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Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Christopher J. McCormick
cjm{at}soton.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Molecular Microbiology, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Reed & Rice, 2000), which are respectively proteolytically processed as a result of host cell (Hijikata et al., 1991) and viral (Bartenschlager et al., 1993; Grakoui et al., 1993; Hijikata et al., 1993) proteases. Flanking the ORF are 5' and 3' untranslated regions (UTR), both of which possess cis-acting elements necessary for virus replication (Friebe et al., 2001; Friebe & Bartenschlager, 2002; Kolykhalov et al., 2000; Luo et al., 2003; Yi & Lemon, 2003), with the 5' UTR also containing an internal ribosome entry site (IRES) (Tsukiyama-Kohara et al., 1992; Wang et al., 1993).
Unusually, HCV is capable of establishing a life-long infection, irrespective of the age at which it is acquired. Long-term chronic infection can lead to clinical manifestations ranging from cryoglobulinaemia and liver fibrosis through to end-stage liver disease including cirrhosis and hepatocellular carcinoma (Poynard et al., 2003). Current combination therapy using pegylated IFN-
and ribavirin is only approximately 50 % effective and there remains a need for improved treatment regimes. One part of the virus life cycle that has potential for therapeutic intervention is replication of the viral genome. This area of research has benefited greatly from the development of the HCV replicon (Blight et al., 2000; Lohmann et al., 1999). However, establishing a level of replication within a tissue culture system to allow detection invariably results in the selection of point mutations within the coding regions of the non-structural proteins, leading to a culture-adapted phenotype (Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 2001, 2003). Furthermore, culture adaptation is associated with attenuated virus replication in vivo (Bukh et al., 2002). Therefore, while the HCV replicon is a good model system for assessing the importance of viral and host cell proteins in virus replication per se, the requirement for culture-adaptation makes it less appropriate for the study of mechanisms that might determine replicative strategies adopted by the virus during a natural infection. We have previously reported the use of a baculovirus delivery system to introduce full-length and subgenomic HCV transcripts into hepatocyte-derived cell lines (McCormick et al., 2002, 2004). Advantages of this system include the facts that transduction is extremely efficient, with almost 100 % of cells showing expression of viral proteins, and that the level of transcript can be controlled in the transduced cells by virtue of a tetracycline-responsive promoter. Furthermore, unlike the HCV replicon, it is also possible to achieve high levels of HCV viral transcript and protein in transduced cells without the need for robust virus replication or culture-adaptation.
The basis for the study reported here was the observation that a single point mutation blocking the activity of the viral polymerase, NS5B, caused an increase in the hyperphosphorylation of NS5A in the context of a non-culture-adapted replicon. This was of interest because some of the more potent culture-adapted mutations tend to cluster within a region of the viral non-structural protein NS5A (Blight et al., 2000; Krieger et al., 2001), leading to speculation that this protein might be critically involved in controlling replication. Moreover, results from more recent studies suggest that the differential phosphorylation of this protein is important, and indicate that the hyperphosphorylated form of NS5A is inhibitory to replication (Appel et al., 2005; Evans et al., 2004; Neddermann et al., 2004). It was also unexpected because previous studies had suggested that neither the coding nor the non-coding regions downstream of NS5A played a significant role in modulating hyperphosphorylation (Asabe et al., 1997; Koch & Bartenschlager, 1999; Neddermann et al., 1999). Our investigation has shown that, in the non-culture-adapted replicon con1, NS5A hyperphosphorylation is influenced by elements in the HCV genome downstream of the NS5A coding region. In addition, it has also revealed the importance of translational activity from the replicon transcript in determining levels of hyperphosphorylation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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DNA constructs.
