Volume 41, Issue 4, Pages 667-672 (October 2004) Interferon-α stimulation of liver cells enhances hepatitis delta virus RNA editing in early infection  Dirk Hartwig, Lutz Schoeneich, Jobst Greeve, Claudia Schütte, Isabel Dorn, Holger Kirchner, Holger Hennig  Journal of Hepatology  Volume 41, Issue 4, Pages 667-672 (October 2004) DOI: 10.1016/j.jhep.2004.06.025 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Expression of ADAR1-L mRNA in IFN-α-treated or transfected (+HDV RNA) Huh-7 cells compared to unstimulated, untransfected control. 24h after stimulation with 200U IFN-α/ml or 1000U IFN-α/ml, the expression of ADAR1-L mRNA is significantly enhanced. Unstimulated, transfected cells showed no increase in ADAR1-L mRNA compared to control. Journal of Hepatology 2004 41, 667-672DOI: (10.1016/j.jhep.2004.06.025) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Results of the relative quantification of ADAR1-S mRNA in Huh-7 cells. 24h after stimulation with 200U/ml IFN-α or 1000U/ml IFN-α, ADAR1-S mRNA expression is not influenced in comparison to the unstimulated control. Unstimulated transfected (+HDV RNA) cells also had similar ADAR1-S mRNA expression. Journal of Hepatology 2004 41, 667-672DOI: (10.1016/j.jhep.2004.06.025) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Huh-7 cells were transfected with plasmid pSVL D3 coding for full-length, replicating HDV RNA on day 0. A proportion of the cells were treated with 1000U/ml IFN-α every 72h. On days 7, 14, 21 and 28, total RNA was isolated and analysed for amber/W site editing and ADAR1-L mRNA expression. (a) The line and scatter plots depict the percentage of edited HDV antigenomic RNA during the course of the transfection experiments. Black points represent mean editing rates in unstimulated Huh-7 cells, while filled triangles represent the results for IFN-α-stimulated host cells. Error bars depict the standard deviation. (b) Editing of antigenomic HDV RNA during the course of the experiment. On days 7, 14, 21 and 28, total RNA of IFN-α-treated (IFN+) and untreated (IFN-) cells was isolated, reverse transcribed and amplified by PCR. PCR products were 32P end-labelled and digested with restriction enzyme NcoI, specific for edited HDV cDNA. Digestion products were analysed by non-denaturing electrophoresis on a 6% polyacrylamide gel. (c) Results of relative quantification of ADAR1-L mRNA in IFN-α-treated Huh-7 cells. Values are normalised with respect to the mRNA expression of the house-keeping gene GAPDH and compared to ADAR1-L mRNA of untreated cells. ADAR1-L mRNA level are approximately five-fold enhanced after permanent stimulation with 1000U IFN-α/ml during the time course of the experiments. Journal of Hepatology 2004 41, 667-672DOI: (10.1016/j.jhep.2004.06.025) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Huh-7 cells were transfected on day 0 with plasmid pSVL D3, expressing full-length replicating HDV RNA. A proportion of the cells were stimulated with 1000U/ml IFN-α every 72h. On days 7, 14, 21 and 28, total protein was isolated and subjected to immunoblotting. (a) Immunoblots for ADAR1-S and -L. A small 150kDa-band representing ADAR1-L was visible only in the IFN-α treated Huh-7 cells. The amount of ADAR1-S was comparable in all the samples. (b) Immunoblots for HDAg-S and-L. Levels of HDAg in unstimulated cells decreased over time to the extent that on day 21 and 28, the HDAg was hardly detectable. An increase in HDAg-L relative to HDAg-S was visible between days 7 and 14. Comparing HDAg concentrations of IFN-α stimulated cells and unstimulated controls, HDAg-S and HDAg-L were lower in the stimulated cells. Already on day 14, HDAg was no longer detectable in the IFN-α-treated Huh-7 cells. (c) The β-actin immunoblot served as control for total protein loading. Journal of Hepatology 2004 41, 667-672DOI: (10.1016/j.jhep.2004.06.025) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions