Comparative analysis of anti-hepatitis C virus activity and gene expression mediated by alpha, beta, and gamma interferons

Abstract

A direct comparison of the inhibitory effects of alpha, beta, and gamma interferons (IFNs) on replication of a hepatitis C virus subgenomic replicon in a hepatoma cell line revealed similarities in antiviral potency. However, alternate IFN-induced antiviral mechanisms were suggested following observations of striking differences between IFN-gamma and IFN-alpha/beta with respect to strength and durability of the antiviral response and the magnitude and pattern of IFN-mediated gene expression.

FIG. 1.

Inhibition of HCV RNA synthesis in the subgenomic replicon cells by IFN-α, -β, and -γ. (A) HCV replicon luciferase reporter cells (I₃₈₉luc-ubi-neo/NS3-3′/5.1) were incubated in the presence of IFN-α, -β or -γ for 24 h and then assayed for luciferase activity. Luciferase signals were plotted as mean percentages of those for the untreated control cells and are representative of three independently derived experiments. (B) Northern blot analysis of HCV replicon RNA derived from I₃₈₉neo/NS3-3′/wt replicon (16) cells treated with log increment doses of IFN-α (lanes 2 to 6) or IFN-γ (lanes 12 to 16) (0.01 to 100 IU/ml) or IFN-β (lanes 7 to 11) (0.003 to 33 IU/ml). A ³²P-labeled, HCV-specific (NS5A-NS5B) probe was used to detect replicon RNA extracted from the replicon cells that had been treated with various doses of IFN for 72 h. Similarly, a probe specific for glyceraldehyde-3-phosphate dehydrogenase mRNA was used to monitor the level of this cellular RNA.

FIG. 1.

Inhibition of HCV RNA synthesis in the subgenomic replicon cells by IFN-α, -β, and -γ. (A) HCV replicon luciferase reporter cells (I₃₈₉luc-ubi-neo/NS3-3′/5.1) were incubated in the presence of IFN-α, -β or -γ for 24 h and then assayed for luciferase activity. Luciferase signals were plotted as mean percentages of those for the untreated control cells and are representative of three independently derived experiments. (B) Northern blot analysis of HCV replicon RNA derived from I₃₈₉neo/NS3-3′/wt replicon (16) cells treated with log increment doses of IFN-α (lanes 2 to 6) or IFN-γ (lanes 12 to 16) (0.01 to 100 IU/ml) or IFN-β (lanes 7 to 11) (0.003 to 33 IU/ml). A ³²P-labeled, HCV-specific (NS5A-NS5B) probe was used to detect replicon RNA extracted from the replicon cells that had been treated with various doses of IFN for 72 h. Similarly, a probe specific for glyceraldehyde-3-phosphate dehydrogenase mRNA was used to monitor the level of this cellular RNA.

FIG. 2.

Differential response rates of HCV replicon reporter cells to IFN treatment and durability of IFN inhibitory effect. (A) HCV replicon reporter cells (I₃₈₉luc-ubi-neo/NS3-3′/5.1) were treated with 20, 160, or 320 IU of IFN-α (diamonds) and IFN-β (squares)/ml or with 20 and 320 IU of IFNγ (triangles)/ml for various lengths of time (2.5 min to 24 h) and assayed for luciferase activity at the 24-h time point from the start of IFN treatment. Luciferase signals were plotted as mean percentages of the untreated control cells and are representative of three independently derived experiments. (B) Reporter cells were treated with IFN doses as described above for 4 h and then assayed for the luciferase signal 24, 48, or 72 h posttreatment. In addition, a parallel and equally treated cell culture was trypsinized at the 72-h mark (see arrow), diluted 1:4 into new microtiter plates, and subsequently assayed either 48, 72, or 96 h from this point (120, 144, and 168 total hours post-IFN treatment). Durability of the IFN HCV replication inhibitory effect was plotted over time as a percentage of the luciferase signal derived from mock-treated cells. Data represent means of triplicate values from a typical assay.