| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Mark Harris
mharris{at}bmb.leeds.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). As part of a study to determine whether such transcripts are replication competent, the transcription start site of the tetracycline-regulable promoter was mapped and three baculovirus transfer vectors containing a neoR-expressing culture adapted replicon cDNA were generated. These vectors either had the first nucleotide of the 5'UTR positioned -2 (mkI) and +1 (mkII) with respect to the transcription start site, or included a hammerhead ribozyme at the 5' end of the transcript (5'HH) that cleaves between the ribozyme–5'UTR boundary. Transfection of all of the culture-adapted replicon constructs into Huh7 cells resulted in the formation of more neomycin-resistant colonies than seen with a polymerase knock-out replicon construct, although this was less pronounced in the mkI group. Furthermore, both the positive- and negative-strands of the replicon could be detected in all neomycin-resistant polyclonal cell lines except for those derived from transfection of the polymerase knock-out construct. Transduction of Huh7 cells with recombinant baculoviruses carrying the same expression cassettes improved replicon delivery, but the relative efficiency of the constructs remained the same. The baculovirus vectors were also used to introduce the replicon transcript into HepG2 cells. Expression of the culture-adapted but not the polymerase knock-out construct induced transcription of the 
-interferon gene, a response that may contribute to this cell line being unable to maintain the replicon over long-term culture. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
), Flaviviridae (Varnavski et al., 2000), Togaviridae (Dubensky et al., 1996) and Coronaviridae (Almazan et al., 2000). These studies have involved either transcription of the viral genome by host cell DNA-dependent RNA polymerases, or relied on expression of exogenous polymerases, such as T7, within the cytoplasm of the cell. A layered delivery approach may prove particularly useful in cases where viruses are able to complete their full life-cycle, albeit at a low level that is ultimately self-limiting. One example is the study of hepatitis C virus (HCV) replication, which until recently was limited to detection of negative-strand production by RT-PCR (Lanford et al., 1994). Indeed, production of a full-length HCV genome in cells expressing T7 polymerase and transfected with a full-length HCV genomic cDNA has already been documented (Chung et al., 2001; Myung et al., 2001), with detection of both negative-strand RNA and genetic drift having been used to infer virus replication. A disadvantage of these delivery regimes is that they are limited by the efficiency with which cells can be transfected with the viral cDNA. We recently described a baculovirus delivery system where an inducible polII promoter was used to drive expression of a full-length HCV genome in HepG2 cells (McCormick et al., 2002). Unlike T7-based systems, the HCV genome is efficiently introduced into the cell as a result of viral transduction. In addition, cells challenged with baculovirus show few signs of cytotoxicity. However, due to the nature of the delivery system, the transcripts produced are both capped and rely on ribozyme cleavage to remove the poly(A) tail. In order to establish whether such transcripts can replicate, we have investigated the ability of the baculovirus system to introduce a culture-adapted HCV replicon (Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 1999) into cells. Our findings demonstrate that it is possible using baculovirus transduction to introduce functional replicons into cells, thus validating the use of this delivery system for studying replication of full-length and other subgenomic HCV constructs. Furthermore, we show that when baculovirus transduction is used to introduce a functional replicon into HepG2 cells, an IFN- response is induced. This response is not observed in Huh7 cells, the one cell line capable of maintaining the replicon, implying that an antiviral response may contribute to preventing establishment of the HCV replicon in other cell lines. |
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The 5.1 replicon-containing Huh7 cell line and a stable polyclonal Huh7 cell line expressing neoR were generated as described previously (Macdonald et al., 2003). Cells were seeded at 3·0x104 cells cm-2 (HepG2) or 2·0x104 cell cm-2 (Huh7) for 20–24 h prior to a 4 h transduction with 1·25x107 p.f.u. ml-1 of both BACtTA and another baculovirus, or transfection of plasmid DNA using Lipofectin (Invitrogen) according to the manufacturer's recommendations. For transfection of T7-derived replicon transcripts Huh7 cells (400 µl of 1x107 cells ml-1 in DEPC-treated PBS) were mixed with 2 µg of RNA, placed in a 0·4 cm cuvette and electroporated using a Bio-Rad Gene Pulser II (270 V, 950 µF). For the IFN- RT-PCR assays, polyinosinic–polycytidylic acid [poly(I)–poly(C)] (Sigma) and yeast tRNA (Invitrogen) were transfected into cells using Lipofectin.
Generation of DNA constructs.