We have recently shown that the commercial Bac-to-Bac system (Invitrogen) allows more rapid generation of recombinant baculoviruses for mammalian expression and dramatically reduces the occurrence of point mutations within clones (unpublished data). For this reason, all HCV-containing baculovirus constructs used in his study were derived from a modified pFastBac1 transfer vector. Firstly, an oligonucleotide pair (FASTBst1, FASTBst2; sequences available on request) was cloned into SnaBI/AvrII-cut pFastBac1, generating pFB(XbaI-HindIII). Replicon-containing mammalian expression cassettes were then transferred into pFB(XbaI-HindIII) by sequential cloning of the XbaI–HindIII and HindIII–HindIII fragments from pBACrep5.1neo(mkII) and pBACrepGNDneo (McCormick et al., 2004), generating pFBrep5.1neo and pFBrepcon1GNDneo, respectively. Other full-length replicon constructs, pFBrepcon1neo and pFBrep5.1GNDneo, were produced by exchange of SfiI–SfiI fragments between pFK-I389neo/NS3-3'/con1 (Lohmann et al., 1999) and pFBrep5.1neo and between pFK-I389neo/NS3-3'/5.1 (Krieger et al., 2001) and pFBrepcon1GNDneo, respectively.
To generate replicon-constructs lacking part of the 3' end of the genome, the SfiI–SfiI fragment of pFK-I389neo/NS3-3'/con1 was introduced into SfiI-cut pBACrep5.1neoT7/NotI (McCormick et al., 2004), generating pBACrepcon1neoT7/NotI. The primer pairs NS9con1 and NS5A(rev) or NS9con1 and NS5B(rev) were used in PCRs with pFBrepcon1neo or pFBrepcon1GNDneo as template to generate DNA fragments which were cloned into XhoI/NotI-cut pBACrepcon1neoT7/NotI. The HindIII–HindIII fragment from the resultant vectors were subsequently transferred to HindIII-cut pFBrepcon1neo, generating pFBrepcon1neo(
5B3'U), pFBrepcon1neo(3'U) and pFBrepcon1GNDneo(3'U).
To generate the encephalomyocarditis virus (EMCV) defective replicon constructs, the RsrII(polished)–BsrGI fragment from pFK-I389neo/NS3-3'/5.1 was first transferred into LITMUS38 (NEB), generating pLRM-EMCVwt. Mutagenesis was performed using the GeneEditor mutagenesis kit (Promega) in combination with mutagenic oligonucleotide EMCV1(defect). Sequencing was used to identify mutated clones, but also revealed that a small stretch of nucleotides between the end of the neo ORF and the start of the EMCV IRES was consistently deleted (nucleotides 1243–1252). Such clones were deemed acceptable because this region is not expected to affect either neo expression or EMCV IRES activity. A clone containing the mutated EMCV IRES was then restricted with KpnI and StuI and a KpnI–XhoI(polished) fragment from pFK-I389neo/NS3-3'/con1 was cloned into the vector. Finally, the PmeI–MluI fragment from this latter vector was removed and cloned into PmeI/MluI-cut pFBrepcon1neo and pFBrepcon1GNDneo, generating pFBrepcon1neo(ED) and pFBrepcon1GNDneo(ED), respectively.
Northern blot analysis.
RNA was harvested from cells using Trizol (Invitrogen), electrophoresed through a MOPS/formaldehyde gel and transferred to BrightStar Nylon Plus membrane (Ambion) using standard procedures. 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 the BrightStar detection kit (Ambion).
Western blot analysis.
