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Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Via Pontina Km 30600, I-00040 Pomezia (Roma), Italy
Correspondence
Giacomo Paonessa
giacomo_paonessa{at}merck.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated. Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons. This feature was characterized as a dominant negative effect. Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon. In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Approximately 70 % eventually develop a chronic infection, which can lead to fibrosis, liver cirrhosis and hepatocellular carcinoma (Theodore & Fried, 2000). Currently, HCV represents a significant medical problem still not completely resolved. In fact, until a vaccine becomes available the only treatment is alpha interferon (IFN-
), either alone or in combination with ribavirin (for review see Foster & Thomas, 2000). Unfortunately, sustained response is observed in only about 50 % of treated patients, which also relates to the infecting HCV genotype (Manns et al., 2001). The situation is further complicated by the lack of any suitable animal model, which has hampered the development of new antiviral drugs. A major step forward in the discovery and validation of therapeutic antivirals was the development of robust, cell-based replicon systems for HCV (Lohmann et al., 1999). The minimal version of these subgenomic RNA replicons is composed of the HCV internal ribosome entry site (IRES) [the 5' non-coding region (NCR)] driving the neomycin phosphotransferase (neo) gene and the encephalomyocarditis virus (EMCV) IRES, which drives the translation of HCV non-structural (NS) proteins from NS3 to NS5B, ending with the HCV 3' NCR. Despite the fact that HCV replicons are able to accomplish all the molecular and enzymic steps needed for their own RNA amplification, some drawbacks have emerged. The sequence used for construction of the original replicon (Lohmann et al., 1999) was derived from the Con1 isolate (genotype 1b), but replicon RNAs recovered from G418-resistant cellular clones have been shown to contain a variety of mutations within HCV non-structural proteins. The presence of these adaptive mutations has been demonstrated as essential for efficient replication of Con1 replicons (Blight et al., 2000; Guo et al., 2001; Krieger et al., 2001; Lohmann et al., 2001) as well as other isolates, BK and H77 (Grobler et al., 2003; Blight et al., 2003). The most efficient adaptive mutations lie in the NS5A protein and, interestingly, one other sequence that has been successfully used for replicon (HCV-N) contains a four amino acid insertion within the NS5A protein (Ikeda et al., 2002). This suggests that expression of the natural form of NS5A in cell culture could interfere with replicon RNA replication. Whether adapted HCV replicons can thus be considered an authentic model for HCV replication in human liver cells is a reasonable question. Indeed, the main concern is whether anti-replicon compounds could be considered as true antivirals and therefore efficacious in humans. Moreover, it emerged that, contrary to the original Con1 sequence, the full-length HCV RNA Con1 genome containing adaptive mutations is not infectious when transfected intrahepatically into chimpanzees (Bukh et al., 2002). Although many efforts have been made in this direction, for example mapping adaptive mutations using three-dimensional models or structures of viral proteins (Lohmann et al., 2001), no valid interpretation has been given of their real role (besides a descriptive list), nor about the uniqueness of the hepatoma-derived cell line Huh7 as a recipient.
Here we exploited a new approach to specific aspects of this issue. In addition to Neo-replicons, we employed -lactamase (Bla) as a reporter gene (Zlokarnik et al., 1998; Murray et al., 2003) and used a subpopulation of Huh7 showing high permissiveness for HCV replication (Blight et al., 2002; Murray et al., 2003). Our experimental strategy relied on the appropriate combination of Bla and Neo-replicons in cotransfection experiments. This led to a simple and sensitive way to observe the replication efficiency of one replicon in opposition to the other at an early time after transfection. Our observations were further supported by using quantitative PCR (TaqMan). Surprisingly, this strategy uncovered a peculiar dominant negative effect of the replicon containing the original Con1 isolate sequence [referred to here as wild-type (wt)] on the NS5A-adapted replicon. In fact, cotransfection of a three- to fivefold excess of the wt replicon RNA is sufficient to shut down the replication of an NS5A-adapted replicon almost completely. Our experiments revealed that this effect is exerted by wt NS5A exclusively when expressed as part of a continuous NS3–NS5A polyprotein. NS5A expressed alone in an IRES-dependent manner, or trans-complemented with one or several other viral proteins, has no effect. These results, together with the analysis of an NS5B-adapted replicon, suggest interesting interpretations of the meaning of the adaptive mutations.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2b ml–1 (Intron-A; Schering-Plough) to the cell culture medium.
