Analysis of interferon signaling by infectious hepatitis C virus clones with substitutions of core amino acids 70 and 91

Abstract

Substitution of amino acids 70 and 91 in the hepatitis C virus (HCV) core region is a significant predictor of poor responses to peginterferon-plus-ribavirin therapy, while their molecular mechanisms remain unclear. Here we investigated these differences in the response to alpha interferon (IFN) by using HCV cell culture with R70Q, R70H, and L91M substitutions. IFN treatment of cells transfected or infected with the wild type or the mutant HCV clones showed that the R70Q, R70H, and L91M core mutants were significantly more resistant than the wild type. Among HCV-transfected cells, intracellular HCV RNA levels were significantly higher for the core mutants than for the wild type, while HCV RNA in culture supernatant was significantly lower for these mutants than for the wild type. IFN-induced phosphorylation of STAT1 and STAT2 and expression of the interferon-inducible genes were significantly lower for the core mutants than for the wild type, suggesting cellular unresponsiveness to IFN. The expression level of an interferon signal attenuator, SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type. Interleukin 6 (IL-6), which upregulates SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type, suggesting interferon resistance, possibly through IL-6-induced, SOCS3-mediated suppression of interferon signaling. Expression levels of endoplasmic reticulum (ER) stress proteins were significantly higher in cells transfected with a core mutant than in those transfected with the wild type. In conclusion, HCV R70 and L91 core mutants were resistant to interferon in vitro, and the resistance may be induced by IL-6-induced upregulation of SOCS3. Those mechanisms may explain clinical interferon resistance of HCV core mutants.

Fig. 1.

Comparison of interferon sensitivity between HCV wild type and core mutant clones. The wild type and core mutants were transfected into Huh7 cells and cultured in the presence of IFN-α2b at concentrations ranging from 0 to 100 U/ml. (A) The culture supernatant of HCV-transfected Huh7 cells was collected 72 h after transfection, and the levels of HCV core antigen in the culture supernatant were measured. The values are displayed as percentages of those for the IFN-untreated control. The experiments were repeated three times, and representative results are shown. (B) Expression of intracellular HCV RNA. Cellular RNA was harvested at 72 h posttransfection. HCV RNA was quantified by real-time RT-PCR. The values are displayed as percentages of those for the IFN-untreated control. (C) Expression of intracellular HCV RNA. Cellular RNA was harvested at 72 h postinfection. HCV RNA was quantified by real-time RT-PCR. The values are displayed as percentages of those for the IFN-untreated control. In panels A through C, asterisks indicate P values of less than 0.05, compared to results for the interferon-negative control. (D) Western blotting was performed to assess intracellular suppression of HCV core protein. Ten micrograms of harvested cell lysates were subjected to Western blotting using anti-HCV core antibodies. Densitometry of core protein was performed, and results are shown as percentages of the results for an IFN-negative sample.

Fig. 2.

Analysis of intracellular and supernatant HCV RNA levels in core 70/91 mutants. In vitro-transcribed mutant and wild-type RNAs were transfected into Huh7 cells. Three days after transfection, RNA was extracted from cells (A) or culture supernatant (B) and quantified by real-time RT-PCR. Asterisks indicate P values of less than 0.05 compared to results for the wild type. (C) Western blotting. Expression of core proteins in HCV-transfected cells. Total cellular protein was prepared from HCV RNA-transfected cells, and Western blotting was performed using anticore and anti-beta-actin antibodies. Densitometry was performed, and results are shown as percentages of that for an HCV-negative sample.

Fig. 3.

Interferon-induced expressional induction of the ISGs, P56, PKR, and 25AS in Huh7 cells transfected or infected with wild-type and core mutant JFH1 clones. Two days posttransfection, cells were treated with 50 IU/ml of IFN-α. After 8 h, total cellular RNA was extracted and mRNAs of P56 (A), PKR (B), or 25AS (C) were quantified by real-time RT-PCR analyses. The values are displayed as ratios of IFN-untreated control values. Experiments were repeated three times, and representative results are shown. Asterisks indicate P values of less than 0.05 compared to results for the wild type. (D) Western blotting. Expression of total and phosphorylated STAT1 and STAT2 proteins in cells transfected with the wild type and core mutant HCV clones. (E) Western blotting. Expression of total and phosphorylated STAT1 and STAT2 proteins in cells infected with the wild type and core mutant HCV clones. Densitometries for pSTAT1 and pSTAT2 were performed, and results are shown as percentage of results for HCV-negative samples.

Fig. 4.

Effects of core mutant HCV on SOCS1 and SOCS3 expression in Huh7 cells. Expression levels of SOCS1 (A) or SOCS3 (B) in Huh7 cells transfected with the wild type or the core mutant JFH1. Three days posttransfection, total cellular RNA was isolated and the mRNA was quantified by real-time RT-PCR analyses. The experiments were repeated three times, and representative results are shown. The values are displayed as values relative to beta-actin levels. Each experiment was repeated three times, and the representative results are shown. Asterisks indicate P values of less than 0.05 compared to results for the wild type.

Fig. 5.

Expression of phosphorylated STAT3 and IL-6 in cells transfected with the wild type and core mutant HCV-JFH1 clones. (A) Western blotting. Expression of total and phosphorylated STAT3 and beta-actin proteins in cells transfected with the wild type or core mutant HCV clones. (B) Two days posttransfection, total cellular RNA was extracted and mRNAs of IL-6 were quantified by real-time RT-PCR analyses. The values are displayed as the ratio of values of the HCV-untreated control. Asterisks indicate P values of less than 0.05 compared to results for the wild type.

Fig. 6.

Expression of GRP78 and CHOP UPR genes in cells transfected with the wild type and core mutant HCV-JFH1 clones. Western blotting was performed to assess UPR following transfection with HCV core mutants. Ten micrograms of harvested cell lysates were subjected to Western blotting using anti-GRP78 and anti-CHOP antibodies. Densitometries for GRP78 and CHOP were performed, and results are shown as percentages of results for uninfected cells.

Fig. 7.

Schematic diagram of signaling pathway involved in HCV core mutant infection and IFN resistance.