The construction of pBAC
H77lacZ(H
V)tet [referred to from now on as pBACH77lacZ(HV)(mkI)] and pBACtTA has already been described (McCormick et al., 2002). In order to generate pBACH77lacZ(HV)(mkII), the tetracycline-responsive promoter (Ptet) from pUHD13-1 (Gossen & Bujard, 1992) was coupled to the HCV 5'UTR by means of a two-step PCR using the primers tet-cmv(fwd), tet-cmv(rev)mk2, 5'UTR(fwd/tet)mk2 and 5'UTR(out) (Table 1) and the DNA cloned into pCR-Blunt (Invitrogen) to generate pT5U(mkII). The DNA was subsequently excised using XbaI and AgeI and cloned into XbaI/AgeI-digested pBACH77lacZ(HV)(mkI).
|
) and pFK-I389luc/NS3-3'/GND (gifts from R. Bartenschlager, Heidelberg, Germany). To generate pBACrep5.1neo(mkI) and (mkII) the entire ORF from pFK-I389neo/NS3-3'/5.1 was transferred into pBACH77lacZ(HV)(mkI) and (mkII) by means of common AgeI and NheI sites found in the 5' and 3'UTRs of the constructs. pBACrep5.1neo(mkII) was then used as template to generate pBACrepGNDneo by replacing both the BglII–BglII fragment (nucleotides 6286–7331) and the SfiI–SfiI fragment (nucleotides 2018–6895) with that from pFK-I389luc/NS3-3'/GND. The 5' hammerhead ribozyme sequence was placed between the tetracycline promoter (Ptet) and the 5'UTR by PCR using the primers tet-cmv(fwd) and tet-cmv(rev)BamHI to introduce a BamHI site at the end of Ptet. This DNA fragment was cloned into pCR-Blunt, generating pPtetBamHI. Next, the 5' hammerhead ribozyme sequence was fused to the 5'UTR by PCR using the primers 5'UTR(fwd/HH) and 5'UTR(out2) and the fragment cloned into pCR-Blunt generating p5U(HH). This vector was digested with BamHI and the insert cloned into BamHI-digested pPtetBamHI to generate pT5U(HH). A further AgeI–HindIII fragment from pFK-I389neo/NS3-3'/5.1 was then inserted into AgeI/HindIII-digested pT5U(HH) to generate pT5U(HH)neo. The XbaI–AscI fragment from pT5U(HH)neo was then cloned into XbaI/AscI-digested pBACrep5.1neo(mkII), generating pBACrep5.1neo(5'HH).
To produce in vitro RNA transcripts from the baculovirus transfer vectors, PCR was used to place a T7 promoter immediately upstream of the HCV 5'UTR using the primers T7-5'UTR and 5'UTR(out). The resulting DNA fragment was digested with XbaI and AgeI, cloned into XbaI/AgeI-digested pT5U(HH)neo and the XbaI–AscI fragment from the resulting construct cloned into XbaI/AscI-digested pBACrepGNDneo and pBACrep5.1neo(mkII). To linearize these two constructs at an appropriate position, the oligonucleotide RIst(NotI) containing a unique NotI restriction site, was introduced at an EcoRI site downstream of the hepatitis delta virus (HV) ribozyme using RecA (Koob et al., 1992) to specifically target this latter restriction site. The resultant plasmids are referred to as pBACrep5.1neoT7 and pBACrepGNDneoT7.
For production of strand-specific probes and target RNA used in Northern analysis, a fragment of the NS3 coding region from pFK-I389neo/NS3-3'/5.1 (nucleotides 2537–3431) was cloned in both orientations into PCR-Script (Stratagene). Single-stranded phagemid DNA was produced as described previously (Kunkel, 1985). For production of T7 transcripts, the vectors were linearized with HindIII.
G418-resistant colony formation assays.
For polII-directed delivery of the replicon, Huh7 cells were allowed to express the replicon transcript for 24 h and then seeded into six-well plates along with naïve Huh7 cells such that the final cell density was 2x104 cm-2. Cell cultures were maintained in the presence of 5 µg tetracycline (Sigma) ml-1 with the addition of 1 mg G418 ml-1 after 48 h. Selection was maintained for 14 days with a change of medium twice a week before cell colonies were counted. Under circumstances where the replicon was directly introduced by RNA transfection, the assay was essentially the same except that the cells were seeded immediately after transfection and tetracycline was omitted from the selection medium. The efficiency with which the replicon was introduced into cells was measured as the percentage of viable cells that formed G418-resistant colonies.
Northern blot analysis.
RNA was harvested using Trizol (Invitrogen), electrophoresed through a MOPS/formaldehyde gel and transferred to Bright-Star Nylon Plus membrane. Biotinylated probes and markers were generated using Biotin-Chem-Link reagent (Roche). Hybridization was performed overnight at 42 °C in Ultrahyb (Ambion) and bound probe was detected using Bright Star Detection Kit (Ambion).