Manipulation of nucleic acids and construction of recombinant plasmids.
Nucleic acids were manipulated according to standard protocols (Sambrook et al., 1989). Plasmid DNA was prepared from overnight cultures in Luria–Bertani broth using Qiagen 500 columns according to the manufacturer's instructions. Neo-wt was the progenitor of all replicons described herein, and contained the cDNA for an HCV bicistronic replicon identical to replicon I377neo/NS3-3'/wt described by Lohmann et al. (1999) (EMBL/GenBank accession no. AJ242652). The Bla gene (bla) form of the replicons was constructed by replacing the AscI–PmeI fragment spanning the neo gene of HCV replicons with an AscI–PmeI fragment, including bla from a plasmid kindly provided by Dr J. Grobler. Mutants in the polymerase active site GDD motif were obtained by first constructing a GDD to GAA mutated Neo-wt clone by means of primer-based mutagenesis, and subsequently replacing a restriction fragment spanning the mutation in other replicons. Mutant 5Astop was obtained by means of primer-based mutagenesis, substituting the TCG triplet encoding the first serine amino acid of NS5B with a TGA stop codon triplet. The various NS proteins were cloned singly and in combination by PCR amplification with an appropriate 5' oligonucleotide containing an NcoI restriction site containing an ATG starting codon, and an appropriate 3' oligonucleotide containing a TGA stop codon followed by a NotI restriction site. The resulting fragments were cloned in pCITE-2b vector (Novagen) at the NcoI and NotI sites. All the mutagenized and PCR-amplified fragments used in the cloning steps were verified by sequence before and after their replacement in the final constructs.
In vitro transcription.
ScaI- or BglII-linearized plasmids encoding HCV replicons were transcribed in vitro by T7 RNA polymerase using an Ambion Megascript kit under nuclease-free conditions following the manufacturer's instructions. The reaction was terminated by incubation with DNase I and precipitation with LiCl, according to the manufacturer's instructions. RNA was resuspended in nuclease-free water, quantified by absorbance at 260 nm, immediately frozen in dry ice in 30 µg aliquots and stored at –80 °C.
Transfection of HCV replicon RNA and assays.
Human hepatoma Huh7 and Huh7-derived cell lines were used to test replication of HCV replicon constructs. Confluent cells from 15 cm diameter plates were split 1 : 2. Cells were recovered after 24 h in 5 ml medium, washed twice with 40 ml cold DEPC-treated PBS, filtered with Cell Strainer filters (Falcon) and diluted in cold DEPC-treated PBS at a concentration of 107 cells ml–1. Cell samples (2x106) were subjected to electroporation with the appropriate amount of in vitro transcribed RNA by two pulses at 0·35 kV and 10 µF using a Bio-Rad Genepulser II. Immediately after the electric pulses, the cells were diluted in 8 ml complete DMEM and processed with different protocols depending on the selection/tracer used. When the bla reporter gene was used, the appropriate number of transfected cell suspensions were plated in each well of multiwell-6-wells plates (Falcon) to be stained at different times with the CCF2 substrate system (Aurora Biosciences Corporation) following the manufacturer's instructions, and subsequently photographed using UV light with a digital camera. When quantitative PCR was used to measure transient replication after transfection of the replicon RNA, 105–2x105 cells per well were plated in six-well plates. After 3 days, total RNA was purified as described in the TRIzol protocol (Life Technologies) and 10 out of 100 µl total RNA recovered was used in each TaqMan reaction.
