Replication studies using genotype 1a subgenomic hepatitis c virus replicons

These subgenomic RNAs replicate persistently and efficiently in Huh-7 cells. Our attempts to introduce in vitro-transcribed replicon RNA into nonhepatic cell types were unsuccessful. We tried to introduce Huhadapted replicons into human embryonic kidney cells through transfection of in vitro-synthesized RNA by electroporation or by using multiple cation-based transfection reagents; however, no neomycin-resistant cell colonies with the replicon were obtained.

A possible interpretation of this result is that the cells were nonpermissive for HCV replicons because either i the cells express a dominant activity or factor that prevents replication or ii the cells lack an essential activity that is necessary for replication To investigate the potential nonpermissiveness of non-Huh-7 cells, we examined heterokaryon formation between replicon-containing Huh-7 cells clone S Successful formation of heterokaryons between and S Alternatively, failure to generate heterokaryons would be consistent with the hypothesis that cells express a dominant inhibitor of HCV RNA replication.

We used cells that have a stably integrated chromosomal copy of the zeocin resistance gene Zeo. As controls, we also constructed a Huh-7 Neo cell line, which has the neomycin resistance gene stably integrated, and a Huh-7 Zeo cell line, which has the zeocin resistance gene stably integrated. PEG-mediated cellular fusions between S In parallel, control fusions were performed with combinations of control cell lines that have chromosomally integrated markers Fig.

The control fusion of Huh-7 Neo with Huh-7 Zeo homologous cell lines with different chromosomally integrated resistance markers resulted in no colony formation without the addition of PEG and in colonies for the PEG-treated sample Fig. Hence, Huh-7 cells and cells, with appropriate markers, could be used to select for potential heterokaryons that harbor markers from both cells, and the addition of PEG increased the efficiency of colony formation.

In order to examine the proficiency for such experiments with neomycin-resistant Huh-7 cells S Throughout these experiments, we obtained significant variations in colony formation up to fivefold between repeated experiment sets; however, the relative efficiency of colony formation among the samples was consistent with the experiment presented in Fig.

Neomycin- and zeocin-resistant colony formation with Huh and derived cell lines. A Schematic of somatic cell fusion between replicon-containing Neo r S The generation of heterokaryons from S B Cellular fusions performed with Huh-7 Neo and Huh-7 Zeo , two distinct Huhderived cell lines with chromosomally integrated markers i ; Huh-7 Neo and Zeo cells, each with the respective chromosomally integrated marker ii ; S Cells were then transferred to mm-diameter plates and selection was done with neomycin and zeocin for 21 days.

Colonies obtained were stained with crystal violet and counted numbers in lower right corners. Invariably, PEG-enhanced fusion between S However, coculturing of the two cell lines without PEG treatment, followed by neomycin and zeocin selection, produced rare resistant colonies. In the first trial, we obtained three clones clones 5, 6, and 7. Upon repeating the coculturing experiment four times, we obtained four other clones clones 15, 16, 17, and The result presented Fig. Therefore, the frequency of obtaining doubly antibiotic-resistant colonies was low and independent of PEG-enhanced fusion.

The neomycin- and zeocin-resistant colonies, termed Rep cells, were expanded into cell lines. In order to assess whether Rep cells contain the HCV subgenomic replicon acquired through the coculturing process with S Two representative cell lines resulting from coculturing experiments, Rep5 and Rep17, respectively, were chosen for the metabolic labeling experiment.

In addition, parental Huh-7 and cells and our original replicon-containing S Total cellular RNA was extracted from cells treated with 33 P and actinomycin D for 16 h and was resolved in a 0. B Northern blots detecting the negative strand of HCV subgenomic replicons. Total cellular RNA was extracted and resolved in a glyoxal-dimethyl sulfoxide agarose gel. The first three lanes are positive controls of in vitro-synthesized negative strand RNA, with 10 8 , 10 7 , and 10 6 copies used, as indicated.