In vitro synthesis of RNA transcripts.
Transcripts were produced using T7 polymerase (New England Biolabs), treated with RQ1-DNase (Promega), extracted with acid-phenol/chloroform and chloroform and spun through a mini QuickSpin Column (Roche). RNA integrity was confirmed by MOPS/formaldehyde gel electrophoresis.
RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE).
This was performed using the First Choice RLM-RACE kit (Ambion) according to the manufacturer's recommendations. In addition to those provided, primers used during the PCR and nested PCR steps included lacZ5'RACE and 5'UTR(out). In order to determine the transcription start sites, PCR products were cloned into pCR-Blunt and four clones from each RLM-RACE reaction sequenced.
Detection of IFN- by RT-PCR.
RNA was harvested using Trizol, treated with RQ1-DNase, extracted with acid-phenol/chloroform and chloroform, and ethanol precipitated. Reverse transcription was performed on 1 µg cellular RNA at 42 °C using Superscript (Invitrogen) according to manufacturer's recommendations. cDNA (2·5 µl) was used a template in a 25 µl PCR reaction containing 1·25 units HotStarTaq DNA polymerase (Qiagen), 1x PCR buffer, 2mM MgCl2, 0·8 mM dNTPs and 0·25 µM of each primer. For detection of IFN- and GAPDH primer pairs used were IFNbeta(+) and IFNbeta(-), and GAPDH(+) and GAPDH(-), respectively. The parameters of the reaction were as follows: 95 °C for 15 min; 22 (GAPDH) or 35 (IFN-) cycles of 94 °C for 30 s, 72 °C for 90 s; 72 °C for 10 min.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). This can be accomplished by either positioning the viral cDNA precisely with respect to the promoter, or by including a ribozyme that cleaves to produce the correct 5' end. We decided to assess both approaches, as it was unclear whether the cap structure at the end of a polII-derived replicon transcript would inhibit replication. To define the 5' end of the replicon transcript by positioning it with respect to the tetracycline-regulable promoter (Ptet), it was first necessary to locate the transcription start site. Therefore, HepG2 and Huh7 cells were co-transduced with BACtTA and BACH77lacZ(HV)tet(mkI), a -galactosidase-expressing HCV minigenome construct where the first nucleotide of the 5'UTR was positioned to coincide with the predicted transcription start site (Fig. 1a) (Akrigg et al., 1985; Gossen & Bujard, 1992). Twenty-four hours after transduction, the cellular RNA was subjected to RLM-RACE, resulting in the production of a PCR product of about the expected size. However, the sequence of this PCR product revealed that the HCV minigenome lacked the first two nucleotides of the 5'UTR. A further construct, BACH77lacZ(HV)tet(mkII), was created where the 5'UTR was repositioned a further two nucleotides downstream from the TATA box. RLM-RACE analysis of RNAs from HepG2 and Huh7 cells transduced with this second construct confirmed that the transcription start site coincided with the first nucleotide of the 5'UTR.
|
Delivery of functional replicons by DNA transfection
A series of vectors containing a culture-adapted replicon [pBACrep5.1neo(mkI), pBACrep5.1neo(mkII) and pBACrep5.1neo(5'HH)] was generated, along with a control polymerase knock-out replicon (pBACrepGNDneo) (Fig. 1b
). As a consequence of the cloning strategy, the first 160 bp of the 5'UTR and the SL1 of the 3'UTR X region are derived from the HCV H77 genome (Yanagi et al., 1997), resulting in five and two nucleotide substitutions in the 5'UTR and 3'UTR respectively. Previous work (Gu et al., 2003; Ikeda et al., 2002; Luo et al., 2003; Yanagi et al., 1998) indicates that the HCV replicon is likely to tolerate these small changes. However, to confirm this, a fifth vector (pBACrep5.1neoT7) that allowed in vitro transcription of this modified replicon was generated, along with an appropriate negative control vector (pBACrepGNDneoT7) (Fig. 1c). Transfection of in vitro transcripts derived from either pBACrep5.1neoT7 or the original culture-adapted replicon plasmid pFK-I389neo/NS3-3'/5.1 resulted in similar numbers of G418-resistant colonies (Fig. 2), indicating that the nucleotide substitutions within the 5' and 3'UTRs did not affect replication. As expected, the efficiency of colony formation by the control transcript derived from pBACrepGNDneoT7 was negligible.
