Volume 144, Issue 1, Pages 59-61.e6 (January 2013) Development of Robust Hepatitis C Virus Genotype 4 Subgenomic Replicons  Betty Peng, Mei Yu, Simin Xu, Yu–Jen Lee, Yang Tian, Huiling Yang, Katie Chan, Hongmei Mo, John McHutchison, William Delaney, Guofeng Cheng  Gastroenterology  Volume 144, Issue 1, Pages 59-61.e6 (January 2013) DOI: 10.1053/j.gastro.2012.09.033 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 NS3 and NS4A mutations were selected to enhance GT4a replicon replication. (A) GT4a ED43 strain replicons encode a neomycin phosphotransferase gene (neo) or a Renilla luciferase (Rluc)-neo fusion reporter. , NS5A S232I mutation. (B) Ten-microgram GT4a replicon RNA as indicated was in vitro–transcribed and electroporated into 1C cells. Forty-eight hours after seeding, medium was replaced with complete Dulbecco's modified Eagle medium supplemented with 0.5 mg/mL G418 and refreshed twice per week. (C) Individual mutations were introduced into the ED43 Rluc-neo construct by site-directed mutagenesis, respectively. All replicon RNAs were transfected into 1C cells and 10,000 transfected cells were plated into wells in a 96-well plate without G418. At indicated times, cells were analyzed for luciferase activity. Dotted line indicates the background relative light units (RLU) value. Data represent mean of 2 independent experiments with error bars. Gastroenterology 2013 144, 59-61.e6DOI: (10.1053/j.gastro.2012.09.033) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 1 (A) Intracellular HCV replicon RNA expression level. Selected genotype 4a replicon cell lines were measured for their intracellular HCV replicon RNA level by using HCV-specific real-time RT quantitative PCR (RT-QPCR), normalized by cellular glyceraldehydes-3-phosphate dehydrogenase (GAPDH) level as described in the Supplementary Material and Methods. Genotype 1a H77 Rluc-neo stable replicon cells were included as a reference. (B) NS5A expression in selected replicon cells. The selected replicon cell lines were pelleted and completely lysed in sodium dodecyl sulfate loading buffer. The Western blot was analyzed with primary anti-NS5A antibody (clone 9E10; Apath, Brooklyn, NY), which was co-stained with anti-BiP antibody as a loading control. The staining was analyzed by Odyssey Imaging (LI-COR, Lincoln, NE). (C) NS5A immunostaining of the selected replicon cells. The genotype 4a replicon cell pool was stained with the same primary anti-NS5A antibody as above (red) and Hoechst 33342 (blue, indicating nuclei). 1C cells were stained as a negative control. Gastroenterology 2013 144, 59-61.e6DOI: (10.1053/j.gastro.2012.09.033) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 2 Efficient colony formation of stable GT4a replicon clones. Total cellular RNA was extracted from the GT4a replicon pool cell line and then electroporated into Huh-7 Lunet cells at the indicated amounts. The efficiency of colony formation was determined as described in the Supplementary Materials and Methods. In vitro transcribed parental GT4a replicon RNA was transfected in parallel as a control. Gastroenterology 2013 144, 59-61.e6DOI: (10.1053/j.gastro.2012.09.033) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 3 Selected NS3 or NS4A mutations enhance GT4a Rluc-neo replicon colony formation. All GT4a replicon constructs were prepared by introducing indicated mutations into wild-type GT4a Rluc-neo replicon plasmid using site-directed mutagenesis. Ten micrograms each GT4a replicon RNA was in vitro–transcribed and electroporated into 1C cells. Forty-eight hours after seeding in 150-cm dishes, medium was replaced with complete Dulbecco's modified Eagle medium supplemented with 0.5 mg/mL G418 and refreshed twice per week. After 3 weeks of G418 selection, cells were stained with 0.05% crystal violet. GT1a H77 Rluc-neo replicon was included as a reference. The colony-formation efficiency (CFU/μg RNA) was determined and the data are summarized in Supplementary Table 2. Gastroenterology 2013 144, 59-61.e6DOI: (10.1053/j.gastro.2012.09.033) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 4 1C cells are more permissive to GT4a replicon replication than Lunet cells. GT4a replicon RNA was in vitro transcribed and electroporated into 1C and Lunet cells. Forty-eight hours after seeding, medium was replaced with complete Dulbecco's modified Eagle medium supplemented with 0.5 mg/mL G418 and refreshed twice per week. After 3 weeks of G418 selection, cells were stained with 0.05% crystal violet. Colony-formation efficiency (CFU/μg RNA) was determined and the data are summarized in Supplementary Table 2. Gastroenterology 2013 144, 59-61.e6DOI: (10.1053/j.gastro.2012.09.033) Copyright © 2013 AGA Institute Terms and Conditions