Further confirmation that Rep cells harbor the subgenomic replicon was obtained from a Northern blot specifically probing for negative-strand RNA. Hence, Rep cells contained the subgenomic negative-strand replicative intermediate of the replicon RNA.

Based on the intensities of standards loaded in lanes 1 to 3 Fig. Polyclonal antibodies specific for albumin or AFP, or monoclonal antibodies against E1A, showed that Huh-7 and its derived cell lines S In contrast, cells, as well as the Rep cells, expressed the E1A marker but lacked expression of Huh-7 cell protein markers albumin and AFP.

Western blots examining HCV protein expression were performed with a polyclonal antibody against NS3 protein or a monoclonal antibody against NS5A protein. A Western blot analysis of Rep cells. Equivalent concentrations of total cellular proteins, obtained from cell lysates and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were immunoblotted with either anti-E1A monoclonal antibody or anti-AFP polyclonal antibody.

The anti-AFP blot was stripped and then reblotted with anti-albumin polyclonal antibody. B Western blot analysis of proteins from Rep cells.

Total cellular proteins, prepared as for panel A, were immunoblotted with either anti-NS3 polyclonal antibody or anti-NS5A monoclonal antibody. Huh-7, S Moreover, and Rep cells lacked the distinctive lipid droplets that are readily detectable in Huh-7 and S Detection of HCV proteins in Rep cells by immunofluorescence. Fixed and permeabilized cells were incubated with either a polyclonal antibody against the NS3 protein A or a monoclonal antibody against the NS5A protein B and appropriate secondary antibodies.

Arrows define the characteristic cytoplasmic lipid droplets of Huh-7 cells. PI staining of parental and Huh-7 cells Fig. PI staining of Rep5 cells Fig. In contrast, PI staining of Rep17 cells Fig.

Huh-7, , S In order to determine whether the Rep cell replicons were uniquely adapted for replication in cells, we isolated total cellular RNA from Rep5, Rep17, and S Transfection of total RNA from S Similarly, transfection of total RNA from Rep5 cells also failed to establish colonies. The sequences were compared to those of the replicons of S Most of the mutations resulting in amino acid substitutions were within the NS5A region.

We also sequenced four replicon clones derived from cells that were transfected with early-passage Rep17 total RNA and only identified minor deviations i. These predominantly silent mutations encoding no more than 2 amino acid substitutions were unique to each clone and were not found in other replicons. Nucleotide and amino acid mutations found in Rep replicons relative to the Con1 replicon sequence.

Thus, the cloned and in vitro-transcribed Rep17 RNA, though active in Huh-7 cells, failed to establish colonies and differed in functionality from the RNA directly isolated from Rep17 cells.

We characterized some of these cell lines to confirm the presence of replicon RNA and the expression of viral proteins. Metabolic labeling with 33 P in the presence of actinomycin D was performed with Huh-7, , S Analysis of cell lines generated by transfection of total RNA from Rep17 cells. Western blot analysis performed as described for Fig. In a preliminary effort, we examined the characteristics of these replicon-containing cells in response to various inhibitors and compared them to Huh-7 replicon cells.

Cells were incubated for 3 days in the presence of protease inhibitor 4C concentrations ranging from 0. Huh-7 and cells were also included as controls. Metabolically labeled RNAs indicated that treatment of S Inhibition of subgenomic RNA replicon in cells. Protease inhibitor was tested at 0, 0. Total cellular RNA was extracted and the replicon copy number was quantified by Taq -Man real-time RT-PCR to obtain the percent inhibition of treated samples with respect to untreated control levels.

Circles, S C Inhibition of replicon in cells as described for panel B, but cultured in the presence of different doses 0. The plotted percentages of inhibition represent average values with error bars from at least three determinants. We next examined the effect of treating replicon-containing cells with biological reagents that induce antiviral responses. Treatment of S We also assessed the response of replicon-containing cells to treatment with the double-stranded RNA mimic polyinosine-poly C [poly I -poly C ] This agent is proposed to stimulate the activation of cellular antiviral responses through the Toll-like receptor 3 TLR-3 18 , although the detailed mechanism of activation and the upstream link to TLR-3 remain unknown.

