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. 2003 May;77(9):5352-9.
doi: 10.1128/jvi.77.9.5352-5359.2003.

Replication studies using genotype 1a subgenomic hepatitis C virus replicons

Baohua Gu  1 , Adam T Gates, Olaf Isken, Sven-Erik Behrens, Robert T Sarisky
Affiliations

Replication studies using genotype 1a subgenomic hepatitis C virus replicons

Baohua Gu et al. J Virol. 2003 May.

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. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-alpha), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-alpha may be attributed directly to the type 1a polymerase.

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Figures

FIG. 1.
FIG. 1.
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. Open boxes, type 1a HCV pCV-H77C sequence. The NS3 gene starting codon, AUG, and the NS5A adaptive mutation S2204I are shown above the genome (arrows). The 5′ NTR, the 3′ NTR, and the EMCV IRES structures are schematically shown.
FIG. 2.
FIG. 2.
Rate of translation of H77 and H771B75 replicon RNAs. The translation assay was carried out as described by Grassmann et al. (6) by using the full-length replicon RNAs and cytoplasmic S10 extracts of Huh7 cells. The positions of the major translation products, i.e., of the neomycin phosphotransferase and of NS3, are indicated. 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.
FIG. 3.
FIG. 3.
Replication of a chimeric H77 type 1a subgenomic replicon in Huh7 cells. (A) PCR analysis of the chimeric type 1a replicon cell genomic DNA. Genomic DNA was isolated from chimeric type 1a replicon cells (lanes 1 and 3), parental Huh7 cells (lanes 2 and 4), and a G418-resistant cell line (lane 5) and was used in PCRs with primer pairs that amplify a 380-bp neoR fragment (lanes 1, 2, and 5) or primer pairs that amplify a 400-bp GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene fragment (lanes 3 and 4). (B) Northern blot analysis of total RNA extracted from chimeric type 1a replicon cells. Total RNA (5 μg) was electrophoresed through denaturing formaldehyde agarose gel. The blot was probed with in vitro-transcribed replicon RNA. Lane 1, in vitro-transcribed chimeric replicon RNA; lane 2, RNA from parental Huh7 cells; lane 3, RNA from chimeric type 1a replicon cells; lane 4, RNA from type 1b replicon cells. (C and D) Western blot analysis of NS protein expression in chimeric type 1a replicon cells. Chimeric type 1a cells (lanes 1), parental Huh7 cells (lanes 2), and type 1b replicon cells (lanes 3) (104 cells each) were lysed and separated by SDS-polyacrylamide gel electrophoresis, and the blot was probed for NS5A and NS5B proteins by using antibodies against NS5A and NS5B peptides. (E) Indirect immunofluorescence detection of NS3 (section B), NS5A (section C), and NS5B (section D) proteins in chimeric type 1a replicons. Section A shows NS3 staining for parental Huh7 cells. Similar backgrounds were seen with NS5A and NS5B antibodies.
FIG. 3.
FIG. 3.
Replication of a chimeric H77 type 1a subgenomic replicon in Huh7 cells. (A) PCR analysis of the chimeric type 1a replicon cell genomic DNA. Genomic DNA was isolated from chimeric type 1a replicon cells (lanes 1 and 3), parental Huh7 cells (lanes 2 and 4), and a G418-resistant cell line (lane 5) and was used in PCRs with primer pairs that amplify a 380-bp neoR fragment (lanes 1, 2, and 5) or primer pairs that amplify a 400-bp GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene fragment (lanes 3 and 4). (B) Northern blot analysis of total RNA extracted from chimeric type 1a replicon cells. Total RNA (5 μg) was electrophoresed through denaturing formaldehyde agarose gel. The blot was probed with in vitro-transcribed replicon RNA. Lane 1, in vitro-transcribed chimeric replicon RNA; lane 2, RNA from parental Huh7 cells; lane 3, RNA from chimeric type 1a replicon cells; lane 4, RNA from type 1b replicon cells. (C and D) Western blot analysis of NS protein expression in chimeric type 1a replicon cells. Chimeric type 1a cells (lanes 1), parental Huh7 cells (lanes 2), and type 1b replicon cells (lanes 3) (104 cells each) were lysed and separated by SDS-polyacrylamide gel electrophoresis, and the blot was probed for NS5A and NS5B proteins by using antibodies against NS5A and NS5B peptides. (E) Indirect immunofluorescence detection of NS3 (section B), NS5A (section C), and NS5B (section D) proteins in chimeric type 1a replicons. Section A shows NS3 staining for parental Huh7 cells. Similar backgrounds were seen with NS5A and NS5B antibodies.
FIG. 4.
FIG. 4.
IFN response curves for type 1b replicon (clone A) and chimeric type 1a cells (clone 1 and clone 2 cells). IFN-α was titrated by using a threefold serial dilution, and cells were treated for 48 h before RNA harvest. RNA levels were quantified by Taqman analysis, and data represent averages from four experiments. y axis, relative response of viral RNA normalized to cellular RNA.
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