MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
Correspondence
Paul Targett-Adams
p.targett-adams{at}vir.gla.ac.uk
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ABSTRACT |
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Quantitative RT-PCR results are available as supplementary material in JGV Online.
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MAIN TEXT |
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RNAs transcribed from pSGR-Luc-JFH1 and pSGR-Luc-JFH1/GND were electroporated into Huh-7 cells and luciferase activity within cell extracts was assayed 4, 24, 48 and 72 h later. Genotype 1b subgenomic replicons containing the firefly luciferase gene were used for comparative analysis (Fig. 1a; Krieger et al., 2001
; Friebe et al., 2001
; Lohmann et al., 2003
). Luciferase activities for the wild-type (wt) and GND mutants of both genotype 1b and 2a replicons were comparable at 4 h and taken to represent translation of input RNA. The pattern of luciferase activity displayed by the genotype 1b replicons beyond 4 h was similar to previously published data (Fig. 1b
; Krieger et al., 2001
; Lohmann et al., 2003
). For the wt genotype 1b replicons, luciferase activity gradually decreased after the initial time point, but stabilized by 72 h. By comparison, activity displayed by the genotype 1b subgenomic replicon encoding the GND mutation in the NS5B sequence decreased sharply between 4 and 24 h and was undetectable by 72 h (Fig. 1b
). In contrast to the pattern shown by the wt genotype 1b replicons, luciferase levels generated by the JFH1 replicon rose steadily from 4 h and, by 72 h, were approximately 10-fold higher than initial values (Fig. 1b
). From 48 h onwards, we consistently observed that luciferase activity from the JFH1 replicon was about 200- to 400-fold higher than that exhibited by either the wt genotype 1b replicon or the improved version containing the poliovirus IRES (Fig. 1b
). Indeed, the GND mutant of the JFH1 subgenomic replicon frequently gave enzyme levels that were comparable to those for the wt genotype 1b replicons. However, by day 7 after electroporation, luciferase activity was reduced to background levels (data not shown). Therefore, we did not consider that the comparable levels in activity between the GND mutant of JFH1 and the wt genotype 1b replicons arose from replication of the JFH1 mutant. It is possible that either greater stability of JFH1 RNA in cells or differences in the translational efficiencies of the JFH1 and Con-1 5'NTR elements could contribute to the luciferase activity obtained with the GND mutant for JFH1. Either of these possibilities could combine with other, as yet unknown, characteristics of the wt JFH1 replicon to account for its greater replication efficiency over the genotype 1b replicon. In a previous report, different passages of Huh-7 cells from the same stock gave fluctuations in replication efficiency in short-term assays for the genotype 1b replicons (Lohmann et al., 2003
). With the JFH1 replicon, the absolute levels of luciferase activity were consistent over several cell passages. Therefore, passage number did not appear to influence the efficiency of RNA synthesis from the JFH1 replicon in the same manner as for genotype 1b replicons. We also found that enzyme levels from pSGR-Luc-JFH1 were not enhanced using 2/1C, a Huh-7 cell line that had been cured of the JFH1 subgenomic replicon by IFN-
treatment (data not shown).
To determine whether there was a correlation between luciferase activity and viral protein synthesis, the production of NS5A was examined. Western blot analysis revealed that NS5A could be identified in extracts from cells electroporated with pSGR-Luc-JFH1 RNA at 24 h, and the abundance of the protein had increased by 48 and 72 h (Fig. 1c). By contrast, NS5A was either difficult to detect or undetectable with both of the wt genotype 1b subgenomic replicons and the GND mutants. From indirect immunofluorescence studies, intracellular NS5A protein could be detected up to 24 h post-electroporation for the wt genotype 1b and JFH1 subgenomic replicons, but not at later times for the genotype 1b replicons (Fig. 1d
and data not shown). By contrast, high levels of NS5A were present in 8090 % of cells by 72 h for the JFH1 replicon (Fig. 1d, viii
). The levels of luciferase activity generated by the JFH1 replicon were also reflected in an accumulation of intracellular replicon RNA, as judged by quantitative RT-PCR (see Supplementary Fig. S1, available in JGV Online). Taken together, our data indicate that the high levels of luciferase activity generated by the JFH1 subgenomic replicon in this transient-replication assay represented efficient RNA replication in Huh-7 cells in the absence of any selective pressure.