We treated the replicon-containing S Treatment of the S In contrast, the replicon-containing Rep17 and LR1 cell lines responded effectively even to relatively low doses of poly I -poly C treatment.

In an attempt to examine and extend the tropism of HCV replicons, we conducted a series of somatic cell fusion experiments between replicon-containing human hepatoma Huh-7 cells S From the fusion experiments, we determined that homologous fusion of Huh-7 cells as well as heterologous fusion of Huh-7 and cells could be achieved through selection for two antibiotic resistance markers.

The selection of heterokaryons from S The higher efficiency may reflect a facilitated fusion process between a nuclear marker and a cytoplasmic RNA marker relative to the conventional cell fusion that involves two nuclear DNA markers. Alternatively, as subgenomic replicon RNA can be produced and released into the extracellular medium by Huh-7 cells in ill-defined RNase-resistant particles 30 , the zeocin-resistant cells may have acquired the replicon independently of direct cell fusion.

In contrast to the successful fusion between Huh-7 cells and cells, fusion of S One interpretation from these results is that cells restrict replication of the Huh-7 cell-adapted replicons and that the restrictive phenotype of cells is dominant over the permissive phenotype of Huh-7 cells.

This phenotype of cells may be due to the expression of an inhibitor that is absent or differentially expressed in Huh-7 cells. Due to the fact that we observed a difference between and Huh-7 cells in responding to treatment with activators of antiviral responses discussed below , we suspect that cells may encode a replicon inhibitor that is less active in or absent from Huh-7 cells.

Whether a common inhibitory activity accounts for restriction in and some Huh-7 cells is unknown, and the components of a potential repressive pathway s and their role in HCV subgenomic replication remain to be determined. The nonpermissive phenotype of cells for Huh-7 cell-adapted replicons was suggested from the repeated attempts that failed to produce cell fusions between Zeo and S When replicon-containing S We suspect that a PEG-independent fusion occurred between the two cell types or that Zeo cells acquired an extracellular form of the RNA marker produced by S The analysis revealed that Rep5 cells contain more than the normal diploid, but less than tetraploid, DNA content observed for either parental or Huh-7 cells.

This suggests that Rep5 cells originated from a fusion of the two cocultured parental cell types and then lost chromosomes but maintained the replicon. Hence, either of the two proposed methods of replicon acquisition could apply for Rep17 cells.

Both Rep5 and Rep17 cells expressed the E1A protein, a marker for the adenovirus Ad5 immortalized cells 20 , and lacked expression of the albumin and AFP protein markers of Huh-7 cells; the latter marker may not represent an ideal marker for Huh-7 cells, since its expression is commonly lost in somatic cell hybrids Replicon RNA was detected in these cells by metabolic labeling with 33 P and by Northern blots that confirmed the presence of the negative-strand replicative intermediate.

Our data supported the proposal that Rep17 has a cell background and provided us with the means to isolate replicon HCV RNA that may have adapted to cells. The consensus sequence of Rep17 replicon RNA differs from that of Rep5 replicon RNA by 41 nucleotides that encode five amino acid substitutions within the nonstructural region, some of which may represent adaptive mutations required for replication in cells.

We currently have not identified the cause of this difference, and a similar discrepancy was also noted by Zhu and colleagues 39 in replicons isolated and cloned from HeLa cells and a murine liver cell line. Alternatively, unidentified accessory RNA molecules, coisolated with the replicon from Rep17 cells, may play a role in RNA replication.

A third possibility is that the replicon isolated directly from Rep17 cells has epigenetic modifications lacking in vitro-transcribed RNA that are essential.