|
a). Colonies formed during selection were subsequently grown as polyclonal cell lines for Northern analysis to look for the presence of both genomic and negative-strand replicon transcripts. The single-stranded DNA probes used to detect positive- (Fig. 3b) or negative- (Fig. 3c) strand transcripts showed approximately a 20 and 50-fold greater specificity towards their respective RNAs compared to the complementary sequence. Consistent with the introduction of a replication-competent replicon, both probes detected an 8 kb transcript in the cell lines derived after transfection with the mkI, mkII and 5'HH constructs but failed to identify a similar transcript in cell lines derived after transfection of pBACrepGNDneo and naïve Huh7 cells. In the cell lines containing the replicon it was estimated that there was approximately 10-fold more positive-strand than negative-strand transcript (up to approximately 108 and 107 copies per 5 µg cellular RNA respectively), values which are comparable with previous reports (Lanford et al., 2003; Lohmann et al., 1999). One further observation was the detection of a transcript much larger than 8 kb, most readily seen using the negative-strand specific probe (Fig. 3c).
|
). Consistent with the delivery of a functional replicon, almost all cell lines derived from transduction with BACrep5.1neo clones, but not the BACrepGNDneo clones, contained a replicon transcript. However, there was considerable variation in the efficiency with which individual clones introduced the replicon into Huh7 cells, and sequence analysis of functional BACrep5.1neo clones revealed a number of point mutations (mean one to two mutations per clone). Despite this, it was possible to identify a clone for each construct that possessed no changes within the replicon 5' and 3'UTRs, and if any mutations were present in the ORFs, then these were silent. These clones, designated G1 (BACrepGNDneo), 1.5 [BACrep5.1neo(mk I)], 2.5 [BACrep5.1neo(mk II)] and H1 [BACrep5.1neo(5'HH)], were used in the remainder of this study.
|
). However, the efficiency with which the culture-adapted replicon was delivered into the Huh7 was approximately 10-fold greater than seen by DNA transfection, resulting in up to 0·8 % of cells proceeding to form G418-resistant colonies.
|
). In this instance, Huh7 cells were allowed to recover for 12 h after transduction before tetracycline was introduced into the medium to suppress Ptet. In cells transduced with each of the BACrep clones, an 8 kb transcript was observed, which decreased in abundance after the addition of tetracycline into the medium. In the case of the mkII (2.5) and 5'HH (H1) clones, this decline halted after 24 and 48 h respectively, after which time the replicon transcript increased in abundance. In contrast, cells transduced with the mkI (1.5) and GND (G1) clones produced a transcript that continued to decline and became undetectable 48 h after tetracycline had been introduced into the medium. The observation that the mkII (2.5) and 5'HH (H1) clones generated G418-resistant colonies that harboured self-replicating HCV-derived RNA transcripts is indicative of the existence of negative-strand transcripts in these cells; however, this remains to be formally proven as a double-stranded probe was used in these experiments.
|
production (Fredericksen et al., 2002; Pflugheber et al., 2002). This same series of studies showed that another replicon lacking this mutation suppressed activation of this antiviral response in Huh7 cells. What remains unclear is whether the ability of some replicons to suppress activation of the innate antiviral response is reliant on the recently described inability of Huh7 cells to produce IFN- in response to dsRNA (Lanford et al., 2003). The ability to efficiently transduce HepG2 cells, a hepatocyte-derived cell line that is responsive to dsRNA (S. Lemon, personal communication), with the replicon-containing baculovirus constructs provided an opportunity to address this issue. However, it was first necessary to show that the culture-adapted replicon used in our study did not trigger an antiviral response in Huh7 cells. Analysis of the levels of IFN- transcript by RT-PCR demonstrated that low basal levels were present in a Huh7 polyclonal cell line containing the 5.1 replicon as was also seen in naïve Huh7 cells and a Huh7 cell line expressing neoR (Fig. 7a). In contrast, Huh7 cells infected with Semliki Forest virus showed up-regulation of IFN- transcription.