Preparation of cells cured of endogenous replicon.
cl60/591 was obtained by curing a Huh7 cell line clone 60 of HCV replicons using a dihydroxypyrimidine carboxylic acid inhibitor of the NS5B RNA-dependent RNA polymerase (compound 20 of De Francesco & Rice, 2003). Clone 60 cells were cultured for 11 days in the presence of 30 µM inhibitor, and subsequently for 4 days in the absence of inhibitor. The presence of HCV RNA was determined using PCR (TaqMan) at 4, 9, 12 and 15 days. From day 9, the amount of HCV RNA was below the detection limit of the assay. To test further the disappearance of the replicon, 4x106 cells of cl60/591 cells (after 15 days' treatment) were plated in the presence of 1 mg G418 ml–1. After 2 weeks' culture no viable cells were observed, confirming the absence of HCV replicons. Inhibitor-treated cells were stored in liquid nitrogen until they were used for transfection experiments.
TaqMan quantification of HCV, Neo and Bla RNA.
RNAs were quantified by a real-time, 5' exonuclease PCR (TaqMan) assay using specific primer/probe sets. For HCV the set recognized a portion of the HCV 5' untranslated region (nt 130–290) required for efficient RNA replication (Friebe et al., 2001): HCVfor, 5'-CGGGAGAGCCATAGTGG-3'; HCVrev, 5'-AGTACCACAAGGCCTTTCG-3'; HCVprobe, 5'-6-FAM-CTGCGGAACCGGTGAGTACAC-TAMRA-3'. This primer/probe set was originally described and characterized by Takeuchi et al. (1999), and enables detection of as few as 10 copies of HCV RNA. For neomycin: NEO1, 5'-GATGGATTGCACGCAGGTT-3'; NEO5, 5'-cccagtcatagccgaatagcc-3'; NEO-probe, 5'-6-FAM-TCCGGCCGCTTGGGTGGAG-TAMRA-3'. For bla: BLA9FOR, 5'-CGAACTGGATCTCAACAGCG-3'; BLA10REV, 5'-ATAGTGTATGCGGCGACCG-3'; BLA-probe, 5'-6-FAM-CGTATTGACGCCGGCAAGAGC-TAMRA-3'. Human GAPDH mRNA was quantified with a specific primer set (Pre-Developed TaqMan Assay Reagents, Endogenous Control Human GAPDH; PE Applied Biosystems), and used as an endogenous reference. The primers were used at 10 pmol per 50 µl reaction and the probe was used at 5 pmol per 50 µl reaction. All reactions were run in duplicate with Applied Biosystems ABI PRISM 7700 or 7900 under the following conditions: 30 min at 48 °C (RT step), 10 min at 95 °C and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Quantitative calculations were obtained using the Comparative CT method (described in User Bulletin #2, ABI PRISM 7700 Sequence Detection System, Applied Biosystems, December 1997) holding the level of GAPDH mRNA constant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, and identified several adaptive mutations. We concentrated our present work on one adaptive mutation in NS5A: S2204R (referred to below as SR), also reported by Bartenschlager & Lohmann (2001). As described previously (Murray et al., 2003), we substituted the neo reporter gene with the bla reporter gene (Zlokarnik et al., 1998) in various replicons (wt, SR and the polymerase-deficient SR-GAA) and tested them in naive Huh7 cells (Fig. 1). Cells were stained at various times following RNA transfection. In all constructs Bla expression was detected very early, 4 h post-transfection, in which the percentage of positive (blue) cells exceeded 85–90 % (Fig. 1, left column). The identical percentage of blue cells present in the non-replicating GAA mutant experiments suggests that this is the transient expression of bla driven by the HCV IRES of transfected RNA, indicating transfection efficiency. In agreement with results described previously (Murray et al., 2003), at 72 h post-transfection blue positive cells were present only in Bla-SR experiments (approx. 10 %) (Fig. 1, central column). Blue cells indicate the active amplification of the HCV replicon. Consistently, on treatment with IFN-2 the blue staining disappears. In agreement with the extremely low frequency of stable clones emerging from Neo-wt transfected replicon, a very small number of blue cells (one to five in 2x106 transfected cells) were observed in the Bla-wt transfection. We also transfected a subpopulation of Huh7 with increased permissiveness for the HCV replicon (Blight et al., 2002; Murray et al., 2003). This cell line, called cl60/591, was obtained by curing an HCV-replicon stable clone 60 (from which the mutation SR was derived) as described under Methods. Transfection of Bla-SR in the cl60/591 subpopulation resulted in an appreciable increase (up to 30–40 % versus 10 % of naive Huh7) of the percentage of HCV-positive blue cells at 72 h (Fig. 1, right column). No increase in replication efficiency was shown by the Bla-wt replicon. This enhanced Huh7 subpopulation was used throughout the rest of the experimental work.
