of expression from pIRES-CAT and pCAP-LUC in RecVV-HBC-infected cells was similar to that in cells infected with the WR strain of vaccinia virus (data not shown). The effect of the expression of the core protein was examined in three different cell lines, CV-1, HuH7 and HepG2. The cells were infected with the respective viruses at an m.o.i. of 10, transfected with the different RNA reporter molecules after overnight infection and the expression of the reporter proteins was measured 5 h later.
Initially, we co-transfected the RecVV-infected cells with pIRES-CAT and pCAP-LUC RNA. The levels of CAT and luciferase expressed in the RecVV-HBC-infected cells were normalized to 100 % (Fig. 6A). By comparison, the expression of CAT in all three cell lines infected with RecVV-HCC was reduced by 4570 %. The expression of luciferase in RecVV-HCC-infected CV-1 and HuH7 cells showed a similar reduction, but no such reduction was noted in the RecVV-HCC-infected HepG2 cells. Thus, expression of the HCV core protein appeared to inhibit both HCV IRES- and cap-dependent translation in the CV-1 and HuH7 cells, but was specific for the HCV IRES in HepG2 cells
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The above data were derived using the HCV IRES from a genotype 1b virus (Trowbridge & Gowans, 1998). We then extended the study to examine the effect of expression of the HCV core protein (1b) on an IRES element derived from a genotype 1a virus and compared this against IRES elements derived from EMCV and CSFV. These bicistronic vectors have been described previously (Lott et al., 2001
). As described above, RecVV-HBC was used as a negative control. In CV-1 and HuH7 cells, the HCV core protein appeared to inhibit translation from all three IRES elements and cap-dependent translation to similar degrees (Fig. 7
). In contrast, in HepG2 cells, the HCV core protein only inhibited translation from the HCV IRES and had no effect on the EMCV and CSFV IRES elements, or on cap-dependent translation.
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DISCUSSION |
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Our finding, that a synthetic peptide representing aa 120 of the HCV core protein, can inhibit cap- and IRES-translation is novel. Indeed, we have recently demonstrated (data not shown) that the addition of this peptide to a cell monolayer resulted in a reduction in cellular RNA synthesis. In contrast, a 20 aa synthetic peptide derived from the HBV core protein, which encapsidates HBV RNA during replication and which can also bind RNA in a non-specific manner (Cohen & Richmond, 1982), was unable to inhibit translation. This peptide contained the same proportion of basic residues as the HCV core peptide. This suggests that a basic residue-rich domain is necessary, but not sufficient, for the inhibitory phenotype shown by the HCV core peptide. The peptide sequence itself is important.
The full-length HCV core protein was able to inhibit translation in cell cultures in a reproducible manner, whereas the protein which lacked aa 120 was unable to do so. In contrast, the full-length HBV core protein was unable to inhibit cap- or HCV IRES-dependent translation in cells. Thus the in vitro data can be related to events in vivo. The specific effect of the HCV core protein in HepG2 cells as opposed to HuH7 and CV-1 cells may be related to differences in the intracellular distribution of the protein (Wang et al., 2000), although we were unable to demonstrate any difference in the localization patterns in the three cell lines used in our study (data not shown). If the HCV core protein inhibits translation and/or transcription (Ray et al., 1995
, 1997
; Shrivastava et al., 1998
) in human hepatocytes, then this may influence cell viability. A recent study (Bantel et al., 2001
) reported that a high proportion of hepatocytes in HCV-infected livers showed evidence of apoptosis, although it is not known if this is related to the expression of core or other HCV proteins. It has been suggested that the HCV core protein can sensitize the cell to apoptosis, although this may not necessarily be linked to reduced translation efficiency (Lai & Ware, 1999
).
In HepG2 cells, the HCV core protein specifically inhibited translation from the HCV IRES, irrespective of whether the IRES element was derived from a 1a or 1b genotype, but had no effect on translation from the EMCV and CFSV IRES elements. These results are consistent with previous data in which the translation from the HCV IRES was specifically inhibited by the HCV core protein in HepG2 and HepT cells (Shimoike et al., 1999; Zhang et al., 2002
). Our data also highlight yet another difference between HepG2 and HuH7 cells, as it has previously been demonstrated that the HuH7 cells but not the HepG2 cells support replication of the HCV replicon (Lohmann et al., 1999
; Blight et al., 2000
). We have sought an explanation for our results which differ to those showing that the effect is mediated by RNA (Wang et al., 2000
). In our study, a ratio of 1 : 100 of RNA : peptide was necessary for binding, while 1 : 500 was necessary for complete inhibition of translation in vitro. In contrast, the concentration of HCV core used in vitro in the Wang study was much lower (Wang et al., 2000
). There is an additional important difference between the two studies; in the Wang study, the bicistronic reporter plasmids were transfected 24 h prior to infection of the cells with recombinant baculovirus which encoded the HCV core protein, whereas we transfected our reporter plasmids after overnight infection of the cells with a recombinant vaccinia virus. Thus, it is possible that the level of expression of the HCV core protein in the Wang study was insufficient to inhibit HCV IRES function. Indeed, our study shows that a high concentration of core protein and a high ratio of protein : RNA (Fig. 5
) are necessary for an interaction between the HCV core and the HCV IRES. These features are consistent with our understanding of HCV replication (Bartenschlager & Lohmann, 2000
). Our in vitro data showed that a similar concentration of the core peptide was required for translation inhibition and RNA binding (Figs 4 and 5
respectively). Furthermore, this concentration is similar to that necessary for HCV core protein to induce assembly of an HCV nucleocapsid-like particle in vitro (Kunkel et al., 2001
). Extrapolation of these data suggests that a high threshold concentration of the core protein, as might be found in the typical punctate staining patterns (Gowans, 2000
), is required for RNA binding in vivo. This may either inhibit translation per se or reduce translation by removal of the RNA template through packaging. The latter mechanism may account for the difficulties in detecting HCV products in naturally infected liver samples (Gowans, 2000
). In this respect, HCV resembles bovine viral diarrhoea virus because the bulk of the virus is rapidly secreted and very little remains cell-associated (Gong et al., 1996
).
Other circumstantial evidence supports our conclusion that the HCV core protein inhibits HCV IRES activity; first, the efficiency of colony formation by the full-length replicon which encodes the structural proteins was reported to be 34 logs lower than that of the subgenomic replicons (Ikeda et al., 2002). Second, the level of replication of the full-length replicon in the selected cell lines is around 5-fold lower than that of the subgenomic replicon (Pietschmann et al., 2002
). Together, these data suggest that one or more of the structural proteins regulates HCV replication. As E1/E2 have been shown to have no effect on HCV IRES function (Shimoike et al., 1999
), it can be concluded that the core protein itself inhibits HCV IRES-directed translation. Finally, we have recently expressed the HCV core, E1 and E2 proteins from a Semliki Forest virus replicon (Greive et al., 2002
). In similar studies, the level of expression of the HCV structural proteins was reduced by >100-fold when the expression was controlled by the HCV IRES (Greive, 2001
), again suggesting that one or more of the HCV structural proteins inhibited HCV IRES-related translation. It is likely that the HCV IREScore interaction has evolved to reduce the level of HCV replication consistent with persistent infection.
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ACKNOWLEDGEMENTS |
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Received 8 July 2002;
accepted 26 November 2002.