Cells were lysed in RIPA (50 mM Tris/HCl, pH 8·0, 150 mM NaCl, 1 % (v/v) NP-40, 0·5 % w/v sodium deoxycholate, 0·1 % SDS) supplemented with 2x Complete protease inhibitor cocktail (Roche), 1 mM Na3VO4 and 1 mM NaF and the protein concentration of samples was determined using BCA reagent (Pierce). Samples containing equal amounts of protein (typically 5–10 µg per well) were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). Membranes were blocked with 5 % (w/v) low-fat dried milk, 0·1 % Tween 20 (Merck) in Tris-buffered saline and incubated with a 1 : 10 000 dilution of either sheep anti-NS3, anti-NS5A or anti-NS5B serum (raised against a His-tagged NS5B fusion protein expressed in E. coli) or a 1 : 5000 dilution of a rabbit anti-neomycin phosphotransferase (NPT) polyclonal antibody (Upstate). Bound antibody was detected with an appropriate HRP-conjugated secondary antibody (Sigma) in conjunction with ECL reagent (Amersham Pharmacia Biotech) and light emissions were captured by a Luminescent Image Analyser LAS-1000 (Fujifilm) and analysed using Advanced Image Data Analyser version 2 0 software (Raytek Scientific).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Using this system we expressed a series of different bicistronic replicon transcripts, including a non-culture-adapted HCV replicon (con1) and a culture-adapted HCV replicon (5.1), either with a functional polymerase (FBrepcon1neo and FBrep5.1neo) or with a point mutation within the active site of NS5B that abolished polymerase activity (FBrepcon1GNDneo and FBrep5.1GNDneo) (Fig. 1a). Expression of NPT, NS3 and NS5B was confirmed by Western blot (Fig. 1b), demonstrating that proteins from both cistrons were expressed and processed appropriately. Recent work has highlighted the importance of NS5A hyperphosphorylation in controlling HCV replication, so it was of interest to examine hyperphosphorylation of NS5A following transduction of HepG2 cells with the different constructs. Consistent with previous reports, hyperphosphorylation of NS5A (p58 formation) was found to be reduced in cells transduced with the culture-adapted replicon constructs (FBrep5.1neo and FBrep5.1GNDneo) compared with the non-culture-adapted constructs (FBrepcon1neo and FBrepcon1GNDneo) (Fig. 1c). Unexpectedly, the level of NS5A hyperphosphorylation was increased in HepG2 cells transduced with the non-culture-adapted replicon containing a non-functional NS5B polymerase (FBrepcon1GNDneo) compared with cells transduced with the related construct containing a functional polymerase (FBrepcon1neo). In contrast, disabling polymerase activity in the 5.1 replicon background had little or no effect. Immunofluorescence was also used to examine the cellular localization of NS5A in the four constructs. While there were differences in the localization pattern of NS5A between the 5.1- and con1-based constructs, an observation that has been reported when comparing other replicon constructs (Moradpour et al., 2004), no such difference was seen when comparing FBrepcon1neo and FBrepcon1GNDneo (data not shown). Therefore, differences in NS5A hyperphosphorylation expressed from the con1 and con1GND transcripts do not appear to be due to gross changes in cellular distribution of NS5A, although the effects of subtle changes in distribution can not be discounted.
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Viral elements downstream from NS5A affect hyperphosphorylation
Our observation that a point mutation in NS5B which disabled polymerase activity also increased NS5A hyperphosphorylation conflicted with previous reports suggesting that elements downstream from NS5A had little impact on hyperphosphorylation (Asabe et al., 1997; Koch & Bartenschlager, 1999; Neddermann et al., 1999). In order to address this issue, con1 and con1GND replicons with a series of deletions from the 3' end were transferred into baculovirus constructs. These included a con1 replicon lacking both NS5B and the 3' UTR [FBrepcon1neo(5B3'U)], a con1 replicon missing just the 3'UTR [FBrepcon1neo(3'U)] and a con1GND replicon missing the 3' UTR [FBrepcon1GNDneo(3'U)] (Fig. 2a). Northern analysis of HepG2 cells transduced with these and the original FBrepcon1neo and FBrepcon1GNDneo constructs demonstrated the presence of a single band of the expected size in each case, although all transcripts lacking the 3' UTR were less abundant compared with the intact replicon transcripts (Fig. 2b). By Western analysis, hyperphosphorylation of NS5A from cells transduced with FBrepcon1neo(5B3'U) was comparable to cells transduced with the intact replicon (FBrepcon1neo) (Fig. 2c, d), supporting previous findings that expression of non-structural proteins NS3 through to NS4B in cis with NS5A is necessary and sufficient for effective p58 formation (Koch & Bartenschlager, 1999; Neddermann et al., 1999). In contrast, cells transduced with FBrepcon1neo(3'U) had dramatically reduced levels of NS5A hyperphosphorylation, irrespective of whether polymerase activity was intact or defective. This finding indicates that nucleotide elements downstream from the NS5A coding region and/or NS5B can influence NS5A hyperphosphorylation, with NS5B or the coding region for NS5B suppressing p58 formation and the 3' UTR effectively relieving this suppression. In addition, even though the absence of the 3' UTR reduced hyperphosphorylation, FBrepcon1GNDneo(3'U)-transduced cells still possessed higher levels of hyperphosphorylated NS5A compared with FBrepcon1neo(3'U)-transduced cells. Interestingly, comparison of transcript levels with expression of viral non-structural proteins indicated that translation was reduced from both FBrepcon1neo or FBrepcon1neo(3'U) constructs compared with their polymerase-defective counterparts, although this was most apparent using NS3 to gauge translation and least apparent when using NS5B. Therefore, the increase in p58 formation resulting from inactivation of the viral polymerase by point mutation appears to be independent of the presence of the 3' UTR, but does occur concomitantly with an apparent increase in translation of the HCV ORF.