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, top row) independently from cotransfected RNA. Surprisingly, Bla-SR was strongly inhibited by cotransfection with a Neo-wt replicon (Fig. 2, bottom row). To gain more information about this effect, we quantified Bla-SR replicon RNA by TaqMan in a cotransfection experiment with a wider panel of Neo RNA replicons. Cotransfection of Bla-SR with total Huh7 RNA, showing clear replication, was taken as the 100 % reference point. Replication drops to about 90 % in cotransfection with Neo-SR-GAA, to about 50 % with Neo-SR, and to less than 10 % with Neo-wt or Neo-wt-GAA (Fig. 3). TaqMan analysis confirmed the results observed with Bla staining. These reductions probably represent competition with the transient IRES function in the case of replication-deficient Neo-SR-GAA, and also with the host factors required for HCV replication in the case of Neo-SR. The decrease is much greater in cotransfection with Neo-wt or Neo-wt-GAA. The extent of the resulting signal is similar to that observed in cells transfected with Bla-SR-GAA or on treatment with IFN-2b of Bla-SR-transfected cells (data not shown). In cotransfection experiments with wt replicons, it appears that a specific mechanism is responsible for the shut-off of Bla-SR replication, and this mechanism appears independent (as shown by the Neo-wt-GAA) of any minimal, undetectable level of replication of Neo-wt. This suggests that translation of wt viral proteins is sufficient to stop Bla-SR replication. A concentration range of Neo-wt-GAA was cotransfected with 6 µg Bla-SR. We determined that to obtain 50 % inhibition of Bla-SR, about 1·5 µg of Neo-wt-GAA is sufficient, fourfold less than the adapted replicon (Fig. 4). We defined this behaviour as a dominant negative effect of the wt over the adapted replicon.
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, the replicon containing wt NS5A and the stop codon precluding NS5B translation retained their ability to inhibit the adapted replicon when linearized with ScaI or BglII, essentially to the same extent as the wt replicon. In contrast, the adapted SR replicons, with the stop codon as ScaI or BglII transcripts, essentially had no influence. These results suggest that neither NS5B nor the untranslated 3' HCV region is necessary to achieve inhibition. At this point we cloned the sequences of wt and SR-adapted NS5A alone in a pCITE vector, using the EMCV IRES to drive translation in a manner similar to an HCV replicon with NS proteins. Transcribed RNAs were transfected at a 6 : 1 ratio with Bla-SR to determine whether wt NS5A was sufficient to achieve inhibition. Surprisingly, no dominant negative effect was observed at 72 h or more, even with a very large excess of pCITE/wt5A (up to 20 : 1; data not shown). We also cotransfected a DNA plasmid in which EMCV-IRES-NS5A was driven by the strong cytomegalovirus promoter with the Bla-SR RNA, without obtaining any dominant effect (data not shown). In an attempt to unravel this issue, we hypothesized that wt NS5A might require the presence of other viral protein(s). For this reason we cloned in pCITE the coding sequences of NS3/4A and NS4B, singly and in combination with wt or adapted NS5A (NS3/4A, NS4B, NS3/4A/4B, NS4B/5A and NS3/4A/4B/5A). On cotransfection of the pCITE construct transcripts singly and in combination (Fig. 6a) with Bla-SR, we examined transfected cells at 72 h for Bla activity (not shown) and by TaqMan analysis. A selection of the most significant cotransfections is shown in Fig. 6(b) as TaqMan analyses. Both assays showed clearly that the only pCITE transcript successful in inhibiting the adapted replicon was that in which NS3/4A/4B/5A was expressed as a continuous polyprotein, consistently bearing a wt NS5A sequence. The effect of pCITE/NS3-5A/wt at 1 : 5 ratio shown in this experiment was not as strong as with the wt replicon, but increasing amounts of this competitor RNA augmented the negative effect (data not shown), which we believe was due to the reduced stability of these shorter transcripts. None of the other RNA transcripts had a similar effect.