These possibilities are currently being explored. Preliminary characterization of the replicon-containing cells revealed differences relative to Huh-7 cells. There was a substantial reduction of HCV replicon RNA levels in cells following poly I -poly C treatment, whereas we observed only a limited effect in Huh-7 cells, as previously reported This difference between the two cell types may be due to differences in the expression of a receptor for poly I -poly C , such as TLR-3, or a downstream signaling component 10 , such as IRF-3, which is proposed to regulate subgenomic RNA replication These cells can be used to investigate specific cellular inducers or effectors of HCV RNA replication beyond Huh-7 cells and to expand the available cell culture models to investigate the biology of HCV.

We are grateful to Michael Cordingley for his support and encouragement of this work. National Center for Biotechnology Information , U. Journal List J Virol v. J Virol. Author information Article notes Copyright and License information Disclaimer.

Phone: Fax: E-mail: moc. Received Jul 31; Accepted Sep This article has been cited by other articles in PMC. Abstract Hepatitis C virus HCV infects liver cells and its replication in other cells is incompletely defined.

PEG fusion. Northern blot analysis. Western blot analysis. RNA transfection of cells. Open in a separate window. Genetic content of Rep17 cells. TABLE 1. Protein Mutation Presence of mutation in replicon a nt aa S B Northern blot analysis of total RNA extracted from chimeric type 1a replicon cells. The blot was probed with in vitro-transcribed replicon RNA. C and D Western blot analysis of NS protein expression in chimeric type 1a replicon cells.

Section A shows NS3 staining for parental Huh7 cells. Although qualitative, the relative fluorescence signals were lower than those from wild-type 1b replicon cells data not shown.

Although type 1b replicon cells can have variability in copy number between systems 2 , 13 , most type 1b clones reported to date generally appear capable of supporting higher levels of viral RNA than the type 1a replicon cells reported herein. Furthermore, this difference cannot be attributed to the use of different parental Huh7 sources since the clone 1 cells were generated by using the same parental cell line as that used for clone A cells.

An additional stable clonal replicon, the type 1b replicon expressing type 1a NS5B strain H77 , was also generated clone 2 cells from pBB71a5B. No phenotypic differences between clone 1 or clone 2 cell lines were apparent data not shown. Similar to those in clone A cells, viral RNA levels in clone 1 and clone 2 cells decrease severalfold upon cellular confluence, suggesting similar relationships with respect to cell density or cell cycling parameters in the control of viral RNA replication data not shown.

However, the ratios of positive-strand RNA to negative-strand RNA for all replicons were similar, 26 for clone 1 cells and 39 for clone A cells, and were consistent with the reduced efficiency of replication for type 1a. Adaptive mutations in replicons are thought to be required for efficient type 1b replicon replication 2 , 11 , 12 , although the HCV-N subgenomic replicon contained no such mutations 9.

The inability to select for replication of type 1a sequences in the absence of an adaptive mutation pH77 and pHb75 suggests that type 1a replicon sequences may be less fit to establish or support replication in the subgenomic context. All nine plasmid clones encoding the EV change also encoded the VI mutation.

Last, two silent mutations within the NS5B coding sequence, a C to A at nt and a G-to-A conversion at nt starting from the NS3 coding sequence , were present in all plasmid clones.

No additional changes were found across the entire 1b nonstructural coding sequence. Thus, conserved mutations indeed occurred and may have directly contributed to the stability of replication, although it was unclear which mutation or combination of mutations was responsible for augmenting RNA translation or replication. Furthermore, testing with clone 2 cells showed an IC 50 of 7. IFN response curves for type 1b replicon clone A and chimeric type 1a cells clone 1 and clone 2 cells.

RNA levels were quantified by Taqman analysis, and data represent averages from four experiments. In this report, it is shown that chimeric subgenomic replicons between HCV type 1a strain H77 and type 1b strain Con1 sequences are competent to replicate in Huh7 cells, albeit with reduced colony formation efficiency and viral RNA levels.

Similar to what was found for type 1b replicons 2 , 12 , mutations were identified in the 1a replicon sequences upon replication in Huh7 cells. Our results extended the studies on two type 1b isolates, namely, HCV-N 9 and HCV Con1 13 , and indicate that sequences beyond those of the two reported clinical isolates can support replication in cell culture.