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) or vehicle alone (DMSO). In contrast to the increase in NS5A hyperphosphorylation that was observed by mutating the active site of NS5B (Fig. 1b
), Western analysis revealed that SB-750330-0Z did not increase the formation of p58 in FBrepcon1neo-transduced cells (Fig. 3a). However, several other changes were observed in the presence of the drug. Firstly, an accumulation of what appeared to be an NS5A5B precursor was observed, given that it was detectable by both NS5A and NS5B antisera and migrated on SDS-PAGE with an apparent molecular mass equivalent to NS5A and NS5B combined (
120 kDa) (Fig. 3b). Generation of this precursor was also far more apparent in FBrepcon1neo-transduced cells compared with FBrepcon1GNDneo-transduced cells (data not shown). Secondly, the presence of the inhibitor but not DMSO alone increased expression of NPT, NS3 and NS5A in FBrepcon1neo- but not FBrepcon1GNDneo-transduced cells (Fig. 3a, c; Table 1). This appeared to be as a result of increased translational activity from both IRES elements within the HCV replicon, as Northern analysis (Fig. 3d) demonstrated that neither DMSO nor the polymerase inhibitor affected replicon transcript levels. These data are consistent with the notion that functional polymerase activity suppresses translational activity from both the EMCV and HCV IRES elements. However, intriguingly, the data indicated that, whereas genetic elimination of polymerase function resulted in an increase in NS5A hyperphosphorylation, pharmacological inhibition of the polymerase had no such effect.
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). In cells transduced with FBrepcon1neo, reduction of viral polyprotein expression had a negligible effect on the percentage of total NS5A that was hyperphosphorylated. The situation was slightly different in FBrepcon1GNDneo-transduced cells in that reduction of non-structural protein expression caused a modest increase in overall levels of hyperphosphorylated NS5A. However, the level of hyperphosphorylated NS5A in FBrepcon1neo- and FBrepcon1GNDneo-transduced cells did not coincide over the range of expression levels examined. Therefore we conclude that differences in the overall level of non-structural protein within the cell (as determined by varying replicon transcript levels) cannot account for the phenomenon of increased p58 formation in FBrepcon1GNDneo-transduced cells.
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), the changes in NPT and non-structural protein expression following addition of tetracycline simply reflect alterations in replicon transcript levels. This raised the alternative possibility that the amount of non-structural protein translated from each replicon transcript might influence NS5A hyperphosphorylation. We tested this by utilizing a second approach in which we reduced the translational activity of the EMCV IRES by mutating a tetraloop sequence within the EMCV IRES of the con1 and con1GND replicon from GCGA to GTTA. This change has been shown previously to reduce IRES activity by approximately 80 % (Robertson et al., 1999).