|
response in both HepG2 and Huh7 cells when co-transduced with BACtTA and either BACrep5.1neo(mkII) (clone 2.5) or BACrepGNDneo (clone G1), and when transfected with poly(I)–poly(C) (Fig. 7b). Transfection of the cells with poly(I)–poly(C) triggered production of IFN- in HepG2 but not Huh7 cells, confirming that a typical antiviral response to dsRNA occurs in the former but not the latter cell line. As expected, mock transfection either in the presence/absence of yeast tRNA did not trigger an IFN- response. Transduction of both cell lines with a -galactosidase-expressing baculovirus under the control of Ptet (BACINDlacZtet) failed to elicit a significant IFN- response, demonstrating that baculovirus transduction by itself does not trigger an antiviral response. In contrast, the culture-adapted replicon did trigger a detectable increase in IFN- transcription in HepG2 but not Huh7 cells. As the polymerase knock-out construct failed to elicit an IFN- response, the response seen in BACrep5.1neo(mkII)-transduced HepG2 cells was not simply due to expression of non-structural proteins and so seems likely to involve the formation and subsequent detection of dsRNA. However, under the conditions used in the above experiment there would have been significantly more polII-derived replicon transcript present in transduced HepG2 cells compared to both transduced Huh7 cells and also replicon-containing Huh7 cell lines (compare Fig. 6 and Fig. 7d). Therefore, it is possible that while the IFN- response specifically requires the presence of a replication-competent replicon transcript, it is dependent on higher levels of this transcript than would be attainable in a replicon-containing cell line. For this reason, the IFN- response was examined in HepG2 cells co-transduced with BACtTA and either BACrep5.1neo(mkII) (clone 2.5) or BACrepGNDneo (clone G1), and then maintained in medium containing various concentrations of tetracycline in order to modulate the level of polII-derived replicon transcript (Fig. 7c). In the absence of tetracycline, when Ptet is fully activated, increased IFN- transcription occurred after transduction with BACrep5.1neo(mkII) but not BACrepGNDneo, as observed previously. A detectable increase in IFN- transcription was also seen when the BACrep5.1neo(mkII)-transduced HepG2 cells were maintained in low concentrations of tetracycline (0·02 and 0·04 µg ml-1). However, when higher concentrations of tetracycline were used there was no detectable increase in IFN- transcription, and neither was there an increase in IFN- transcription under any of the concentrations of tetracycline used in cells transduced with BACrepGNDneo. The level of polII-derived replicon transcript was then assessed alongside cellular RNA from a replicon-containing Huh7 cell line using Northern blot analysis (Fig. 7d). This confirmed that for any given concentration of tetracycline, both BACrep5.1neo(mkII)- and BACrepGNDneo-transduced cells contained similar levels of replicon (8 kb) transcript. Two other features were also apparent. The first was the detection of a low abundance high molecular mass RNA species (>8 kb) in HepG2 cells transduced with the culture-adapted but not the polymerase knock-out replicon construct when the cells were maintained in the absence of tetracycline. This band migrated at the same position as the large transcript seen in a replicon-containing cell line, an RNA species earlier shown to hybridize to both negative- and positive-sense probes. In addition, baculovirus-transduced HepG2 cells maintained in the presence of 0·04 µg tetracycline ml-1, conditions where an IFN- response was still observed, had a similar level of replicon transcript compared to the replicon-containing Huh7 cell line. It is therefore unlikely that the IFN- response observed is simply due to overexpression of the replicon in HepG2 cells. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In these cell lines the transcription start site was positioned six nucleotides upstream of the start of the 5'UTR, and it was unclear how far the polII transcript extended beyond the 3'UTR. Moreover, because the full-length HCV genome undergoes limited, if any, replication in tissue culture, evidence for replication relied on detection of negative-strand production as a surrogate marker. By using the HCV replicon in combination with Huh7 cells which, unlike full-length HCV, is capable of self-sustaining replication, we have confirmed that polII-derived HCV transcripts are replication competent. Artificially synthesized RNA transcripts from the cDNA of other positive-strand RNA viruses require that both the 5' and 3' ends correspond to those found in the viral genome in order to maximize infectivity (Herold & Andino, 2000). Therefore, it was not unexpected that correct positioning of the 5'UTR with respect to the transcription start site was required to maximize the delivery of functional replicon transcripts into Huh7 cells. However, our results also indicate that the presence of a 5' cap structure does not impair the ability of the polII-derived replicon transcript to replicate. In fact, transcripts possessing a 5' cap were slightly more effective at establishing the replicon in Huh7 cells compared to transcripts where the 5' end was defined using a hammerhead ribozyme. The reason for this has not been investigated further but could stem from incomplete cleavage by the ribozyme or the fact that the cleaved transcript will have a 5'-hydroxyl group as opposed to a 5'-phosphate group.
Despite the use of baculovirus transduction, the efficiency of replicon delivery into Huh7 cells was still approximately 10-fold less than seen using RNA transfection, maybe because of the low levels of polII-derived transcript present within the cell. Therefore, it was surprising to find between 100- to 1000-fold higher levels of replicon transcript and HCV non-structural protein expression in HepG2 compared to Huh7 cells, particularly as there is only a 2- to 3-fold difference in the level of reporter gene expression between HepG2 and Huh7 cells transduced with -galactosidase expressing baculovirus BACINDlacZtet (McCormick et al., 2002). This higher level of expression has allowed us to detect non-structural protein expression by immunofluorescence and demonstrate that the majority of HepG2 cells do express the replicon construct (data not shown). We have not yet investigated whether the levels of polII-derived replicon transcript can be further increased in Huh7 cells. However, should the replicon cDNA contain cryptic or dysfunctional RNA splicing signals, an approach such as the introduction of an HIV rev-response element into the polII transcript in combination with expression of Rev (Malim et al., 1989) might improve transcript production.
