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. 2001 May;75(10):4614-24.
doi: 10.1128/JVI.75.10.4614-4624.2001.

Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations

N Krieger  1 , V Lohmann, R Bartenschlager
Affiliations

Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations

N Krieger et al. J Virol. 2001 May.

Abstract

Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.

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Figures

FIG. 1
FIG. 1
Sequence analysis of HCV replicons isolated from various cell lines. The structure of the selectable replicon construct is shown in the top, with numbers below the NS3-NS5B polyprotein referring to the P1 positions of the cleavage sites or its carboxy terminus. neo, neomycin phosphotransferase gene; EI, EMCV IRES. Since for optimal HCV IRES activity ∼36 nt of the core ORF are required, 12 amino acid residues of the core protein are fused to the amino terminus of the neomycin phosphotransferase. Coding regions of the NS3-NS5B polyprotein cloned from cell line 5-15-9-2-3 and the founder line 5-15 are drawn below. Differences in the amino acid sequences are indicated by vertical lines. A black line labeled with a star refers to an amino acid substitution that was conserved with the four sequences isolated from 5-15-9-2-3 cell lines, and gray lines labeled with a dot indicate nonconserved mutations. Note that both replicons cloned from the founder cell line 5-15 carried the proline substitution in NS5A at position 2197 but that the glutamate substitution in NS5A at position 2350 (labeled with a gray star) was found in only one of these replicons (clone 5-15/4B). The positions of single nucleotide deletions in the polyprotein coding sequences of clone 5-15/4D are indicated with triangles.
FIG. 2
FIG. 2
Identification of cell culture-adaptive mutations. Huh-7 cells were transfected with the parental replicon 5.1 or replicon RNAs carrying the mutations given above each plate. Numbers below the plates refer to the CFU per microgram of in vitro-transcribed replicon RNA. For comparison, representative plates that were obtained after transfection of each 10 ng of in vitro-transcribed replicon RNA or 1 ng in the cases of the replicons shown in the bottom line are shown. Note that for the determination of the CFU, serial titrations down to 0.25 ng of each RNA were transfected. With the nonadapted replicons, five independent transfections were performed, but with the adapted RNAs, 20 to 50 independent transfections were performed.
FIG. 3
FIG. 3
Transient replication of cell culture-adapted HCV replicons. (A) Huh-7 cells were transfected with selectable replicon RNAs and harvested at time points indicated above each lane. HCV RNAs were analyzed by Northern blotting. The number of replicon RNA molecules was determined by comparison with the serial dilution of in vitro transcripts (lanes 1 to 3). β-Actin RNA (β-act) served as a control to correct for the amount of total RNA loaded in each lane of the gel (∼2 μg). The result obtained with total RNA from naïve Huh-7 cells is shown in lanes 4 and 20. The positions of replicon RNAs, 28S RNA, and β-actin m-RNA are given in the left. The replicon carrying a deletion that spans the active site of the NS5B RdRp served as a negative control (rep5.1/Δ5B). (B) Quantification of the RNA in the Northern blots shown in panel A by phosphorimaging. Values were corrected for the RNA amounts determined 3.5 h after transfection, and they are expressed as percentages. With rep5.1, the 100% value corresponds to 1.3 × 107 replicon RNA molecules per μg of total RNA. ■, replicon 5.1; ▴, replicon 19; ⧫, replicon 9-13F; ○, replicon 5.1/Δ5B. Analogous results were obtained in three further independent experiments.
FIG. 4
FIG. 4
Immunofluorescence analysis of transiently replicating HCV RNAs. A portion of transfected Huh-7 cells (Fig. 3) was seeded onto glass coverslips. NS3 was detected by immunofluorescence at 4 and 96 h posttransfection (left and right sides, respectively). Mock-transfected Huh-7 cells served as a negative control. Bar = 50 μm.
FIG. 5
FIG. 5
Transient replication of HCV replicons carrying a reporter gene. (A) Structure of a replicon construct harboring a firefly luciferase gene (Ffl-luc). GND indicates the position of the amino acid substitution of the NS5B RdRp. For further details, see the legend to Fig. 1. (B) Flow chart of the experimental approach. Huh-7 cells were transfected by electroporation (Epo), and 1/10 of each was seeded in a culture dish. After 4, 24, 48, 72, and 96 h, cells were lysed and luciferase activities were determined. Cells that were cultured for 72 h were passaged at a dilution of 1:3. One-third of each was harvested at time points (t) corresponding to 127 and 144 h after transfection, whereas the remainder were again passaged at a time point corresponding to 168 h posttransfection. Cells of this second passage were lysed at time points corresponding to 216 and 244 h after transfection. (C) Representative results obtained with the replicons specified at the top. Values for each time point correspond to the mean and the error range of quadruplicate results. Note that, owing to low variations, error bars are in most cases not visible in the graph. Values are corrected for transfection efficiency as determined by measuring the luciferase activity 4 h after transfection. Wt, wild type.
FIG. 6
FIG. 6
Transient replication of luciferase replicon RNAs carrying mutations in NS3 and NS5A. Huh-7 cells were transfected with the replicons specified at the left, and luciferase activities were determined in lysates of cells harvested 4, 24, 48, 72, and 96 h (not shown) after transfection. The 4-h value (not shown) was used to correct for different transfection efficiencies. Bars represent the means and the error ranges of quadruplicate results. In most cases, the variations are very small and therefore the error bars are not visible.
FIG. 7
FIG. 7
Determination of replicon RNA levels in selected cell lines, 24-1, 25-1, and 25-2, each harboring the adapted replicon 5.1. Cells were harvested at given times after being seeded, and HCV RNA was analyzed by Northern blotting as described in the legend to Fig. 3. A summary of the data, including analogous quantifications with cell lines harboring other replicon RNAs, is given in Table 2. β-act., β-actin.
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