Having established a short-term replication assay using the JFH1 replicon, we sought to determine whether the amount of RNA used to electroporate cells influenced the levels of replication (Fig. 2a). Huh-7 cells were electroporated with 1, 2·5, 5 or 10 µg pSGR-Luc-JFH1 RNA and the luciferase activity contained within cell extracts was monitored over 72 h (Fig. 2a
). By 4 h, luciferase activities were enhanced as the amount of RNA introduced into cells was increased. However, the enzyme levels reached at the end of the experiment were similar, irrespective of the amount of input RNA. Thus, in agreement with Lohmann et al. (2003)
, overall replication efficiency was highest for 1 µg input RNA and decreased as larger amounts of RNA were introduced into cells. These findings, along with those described above, suggest that Huh-7 cells support efficient replication from the JFH1 replicon, but that there is a finite limit to the levels of replication that can be achieved.
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From previous reports, the JFH1 replicon has been shown to replicate in hepatic-derived Huh-7, HepG2 and IMY-N9 cells and non-hepatic HeLa cells (Kato et al., 2003, 2005
; Date et al., 2004
). In these studies, replication was assessed on the ability of the subgenomic JFH1 replicon to form G418-resistant colonies following drug selection of cells for 34 weeks. To determine whether our assay was suitable for screening the ability of different cell types to support JFH1 replication, we used pSGR-Luc-JFH1 to assess transient replication in various cell lines. Cells tested were HeLa (human cervical adenocarcinoma), U2OS (human osteosarcoma), HUVEC (human umbilical vein endothelial), HFF (human foreskin fibroblasts), BHK (baby hamster kidney) and McA-RH7777 (rat hepatoma) (Fig. 3
a and data not shown). In the case of HeLa cells, any replication as determined by luciferase activity was approximately 1000-fold less efficient than in Huh-7 cells (Fig. 3a
). The JFH1 replicon could establish efficient replication in this stock of cells, as determined by selection of G418-resistant colonies (data not shown), although its colony-forming capacity was about 10-fold less in HeLa cells compared with Huh-7 cells (Kato et al., 2005
). The greater reduction in the transient assay may reflect differences in kinetics for RNA synthesis over short compared with long time periods and the selection of cells within the population that can support replication over an extended period. Among the other cell lines tested, evidence for efficient replication was not detected in either the non-human or non-hepatic cells with the exception of U2OS cells (Fig. 3a
and data not shown). The difference in luciferase activities between wt and GND replicons in this cell line was approximately 1000-fold by 48 h, which was roughly equivalent to the differential observed in Huh-7 cells (Fig. 3a
). Although the absolute enzyme levels for the wt JFH1 replicon cells were about 510 % of those produced in Huh-7 cells, our data indicated that U2OS cells represent another cell line in which HCV replication could be examined. To underline the ability of U2OS cells to support replication, G418-resistant colonies could be readily selected 3 weeks after electroporation of cells with pSGR-JFH1 RNA (Fig. 3b
). Previous studies have shown that U2OS cells support both constitutive and inducible expression of HCV proteins (Moradpour et al., 1998
; Egger et al., 2002
). Moreover, the membranous web, which is induced by NS4B expression and represents the site of HCV RNA synthesis, was identified initially in U2OS cells (Egger et al., 2002
; Gosert et al., 2003
). Such similarities to the morphological changes that occur in Huh-7 cells may indicate that U2OS cells are an alternative model to characterize the processes that contribute to HCV RNA replication.
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ACKNOWLEDGEMENTS |
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Received 12 July 2005;
accepted 1 August 2005.
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