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. 2008 Mar 18;105(11):4370-5.
doi: 10.1073/pnas.0800422105. Epub 2008 Mar 11.

Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

Rodney S Russell  1 , Jean-Christophe Meunier, Shingo Takikawa, Kristina Faulk, Ronald E Engle, Jens Bukh, Robert H Purcell, Suzanne U Emerson
Affiliations

Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

Rodney S Russell et al. Proc Natl Acad Sci U S A. .

Abstract

The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast, both the p7 and NS2 mutations independently increased the amount of virus released.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mutations permit 3- to 4-log higher levels of virus production. One million Huh-7.5 cells were inoculated with day 5 transfection supernatants containing 100 ffu (m.o.i. = 0.0001) of indicated HCVcc. (A) HCV RNA levels in culture fluids at indicated time points postinfection were measured by TaqMan real-time RT-PCR. At day 5 posttransfection, all culture fluids still contained large amounts of transfection-related RNA. Therefore, dilution of infectious virus to produce the desired m.o.i. resulted in a corresponding dilution of residual transfection RNA with the result that at day 0 RNA levels differed even though the amount of each infecting virus was the same. Culture supernatant from an SGR-JFH1 transfection was used as a mock infection control. Data points represent the mean value obtained from duplicate TaqMan amplifications of the same sample. (B) Infectious viral titers were measured at indicated time points postinfection by a limiting dilution assay for ffu. Results are representative of at least two independent transfection/infection experiments. Assays of ffu were performed in triplicate, and the means plus standard error are plotted. The dotted line represents the cutoff of the assay, which was 10 ffu/ml.
Fig. 2.
Fig. 2.
Effects of individual mutations on virus replication capacity. One million Huh-7.5 cells were inoculated with RNase-treated transfection supernatants containing 500 ffu (m.o.i. = 0.0005) of indicated HCVcc. (A) HCV RNA levels in culture fluids at indicated time points postinfection were measured by TaqMan Real Time RT-PCR. (B) Infectious viral titers were measured at indicated time points postinfection by a limiting dilution assay for ffu. See Fig. 1 legend for details.
Fig. 3.
Fig. 3.
Characterization of a cell line that can support virus replication but cannot be infected. Two hundred thousand S29 cells were transfected with either irrelevant vector (Left) or human CD81 (Right) in two-chamber culture slides and inoculated with 500,000 ffu (m.o.i. = 2.5) of JFH-AM2 24 h later. Two days postinoculation, cells were costained by direct IF for CD81 by using a FITC-conjugated murine monoclonal Ab against CD81, and indirect IF for HCV proteins using serum from an HCV-infected chimpanzee in combination with anti-human AlexaFluor 568. Green fluorescence represents CD81, red fluorescence represents HCV-infected cells, and DAPI-stained nuclei are shown in blue as observed using the ×40 objective. White boxes outline two cells that have been enlarged to better display the dual staining. Results are representative of two independent experiments.
Fig. 4.
Fig. 4.
Adaptive mutations enhance the accumulation of infectious virus. One million CD81-deficient S29 cells were transfected with 4 μl of a 20-μl DNase-treated T7 transcription reaction containing indicated wild-type JFH1, JFH1 plus culture-selected mutations, or SGR-JFH1 RNA. (A) Transfected HCV core-positive cells were visualized by indirect IF with murine monoclonal anti-core followed by anti-mouse AlexaFluor 488. Green fluorescence represents HCV core and blue represents DAPI-stained nuclei as observed using the 10X objective. (B) Total extracellular and intracellular infectious virus accumulated by day 2 was measured by assays for ffu in triplicate, and means plus standard errors are plotted. The dotted line represents the cutoff of the assay, which was 10 ffu/ml. (C) Levels of extracellular and intracellular infectious virus present in the cultures of JFH1 mutant viruses (mt) were compared with that of wild-type JFH1 (wt) and the ratios were plotted. Fold increases (mt/wt) are indicated above the respective bars. Results are representative of two independent transfection experiments.
Fig. 5.
Fig. 5.
Adaptive mutations increase the efficiency of infectious virus production. One million CD81-deficient S29 cells were transfected as described in Fig. 4, and total extracellular infectious virus produced each day was measured by assays for ffu in triplicate and means plus standard errors are plotted. The bar representing day 2 for the p7 mutant appears not to have an error bar, because the standard error of the three measurements taken for this sample was zero. The dotted line represents the cutoff of the assay, which was 10 ffu/ml. Results are representative of three independent experiments.
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