An unexpected problem encountered in this study was the stability of the replicon cDNA in the baculovirus genome. Despite Southern blot analysis indicating that the constructs were intact, Western blot and later a functional replicon screen indicated that this was not the case. Even when a functional screen had been used to select replicon clones, sequencing these clones revealed point mutations that in one instance led to a change in the HCV non-structural open reading frame. As the transfer vectors used to generate these viruses do not contain point mutations, it seems likely that these mutations arose during integration into the virus genome or in the initial rounds of virus replication prior to cloning, presumably due to low fidelity of the baculovirus DNA polymerase. For this reason we have transferred our replicon constructs into the FAST-BAC system (Luckow et al., 1993). In this system, propagation of the baculovirus genome, recombination and subsequent cloning are all performed in E. coli and so rely on bacterial DNA polymerase, thereby reducing the chances of a point mutation arising prior to cloning of the virus. Initial results indicate that these FAST-BAC constructs enter and express the culture-adapted replicon constructs in HepG2 and Huh7 cells. Furthermore, there is no discernible clone-to-clone variation in their ability to deliver a functional replicon into Huh7 cells, indicating the absence of point mutations in the consensus sequence of these clones.
An observation made during this study was the presence of a high molecular mass RNA species detected by HCV-specific probes in replicon-containing cell lines. The existence of this RNA species has not been commented on in previous studies, although in the original paper first describing the HCV replicon (Lohmann et al., 1999), a transcript with an apparent size considerably greater than 8 kb is visible by Northern analysis. In addition, a study on the replication of an HCV-related pestivirus, bovine viral diarrhoea virus (BVDV), also observed a similar RNA species, which was considered to be a combination of replicative forms and replicative intermediates (Tomassini et al., 2003). Our data are consistent with this conclusion, as both positive- and negative-strand-specific probes hybridize to this RNA species, and it is seen in HepG2 cells transduced with baculovirus carrying the culture-adapted but not polymerase knock-out replicon. We are further characterizing this RNA species.
Although the replicon system is the best model available to study HCV replication, it is still not possible to examine many aspects of the virus life-cycle. For example, there is as yet no ideal way to study viral particle assembly and entry as replicon cell lines do not form infectious viral particles, even when the replicating RNA expresses the entire HCV genome (Pietschmann et al., 2002). The reason for this failure has not been identified and it has been speculated that the lack of particle formation may stem from a defect in Huh7 cells. The reliance on Huh7 cells also complicates the study of the antiviral response to both full-length and subgenomic HCV replicons, particularly as Huh7 cells have recently been shown to be unable to produce IFN- in response to dsRNA (Lanford et al., 2003). A recent finding that a number of other cell lines are able to maintain the replicon (Zhu et al., 2003) may help address some of these issues. However, our finding that the presence of a functional replicon in HepG2 cells, unlike in Huh7 cells, does activate transcription of IFN- highlights the problem of using Huh7 cells, particularly when studying the innate antiviral response of the cell to genomic and subgenomic HCV RNAs. Indeed, it seems to be the ability to respond to dsRNA that accounts for the discrepancy between the antiviral response to HCV replicons in HepG2 cells compared to Huh7 cells, as expression of the polymerase knock-out replicon construct in HepG2 cells did not trigger IFN- transcription. While it is tempting to speculate that IFN- production in HepG2 cells might prevent long-term establishment of replicons in this cell line, this seems unlikely as replicon-containing Huh7 cell lines exist that constitutively express IFN- (Fredericksen et al., 2002). However, as replicons can be cured with IFN- treatment (Cheney et al., 2002) and constitutive expression of IFN- normally suppresses replicon replication (Pflugheber et al., 2002), it is possible that the production of IFN- in HepG2 cells is a contributory factor in preventing establishment of replicons in this cell line. The interferon response observed in HepG2 cells containing the HCV replicon contrasts with a lack of a response seen in cells infected with BVDV (Baigent et al., 2002). In this instance the non-cytopathic strain of the virus is able to prevent IFN- production in response to dsRNA by blocking IRF3 function. A similar function has also recently been ascribed to NS3/4A of HCV, and therefore, while the presence of the HCV replicon in HepG2 cells did not block an IFN- response completely, it remains possible that the response seen was attenuated. It is also possible that expression of the full-length HCV genome, as compared to the NS3–NS5B component expressed by the replicon, may further contribute in down regulation or blockage of IFN- production. Baculovirus delivery of various HCV constructs along with a more quantitative assay for IFN- production should allow these questions to be addressed. Indeed, production of replication-competent HCV transcripts from a polII promoter may be a superior approach to answering these questions as we have observed that unlike baculoviral transduction, transfection of T7 transcripts into HepG2 triggers a substantial IFN- response (data not shown).
