Novel type I interferon IL-28A suppresses hepatitis C viral RNA replication

Abstract

Interferon alpha (IFN-alpha)-based therapy is the currently approved treatment for chronic hepatitis C viral infection. The sustained antiviral response rate is approximately 50% for genotype-1 infection. The major challenge to the HCV community is to improve antiviral efficacy and to reduce the side effects typically seen in IFNalpha-based therapy. One of the strategies is to identify new interferons, which may have better efficacy and less undesirable side effects. In this report, we examined the role of IL-28A (IFN lambda2), a novel type I IFN, in suppression of human hepatitis C viral RNA replication. We have cloned both the human genomic DNA and cDNA of IL-28A, and evaluated their biological activity using HCV RNA replicon cell culture system. The results show that IL-28A effectively inhibits HCV subgenomic RNA replication in a dose-dependent manner. Treatment of human hepatoma cells with IL-28A activates the JAK-STAT signaling pathway and induces the expression of some interferon-stimulated genes (ISGs), such as 6-16 and 1-8U. We also demonstrate that IL-28A induces expression of HLA class I antigens in human hepatoma cells. Moreover, IL-28A appears to specifically suppress HCV IRES-mediated translation. Although IL-28A receptor shares one subunit with the IL-10 receptor, IL-10 treatment has no detectable effect on IL-28A-induced antiviral activity. Interestingly, IL-28A can synergistically enhance IFNalpha antiviral efficacy. Our results suggest that IL-28A antiviral activity is associated with the activation of the JAK-STAT signaling pathway and expression of ISGs. The effectiveness of IL-28A antiviral activity and its synergistic effect on IFN-alpha indicate that IL-28A may be potentially used to treat HCV chronic infection.

Figure 1

IL-28A gene structure. A) Schematics of the exon-intron structure of the gene. The numbers indicate exon location. B) Complete sequence of IL-28A genomic sequence (Accession number DQ126336). The bold nucleotides are the nucleotide sequence of the exons (Accession number DQ126337).

Figure 2

Effects of IL-28A on HCV RNA replication and protein expression. A) GSB cells were transfected by either control plasmid (TOPO) or IL-28A genomic expression construct (TOPO-hIL28A). After 48 hours, total RNA was isolated, followed by real-time PCR analysis with HCV-specific primers. The data represents the normalization with the internal control GADPH. B) Western blot analysis of GSB1 cells transfected with the control plasmid (TOPO) or IL-28A expression construct (TOPO-IL28A). The monoclonal antibody is specifically against HCV NS5A. The internal control is actin. C) Effect of IL-28A-conditioned medium on HCV RNA replication in GSB cells. The conditioned medium was used to treat the cells for 48 hours, followed by real-time RT-PCR analysis. D) Effect of recombinant IL-28A on HCV RNA replication in GSB cells. The relative HCV RNA levels were normalized with the internal control GADPH. The error bars indicate the variations of three independent assays.

Figure 3

Effects of IL-28A on CAP-dependent and HCV IRES-dependent translation. The GSB1 cells was transfected with control plasmid or plasmid pRL-HL, which has different luciferases directed by either CAP- or HCV IRES. After 48 hours of transfection, the cells were treated with varying doses of IL-28A. Cell extracts were made after 24 hours of incubation, followed by luciferase determination. The data represents the average of three independent experiments. The open column indicates CAP-mediated translation. The shadowed column indicates HCV IRES-mediated translation. The error bars indicate the variations of three independent assays.

Figure 4

The effect of IL-28A on JAK-STAT signaling pathway. GSB1 cells were treated with either IL-28A conditioned medium (A) or recombinant rIL-28A (B) for 30 minutes, followed by protein extraction and Western blot analysis using antibodies as indicated in the figure. Equal amounts (20 ug) of proteins were loaded in each lane and confirmed by detection of actin. STAT1 or STAT3 indicates total STAT protein. p-STAT1 or p-STAT3 indicates tyrosine phosphorylated form (activated STAT protein). The figures are representatives of at least four independent experiments.

Figure 5

Induction of interferon stimulated genes by IL-28A in GSB1 cells. GSB1 cells were treated by either control or 2-ml IL-28A-conditioned medium for 12 hours. Total RNA was isolated, followed by RT-PCR analysis using a pair of gene-specific primers and a pair of DADPH primers. The PCR amplification cycle is 25, which ensures PCR reaction in linear range. The PCR products were analyzed in 1% agarose gel. M indicates the DNA molecular weight marker. The arrow indicates gene-specific products. The bar indicates GADPH DNA fragment. The figure is a representative of two independent assays.

Figure 6

Effect of IL-10 on IL-28A-induced antiviral activity. The GSB1 cells were treated with varying doses of IL-10 or IL-28A as indicated for 72 hours. Total RNA was isolated for real-time PCR analysis using HCV-specific primers. The data represents the normalization with internal control GADPH. The error bars indicate the variations of three independent assays.

Figure 7

Effect of IL-28A on the antiviral activity of IFNα. Varying doses of IL-28A and IFNα2b, either alone or in combination, were added to the GSB1 cells and incubated for 48 hours. Total RNA was isolated for real-time PCR analysis. The vertical axis represents the fold of viral RNA reduction by IL-28A or IFN. The data represents the results of normalization with the internal control GADPH.

Figure 8

Effect of IL-28A on IFNα-induced STAT1 activation in GSB1 cells. GSB1 cells were treated with IL-28A or/and IFN as indicated. After 30 minutes of incubation, total protein was extracted for Western blot analysis using antibodies against total STAT1 (STAT1) or phosphorylation-specific STAT1 (p-STAT1).

Figure 9

Effect of IL-28A on HLA class I antigen expression in Huh7 cells. The Huh7 cells were treated with 2 ml IL-28A-conditioned culture medium for 72 hours. The cells were then harvested and incubated with HLA class I antigen-specific antibody labeled by FITC fluorescence, followed by flow cytometric analysis. The arrow-marked curve indicates control cells. The arrowhead-marked curve indicates cells treated with IL-28A.