HepG2 cells were transduced with baculovirus constructs containing these two replicons [FBrepcon1neo(ED) and FBrepcon1GNDneo(ED)] or with the wild-type FBrepcon1neo and FBrepcon1GNDneo and the cell lysates were subjected to Western analysis (Fig. 5). As expected, expression of NS3, NS5A and NS5B was much lower in cells containing replicons with the mutated EMCV IRES compared with replicons with a wild-type IRES (Fig. 5b). However, in contrast to reducing HCV non-structural protein expression by changing replicon transcript levels, where little effect was seen on NS5A hyperphosphorylation, a decrease in HCV non-structural protein expression as a result of reduced EMCV IRES activity suppressed p58 formation (Fig. 5a). Furthermore, unlike FBrepcon1neo- and FBrepcon1GNDneo-transduced cells, there seemed to be no difference in the extent to which NS5A was hyperphosphorylated between FBrepcon1neo(ED)- and FBrepcon1GNDneo(ED)-transduced cells, nor was there any noticeable difference in overall levels of NPT and non-structural protein expression. Another difference between cells containing replicons with a functional or attenuated EMCV IRES was that, in the latter case, NPT expression was increased, perhaps indicating the existence of competition between the two IRESs within the replicon. Further evidence that all these differences in expression and phosphorylation of proteins from the replicon ORFs were due to translational activity was provided by Northern analysis, which showed that the amount of replicon transcript was comparable between cells transduced with the different baculovirus constructs (Fig. 5c). We conclude that, in the context of the non-culture-adapted replicon, the extent of translation from the viral transcript determines the extent to which NS5A is hyperphosphorylated. Moreover, differences in translation levels resulting from the presence or absence of polymerase activity appear to be suppressed under reduced IRES activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Evans et al., 2004; Neddermann et al., 2004). Perhaps more convincing is the finding that kinase inhibitors that block or reduce NS5A hyperphosphorylation also facilitate replication of a non-culture-adapted replicon (Neddermann et al., 2004). Indeed, a speculative model has been proposed in which hyperphosphorylation disrupts binding of NS5A to human vesicle-associated membrane protein-associated protein A (hVAP-A), a cellular protein considered necessary for efficient replicon replication (Evans et al., 2004). However, if hyperphosphorylation of NS5A does have a regulatory role in virus replication, this also implies it is a dynamic process in which the levels of hyperphosphorylation are in turn altered in response to one or more stimuli that affect HCV replication. To date, no evidence has been put forward to suggest how hyperphosphorylation might be modulated. Instead, studies have been devoted to identifying both the cellular kinases involved in NS5A phosphorylation (Coito et al., 2004; Reed et al., 1997) and the components of the viral genome necessary, when expressed either in cis or in trans, to facilitate hyperphosphorylation (Asabe et al., 1997; Koch & Bartenschlager, 1999; Neddermann et al., 1999). Indeed, it is difficult to utilize replicon-containing cell lines to examine how hyperphosphorylation might be modulated, since culture-adapted mutations are likely to affect the dynamics of hyperphosphorylation. We have developed a baculovirus delivery system which enables efficient delivery of full-length and subgenomic replicon transcripts into HepG2 cells (McCormick et al., 2002, 2004), thus by-passing the need for culture-adaptation. It was therefore of interest to observe that, in cells containing a non-culture-adapted HCV replicon (con1), the presence of a point mutation in NS5B that disabled polymerase activity modulated the degree of NS5A hyperphosphorylation. Subsequent attempts to determine how polymerase activity could affect hyperphosphorylation led us to three conclusions: firstly, that RNA elements downstream of the NS5A coding region, and possibly expression of NS5B as well, influence NS5A hyperphosphorylation (Fig. 2); secondly, that polymerase activity suppresses translation from both the HCV and EMCV IRES within the HCV replicon (Fig. 4); and finally, and more importantly, we also found that reducing the translational activity of the EMCV IRES reduces hyperphosphorylation of NS5A in a non-culture-adapted replicon background. This is not due to a change in overall levels of non-structural protein present within the cell, as reducing the amount of replicon transcript within each cell did not have the same effect. To our knowledge, this represents the first evidence that modulation of NS5A hyperphosphorylation might be controlled in part by factors that influence translational activity from the HCV genome.