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Almazan, F., Gonzalez, J. M., Penzes, Z., Izeta, A., Calvo, E., Plana-Duran, J. & Enjuanes, L. (2000). Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 97, 5516–5521.
Baigent, S. J., Zhang, G., Fray, M. D., Flick-Smith, H., Goodbourn, S. & McCauley, J. W. (2002). Inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism. J Virol 76, 8979–8988.
Blight, K. J., Kolykhalov, A. A. & Rice, C. M. (2000). Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1974.
Cheney, I. W., Lai, V. C., Zhong, W., Brodhag, T., Dempsey, S., Lim, C., Hong, Z., Lau, J. Y. & Tam, R. C. (2002). Comparative analysis of anti-hepatitis C virus activity and gene expression mediated by alpha, beta, and gamma interferons. J Virol 76, 11148–11154.
Chung, R. T., He, W., Saquib, A., Contreras, A. M., Xavier, R. J., Chawla, A., Wang, T. C. & Schmidt, E. V. (2001). Hepatitis C virus replication is directly inhibited by IFN-alpha in a full-length binary expression system. Proc Natl Acad Sci U S A 98, 9847–9852.
Dubensky, T. W., Driver, D. A., Polo, J. M. & 9 other authors (1996). Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer. J Virol 70, 508–519.[Abstract]
Fredericksen, B., Akkaraju, G. R., Foy, E., Wang, C., Pflugheber, J., Chen, Z. J. & Gale, M., Jr (2002). Activation of the interferon-beta promoter during hepatitis C virus RNA replication. Viral Immunol 15, 29–40.[CrossRef][Medline]
Gossen, M. & Bujard, H. (1992). Tight control of gene-expression in mammalian-cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A 89, 5547–5551.
Gu, B., Gates, A. T., Isken, O., Behrens, S. E. & Sarisky, R. T. (2003). Replication studies using genotype 1a subgenomic hepatitis C virus replicons. J Virol 77, 5352–5359.
Herold, J. & Andino, R. (2000). Poliovirus requires a precise 5' end for efficient positive-strand RNA synthesis. J Virol 74, 6394–6400.
Ikeda, M., Yi, M., Li, K. & Lemon, S. M. (2002). Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 76, 2997–3006.
Koob, M., Burkiewicz, A., Kur, J. & Szybalski, W. (1992). RecA-AC: single-site cleavage of plasmids and chromosomes at any predetermined restriction site. Nucleic Acids Res 20, 5831–5836.
Krieger, N., Lohmann, V. & Bartenschlager, R. (2001). Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75, 4614–4624.
Kunkel, T. A. (1985). Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82, 488–492.
Lanford, R. E., Sureau, C., Jacob, J. R., White, R. & Fuerst, T. R. (1994). Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR. Virology 202, 606–614.[CrossRef][Medline]
Lanford, R. E., Guerra, B., Lee, H., Averett, D. R., Pfeiffer, B., Chavez, D., Notvall, L. & Bigger, C. (2003). Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(I)-poly(C), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons. J Virol 77, 1092–1104.
Lim, S. P., Soo, H. M., Tan, Y. H., Brenner, S., Horstmann, H., MacKenzie, J. M., Ng, M. L., Lim, S. G. & Hong, W. (2002). Inducible system in human hepatoma cell lines for hepatitis C virus production. Virology 303, 79–99.[CrossRef][Medline]
Lohmann, V., Korner, F., Koch, J. O., Herian, U., Theilmann, L. & Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113.
Luckow, V. A., Lee, S. C., Barry, G. F. & Olins, P. O. (1993). Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67, 4566–4579.
Luo, G., Xin, S. & Cai, Z. (2003). Role of the 5'-proximal stem-loop structure of the 5' untranslated region in replication and translation of hepatitis C virus RNA. J Virol 77, 3312–3318.
Macdonald, A., Crowder, K., Street, A., McCormick, C., Saksela, K. & Harris, M. (2003). The hepatitis C virus non-structural NS5A protein inhibits activating protein-1 function by perturbing Ras-ERK pathway signaling. J Biol Chem 278, 17775–17784.