However, the interaction of nonstructural genes and viral NTRs from different genotypes may be suboptimal, and this may partially account for the lower replication efficiency within these type 1a replicons. The infectivity of three full-length cDNA clones was tested by direct injection of RNA transcripts into the liver of a chimpanzee, and only one clone was found to be infectious. Our data and those of Guo et al. However, since translation and replication are linked for bovine viral diarrhea virus and likely HCV 6 , we cannot rule out the possibility that there is also a direct effect on replicase activity that contributes to the enhanced ability to select for stable replicons when the type 1b sequence is present.

Similar to what was found for type 1b replicons, conserved mutations occur within type 1a sequences. Interestingly, of the two mutations identified within NS3, both were located in the helicase domain, a preferred site for adaptive change 9 , In particular, the KE mutation was also found to occur in two independent Con 1 replicons This mutation directly enhanced colony-forming efficiency fivefold and conferred additive effects on replication efficiency when combined with other NS3 mutations.

Since this particular amino acid change was observed in several independent laboratories, it will be interesting to test whether KE in NS3 modulates helicase activity in in vitro assays. The other mutation found within NS3, SP, represents a change from the type 1a to the type 1b coding sequence for that position amino acid is proline in Con1. Interestingly, all clones containing the change in NS5A also contained the corresponding mutation in NS5B, indicating a potential interaction between these proteins.

Recently, NS5A was shown to locate in the endoplasmic reticulum membrane through an amphipathic alpha-helix anchor formed by the N-terminal 30 amino acids 3. Since the EV mutation occurred on the hydrophilic side of the amphipathic helix facing the cytoplasm, membrane association of NS5A was not likely affected.

Additional studies to assess this phenotype are ongoing. Although the replicons described in this paper were type 1a-type 1b chimeras, antiviral compounds against type 1a HCV NS5B can now be tested in a cell-based system. Our results support the findings of Blight et al.

Blight, J. McKeating, J. Marcotrigiano, and C. Rice, J. We thank C. Rice and J. We thank J. Guo in Christoph Seeger's laboratory for expert help with Northern blot analysis.

National Center for Biotechnology Information , U. Journal List J Virol v. J Virol. Sarisky 1. Adam T. Robert T. Author information Article notes Copyright and License information Disclaimer. Collegeville Rd.

Phone: Fax: E-mail: ten. Received Dec 19; Accepted Jan This article has been cited by other articles in PMC. Abstract Recently, cell-based replicon systems for hepatitis C virus HCV , in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. Plasmid construction. In vitro transcription. RNA electroporation. Western blot analysis. Northern blotting. Taqman analysis. Negative-strand replicon RNA detection.

Indirect immunofluorescence. Cell-free translation. Open in a separate window. H77 replicon validation. Replication dynamics. TABLE 1. Replicon RNA copy numbers per cell. Adaptation mutations. TABLE 2. Conserved mutations in H77 type 1a chimeric replicons. Changes EV and VI were consistently identified together on the same clone. Ratios of the numbers of clones in which changes were found to the numbers of clones tested are in parentheses.

Drug discovery applications. Acknowledgments We thank C. Alter, H. Recovery, persistence and sequelae in hepatitis C virus infection: a perspective on long-term outcome. The cells were washed three times with TBS for 5 min. Fluorescence was examined with a confocal microscope. Several subgenomic replicons containing sequences from genotype 1a were constructed Fig. Schematic representation of the construction of a chimeric type 1a replicon.

The pHCV1b replicon BB7 structure is shown at the top, with the Con1 type 1b sequence shown in hatched boxes with protein names. Several changes were also introduced into the replicons to facilitate subcloning. Specifically, three restriction sites were created: a Bsr GI site at nucleotide nt , an Eco RI site at nt , and an Mfe I site at nt nucleotide numbering starts from the first codon for NS3. Electroporation of these hybrid replicons into Huh7 cells resulted in enhanced protein synthesis 4 h posttransfection, compared to that with the wild-type 1a replicon RNA control data not shown.