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described an adaptive mutation in NS5B: 2884 Arg to Gly. This was outlined originally as a weaker adaptive mutation than those in NS5A, alone or in combination with NS3 mutations. Nevertheless, the replication efficiency of this adapted replicon was well above that of the wt replicon. A bla version of the HCV replicon containing the NS5B mutation R2884G has been constructed and compared with other NS5A-adapted replicons (Murray et al., 2003). The replicon containing NS5B R2884G expresses a wt form of NS5A, and therefore its behaviour in our cotransfection experiments was of great interest. We therefore used a bla version of the HCV replicon containing the NS5B mutation R2884G (named Bla-RG) and tested it in our experimental strategy to address the following specific issues. In the first experiment we determined whether the Bla-RG replicon was inhibited by wt replicon. Bla-SR and Bla-RG results are shown in Fig. 7 (left column) 72 h post-transfection in cured cells, 6 µg each plus 30 µg of total RNA or Neo-wt-GAA. As shown in Fig. 7 (right column) Bla-RG replicates less efficiently than Bla-SR but, while Bla-SR is clearly inhibited by the Neo-wt-GAA replicon, Bla-RG is mostly unaffected. TaqMan analysis of bla replicon RNA confirmed this result (data not shown). Next we investigated the effect of NS5B-RG on NS5A-SR adapted replicons and, for the trans-complementation studies, the effect of NS5B-RG on wt replicon. In this case we measured the Neo-replicons (Neo-SR and Neo-wt) cotransfected with total RNA, Bla-SR, Bla-RG or Bla-wt (TaqMan). As shown in Fig. 8, Neo-SR is inhibited about 50 % by Bla-SR and more than 90 % by both Bla-wt and Bla-RG (Neo-SR plus total RNA was taken as 100 %). These results mirror those obtained with Neo-replicons as competitors, confirming that the dominant negative effect is indeed reporter-independent. Not surprisingly, Bla-RG has a strong dominant negative effect on Neo-SR, as strong as the Bla-wt replicon, due to its wt NS5A content. No positive effect has been detected on Neo-wt replicon by cotransfection of any of the bla replicons.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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This effect can be explained by at least two hypotheses. The first could explain this phenotype through the existence of protein–protein interactions, such as between NS5A (probably as an NS3–NS5A complex) and NS5B, in the replication complex. This hypothesis suggests that in cell lines wt NS5A is not able to establish replication-competent contacts with NS5B in the same replicon, but also that wt NS5A (again as an NS3–NS5A complex) could interact with an NS5B encoded by another replicon blocking its function. If this exchange of proteins or complexes is possible as postulated, we would expect to find that defective replicons could replicate in trans-complementation assays. So far all attempts at achieving this have failed in our hands (data not shown). However, non-replication-competent NS5A mutants were trans replicated in the case of bovine viral diarrhoea virus (Grassmann et al., 2001). It cannot be excluded, however, that one or more cellular factors directly or indirectly participate in the HCV replication complex, for example bridging NS3–NS5A complex with NS5B. The varying expression of this factor(s) between human liver and Huh7 could be compensated by the emergence of adaptive mutations. The second and more appealing hypothesis is that wt NS5A stimulates a cellular antiviral response that consequently inhibits HCV subgenomic replication. Adaptive mutations in the replicon proteins fail to trigger such a response, thus allowing HCV replication. In any case, the dominant negative effect suggests that this response should be effective not only in the replicon containing a wt NS5A, but also on any other replicon present in the cell at that moment.