Malim, M. H., Hauber, J., Le, S. Y., Maizel, J. V. & Cullen, B. R. (1989). The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338, 254–257.[CrossRef][Medline]
McCormick, C. J., Rowlands, D. J. & Harris, M. (2002). Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors. J Gen Virol 83, 383–394.
Myung, J., Khalap, N., Kalkeri, G., Garry, R. & Dash, S. (2001). Inducible model to study negative strand RNA synthesis and assembly of hepatitis C virus from a full-length cDNA clone. J Virol Methods 94, 55–67.[CrossRef][Medline]
Pflugheber, J., Fredericksen, B., Sumpter, R., Jr, Wang, C., Ware, F., Sodora, D. L. & Gale, M., Jr (2002). Regulation of PKR and IRF-1 during hepatitis C virus RNA replication. Proc Natl Acad Sci U S A 99, 4650–4655.
Pietschmann, T., Lohmann, V., Kaul, A., Krieger, N., Rinck, G., Rutter, G., Strand, D. & Bartenschlager, R. (2002). Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 76, 4008–4021.
Tomassini, J., Boots, E., Gan, L., Graham, P., Munshi, V., Wolanski, B., Fay, J. F., Getty, K. & Lafemina, R. (2003). An in vitro Flaviviridae replicase system capable of authentic RNA replication. Virology 313, 274–285.[CrossRef][Medline]
Varnavski, A. N., Young, P. R. & Khromykh, A. A. (2000). Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors. J Virol 74, 4394–4403.
Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. (1997). Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 94, 8738–8743.
Yanagi, M., St Claire, M., Shapiro, M., Emerson, S. U., Purcell, R. H. & Bukh, J. (1998). Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 244, 161–172.[CrossRef][Medline]
Yap, C. C., Ishii, K., Aoki, Y., Aizaki, H., Tani, H., Shimizu, H., Ueno, Y., Miyamura, T. & Matsuura, Y. (1997). A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA. Virology 231, 192–200.[CrossRef][Medline]
Zhu, Q., Guo, J. T. & Seeger, C. (2003). Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells. J Virol 77, 9204–9210.
Received 23 September 2003;
accepted 5 November 2003.
This article has been cited by other articles:
|
|
 |
|
  C. J. McCormick, O. Salim, P. R. Lambden, and I. N. Clarke Translation Termination Reinitiation between Open Reading Frame 1 (ORF1) and ORF2 Enables Capsid Expression in a Bovine Norovirus without the Need for Production of Viral Subgenomic RNA J. Virol., September 1, 2008; 82(17): 8917 - 8921. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. K. Ward, C. J. McCormick, I. N. Clarke, O. Salim, C. E. Wobus, L. B. Thackray, H. W. Virgin IV, and P. R. Lambden Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA PNAS, June 26, 2007; 104(26): 11050 - 11055. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Muller, R. Geffers, and S. Gunther Analysis of gene expression in Lassa virus-infected HuH-7 cells J. Gen. Virol., May 1, 2007; 88(5): 1568 - 1575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. McCormick, S. Maucourant, S. Griffin, D. J. Rowlands, and M. Harris Tagging of NS5A expressed from a functional hepatitis C virus replicon. J. Gen. Virol., March 1, 2006; 87(Pt 3): 635 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. McCormick, D. Brown, S. Griffin, L. Challinor, D. J. Rowlands, and M. Harris A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A J. Gen. Virol., January 1, 2006; 87(1): 93 - 102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Cai, C. Zhang, K.-S. Chang, J. Jiang, B.-C. Ahn, T. Wakita, T. J. Liang, and G. Luo Robust Production of Infectious Hepatitis C Virus (HCV) from Stably HCV cDNA-Transfected Human Hepatoma Cells J. Virol., November 15, 2005; 79(22): 13963 - 13973. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang, O. Yamada, T. Sakamoto, H. Yoshida, H. Araki, and K. Shimotohno Exploiting cis-Acting Replication Elements To Direct Hepatitis C Virus-Dependent Transgene Expression J. Virol., May 15, 2005; 79(10): 5923 - 5932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Li, Z. Chen, N. Kato, M. Gale Jr., and S. M. Lemon Distinct Poly(I-C) and Virus-activated Signaling Pathways Leading to Interferon-{beta} Production in Hepatocytes J. Biol. Chem., April 29, 2005; 280(17): 16739 - 16747. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Street, A. Macdonald, C. McCormick, and M. Harris Hepatitis C Virus NS5A-Mediated Activation of Phosphoinositid |