Attempts to select for stable replication of pHb75, in the presence of G, failed to result in colony formation. The translation assay was carried out as described by Grassmann et al. The positions of the major translation products, i. The rate of translation of the NS3 protein was determined by phosphorimage analysis of the protein band from three independent experiments, after normalization for the translation of neomycin.

Lanes are labeled at the top of the gel. C, control lysate, no RNA. Many clonal cells failed to survive the initial cell passage from the cloning cylinder.

Nevertheless, several clonal colonies were successfully amplified with Huh7 cell-conditioned media and once established had a doubling time of around 30 h. Cell viability and replicon RNA copy number remained stable throughout long-term passage. Data are shown for clone 1 cells. That authenticity of clone 1 cells to stably express the HCV replicon was first confirmed by showing the absence of neomycin resistance gene DNA integration within the cellular genome Fig.

Next, the presence of full-length subgenomic RNA was analyzed by Northern blotting. As expected, an 8-kb RNA transcript was identified Fig.

Notably, the abundance of the replicon RNA was considerably lower than that found previously with the type 1b replicon clone A cells. Clone A cells constitute a cloned-replicon cell line generated from pHCVrep1b and have RNA copy numbers similar to those of other reported replicon cells.

Western blot and indirect immunofluorescence assays were utilized to confirm the presence of NS5A and NS5B proteins in total-cell lysates. A single polyprotein product with cross-reactivity to antipeptide sera specific for NS5A was detected; however, the hyperphosphorylation state of this protein was not assessed. Use of antisera against NS5B resulted in the detection of multiple bands on a Western blot, most likely due to protein stability Fig.

The relative levels of NS5A and NS5B proteins were approximately 5- to fold lower in the clone 1 replicon cells than in clone A cells, and this may account for the differences in replication efficiency and replicon RNA copy number among cell lines. The antisera reacting with purified type 1a and type 1b proteins had similar efficiencies.

Replication of a chimeric H77 type 1a subgenomic replicon in Huh7 cells. Genomic DNA was isolated from chimeric type 1a replicon cells lanes 1 and 3 , parental Huh7 cells lanes 2 and 4 , and a Gresistant cell line lane 5 and was used in PCRs with primer pairs that amplify a bp neoR fragment lanes 1, 2, and 5 or primer pairs that amplify a bp GAPDH glyceraldehydephosphate dehydrogenase gene fragment lanes 3 and 4.

B Northern blot analysis of total RNA extracted from chimeric type 1a replicon cells. The blot was probed with in vitro-transcribed replicon RNA. C and D Western blot analysis of NS protein expression in chimeric type 1a replicon cells. Section A shows NS3 staining for parental Huh7 cells. Although qualitative, the relative fluorescence signals were lower than those from wild-type 1b replicon cells data not shown. Although type 1b replicon cells can have variability in copy number between systems 2 , 13 , most type 1b clones reported to date generally appear capable of supporting higher levels of viral RNA than the type 1a replicon cells reported herein.

Furthermore, this difference cannot be attributed to the use of different parental Huh7 sources since the clone 1 cells were generated by using the same parental cell line as that used for clone A cells. An additional stable clonal replicon, the type 1b replicon expressing type 1a NS5B strain H77 , was also generated clone 2 cells from pBB71a5B.

No phenotypic differences between clone 1 or clone 2 cell lines were apparent data not shown. Similar to those in clone A cells, viral RNA levels in clone 1 and clone 2 cells decrease severalfold upon cellular confluence, suggesting similar relationships with respect to cell density or cell cycling parameters in the control of viral RNA replication data not shown.

However, the ratios of positive-strand RNA to negative-strand RNA for all replicons were similar, 26 for clone 1 cells and 39 for clone A cells, and were consistent with the reduced efficiency of replication for type 1a. Adaptive mutations in replicons are thought to be required for efficient type 1b replicon replication 2 , 11 , 12 , although the HCV-N subgenomic replicon contained no such mutations 9.