We do not know the nature of this antiviral response, but the R2884G mutation behaviour in NS5B is somewhat peculiar in this scenario. Our results suggest that the R2884G replicon is not susceptible to the inhibitory action of wt NS5A from the wt replicon. On the contrary, it expresses a wt version of NS5A that is able to inhibit replication of a cotransfected NS5A-adapted replicon. Taking these results together, one might presume that NS5B (and Arg 2884 in particular) represents the target of the direct (protein–protein interaction) or indirect (cellular response) action of wt NS5A. It is intriguing to note that our efforts towards combining NS5A and NS5B (R2884G) adaptive mutations, as tried by Lohmann et al. (2001), resulted in a replicon incompetent for replication. It is also interesting to note that NS5A was identified as the key player in the anti-IFN response at the early stage of HCV research (reviewed by Tan & Katze, 2001). It is very tempting to speculate on this issue in the light of the dominant negative effect, but further investigation of the role of NS5A is required to establish a firm relationship between the two phenomena.
On the basis of the above considerations, two classes of adaptive mutation can be envisaged. A first class is represented by the adaptive mutations in NS5A (potentiated or not by mutations in NS3: we show here that it is the NS3–NS5A complex that exerts the dominant negative effect), which either fail to induce cellular antiviral response, or give rise to an active replication complex with NS5B. A second class is in NS5B (so far only one mutant has been described) which, in contrast, is resistant to the antiviral response possibly elicited by wt NS5A or, following the other hypothesis, which forms an active replication complex with wt NS5A. A number of reports have suggested various types of interaction between NS5A and cellular proteins, as well as specific interactions between NS5B and other NS proteins, including NS5A (Chung et al., 2000; Ghosh et al., 2000; He et al., 2002; Lan et al., 2002; Lin et al., 1997; Majumder et al., 2001; Tan et al., 1999; Tu et al., 1999). The two-hybrid system or immunoprecipitations are the most widely used experimental systems, with the majority of cases using the isolated NS5A polypeptide, tagged or not. Regrettably, these observations are not helpful in elucidating the phenomenon described here, because our results strongly suggest that the dominant negative effect occurs only when NS5A is expressed in the context of the NS3–NS5A polyprotein, not alone. Exceptions lie in the reports of the hyper-phosphorylation of NS5A by Koch & Bartenschlager (1999) and Neddermann et al. (1999). The authors found that ‘Hyper-phosphorylation occurs when NS5A is expressed as part of a continuous NS3–NS5A polyprotein, but not when it is expressed on its own or trans complemented with one or several other viral proteins.’ It is worth remarking that the same condition is required to achieve the dominant negative effect. At present we cannot unequivocally link NS5A hyper-phosphorylation with the dominant-negative effect, but it is tempting to speculate that both might result from the same phenomenon. Differences in the post-translational modification of viral and/or cellular proteins (e.g. phosphorylation, ubiquitylation and methylation) might play an important role in the regulation of HCV replication.
In conclusion, this study has shed some initial light on the mechanisms of HCV replicon adaptation, and described the dominant negative effect of the wt form of NS5A. A thorough understanding of this phenomenon will be of great advantage in elucidating HCV biology and developing novel therapeutic strategies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 2 February 2004;
accepted 25 March 2004.
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