Is it now possible to account for the original observation that the non-culture-adapted replicon differed in NS5A hyperphosphorylation depending on whether or not NS5B was inactivated by a point mutation within the active polymerase site? Our results clearly show that removal of elements downstream of the NS5A coding region alter hyperphosphorylation. Therefore, it is possible that the difference in hyperphosphorylation between con1 and con1GND is a result of direct structural changes within NS5B that affect its association with other viral non-structural and cellular proteins, rather than a lack of polymerase function. However, the mutation is present within the active site of NS5B and so is unlikely to be directly accessible to other non-structural and/or cellular proteins, although it is pertinent to note that the level of NS5B protein was reduced relative to the other non-structural proteins when inactivated by the GND mutation, suggesting decreased stability of the non-functional protein. Two further hypotheses may explain the data. The first is based on the result from experiments with the 3' deletion constructs (Fig. 2) which are consistent with, but not proof of, a model where NS5B serves to suppress NS5A hyperphosphorylation. In this case, if NS5B stability is decreased by the GND point mutation, as already mentioned, suppression of NS5A hyperphosphorylation might be relieved. The second explanation is based on the positive correlation that exists between the activity of the EMCV IRES and the level of NS5A hyperphosphorylation. If functional polymerase activity suppresses translation from the replicon transcript, as is observed, this in turn would be expected to reduce NS5A hyperphosphorylation. In support of this hypothesis was the finding that, in the context of a defective EMCV IRES, the con1 and con1GND transcripts displayed similar translational activity and had a reduced but comparable level of NS5A hyperphosphorylation. In other words, under conditions where there was no difference in translational activity from the non-culture-adapted replicon with or without a functional polymerase, the level of NS5A hyperphosphorylation was the same. This was not seen when FBrepcon1neo-transduced cells were treated with the polymerase inhibitor, despite an increase in translation from the two IRESs within the replicon transcript. However, this observation has to be viewed with some caution, as a significant build-up of the NS5A5B precursor also occurred in the presence of inhibitor, suggesting that the inhibitor has global effects on NS5B in addition to its effect on polymerase activity which may in turn alter hyperphosphorylation of NS5A.
Another finding of the study was that replicon-derived translated products increased when a point mutation was introduced into NS5B that disabled polymerase activity or when a polymerase inhibitor was present. The reasons for this are currently being investigated but may be the result of a number of possibilities. Firstly, we have previously shown that the presence of a polymerase-competent but not a polymerase-defective replicon in HepG2 cells triggers an interferon response (McCormick et al., 2004), which may in turn modulate translational activity from the replicon transcript through phosphorylation of eIF2 (Goodbourn et al., 2000). Alternatively, it may be that viral transcripts are recruited into membrane-bound replication complexes as a result of polymerase activity, which then decreases their availability to cellular translational machinery. Indeed, a dependence on polymerase activity for recruitment to such complexes has already been observed with poliovirus (Teterina et al., 2001). Finally, competition may occur between the translating ribosomes moving in the 5' to 3' direction and the replication complex moving in the 3' to 5' direction on the same viral transcript. Such clashes are normally thought to be avoided in positive-strand viral RNA replication, as they have been shown to suppress polymerase activity and therefore would affect viral fitness (Barton et al., 1999). Nonetheless, it is possible that, either in HCV replication generally or in the system that is employed in this study, the switch from translation of the viral transcript to use of the transcript for RNA-dependent RNA polymerase-catalysed production of negative strand is ‘leaky’. Under such circumstances both replication and translation might be suppressed.
One of the most interesting findings of this study was that the introduction of a defective EMCV IRES into the HCV replicon resulted in a significant reduction in the hyperphosphorylation of NS5A. It is difficult at present to speculate on the mechanism whereby the machinery responsible for the hyperphosphorylation of NS5A can respond to the rate at which protein translation is initiated on HCV-related transcripts. However, the ability of the virus to respond to transcript utilization in this way may be very important for the regulation of virus replication and progeny production, given the well-established inverse correlation between hyperphosphorylation of NS5A and replicon replication (Appel et al., 2005; Blight et al., 2000; Evans et al., 2004; Neddermann et al., 2004). It is conceivable that such a mechanism might provide the virus with the means to respond to the intracellular environment so that replication could be modulated in response, for example, to the immune status of the host. Such a facility may help explain the almost unique ability of HCV routinely to initiate life-long chronic infections.
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ACKNOWLEDGEMENTS |
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Received 13 May 2005;
accepted 28 September 2005.
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V), which allow production of polII-derived replicon transcripts with appropriate 5' and 3' ends. (b) Representative Western blots for NPT, NS3 and NS5B in HepG2 cell lysates mock-transduced (lane 1) or co-transduced with BACtTA and either FBrepcon1neo (lane 2), FBrepcon1GNDneo (3), FBrep5.1neo (4) or FBrep5.1GNDneo (5). (c) Representative Western blots for NS5A expression using the cell lysates from (b) (top panel) or from transduced Huh7 cells (lower panel) and maintaining the same lane order. The percentage of NS5A hyperphosphorylated was determined by densitometry and is depicted graphically. The data from HepG2 cells are from five separate experiments (means±SEM); the Huh7 cell data result from a single experiment.