The inability to select for replication of type 1a sequences in the absence of an adaptive mutation pH77 and pHb75 suggests that type 1a replicon sequences may be less fit to establish or support replication in the subgenomic context. All nine plasmid clones encoding the EV change also encoded the VI mutation.

Last, two silent mutations within the NS5B coding sequence, a C to A at nt and a G-to-A conversion at nt starting from the NS3 coding sequence , were present in all plasmid clones. No additional changes were found across the entire 1b nonstructural coding sequence.

Thus, conserved mutations indeed occurred and may have directly contributed to the stability of replication, although it was unclear which mutation or combination of mutations was responsible for augmenting RNA translation or replication. Furthermore, testing with clone 2 cells showed an IC 50 of 7.

IFN response curves for type 1b replicon clone A and chimeric type 1a cells clone 1 and clone 2 cells. RNA levels were quantified by Taqman analysis, and data represent averages from four experiments. In this report, it is shown that chimeric subgenomic replicons between HCV type 1a strain H77 and type 1b strain Con1 sequences are competent to replicate in Huh7 cells, albeit with reduced colony formation efficiency and viral RNA levels.

Similar to what was found for type 1b replicons 2 , 12 , mutations were identified in the 1a replicon sequences upon replication in Huh7 cells. Our results extended the studies on two type 1b isolates, namely, HCV-N 9 and HCV Con1 13 , and indicate that sequences beyond those of the two reported clinical isolates can support replication in cell culture. However, the interaction of nonstructural genes and viral NTRs from different genotypes may be suboptimal, and this may partially account for the lower replication efficiency within these type 1a replicons.

The infectivity of three full-length cDNA clones was tested by direct injection of RNA transcripts into the liver of a chimpanzee, and only one clone was found to be infectious. Our data and those of Guo et al. However, since translation and replication are linked for bovine viral diarrhea virus and likely HCV 6 , we cannot rule out the possibility that there is also a direct effect on replicase activity that contributes to the enhanced ability to select for stable replicons when the type 1b sequence is present.

Similar to what was found for type 1b replicons, conserved mutations occur within type 1a sequences. Interestingly, of the two mutations identified within NS3, both were located in the helicase domain, a preferred site for adaptive change 9 , In particular, the KE mutation was also found to occur in two independent Con 1 replicons This mutation directly enhanced colony-forming efficiency fivefold and conferred additive effects on replication efficiency when combined with other NS3 mutations.

Since this particular amino acid change was observed in several independent laboratories, it will be interesting to test whether KE in NS3 modulates helicase activity in in vitro assays. The other mutation found within NS3, SP, represents a change from the type 1a to the type 1b coding sequence for that position amino acid is proline in Con1. Interestingly, all clones containing the change in NS5A also contained the corresponding mutation in NS5B, indicating a potential interaction between these proteins.

Recently, NS5A was shown to locate in the endoplasmic reticulum membrane through an amphipathic alpha-helix anchor formed by the N-terminal 30 amino acids 3. Since the EV mutation occurred on the hydrophilic side of the amphipathic helix facing the cytoplasm, membrane association of NS5A was not likely affected.

Additional studies to assess this phenotype are ongoing. Although the replicons described in this paper were type 1a-type 1b chimeras, antiviral compounds against type 1a HCV NS5B can now be tested in a cell-based system.

Our results support the findings of Blight et al. Blight, J. McKeating, J. Marcotrigiano, and C. Rice, J. We thank C. Rice and J. We thank J. Guo in Christoph Seeger's laboratory for expert help with Northern blot analysis.

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Isken O ,. Behrens SE ,. Sarisky RT. Affiliations 1 author 1. Share this article Share with email Share with twitter Share with linkedin Share with facebook. Abstract Recently, cell-based replicon systems for hepatitis C virus HCV , in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. Evidence is provided herein to confirm that this differential response to IFN-alpha may be attributed directly to the type 1a polymerase.