31)8 were synthesized in the

Medicinal Chemistry Laboratory of the Institute of Medicinal Biotechnology Chinese Academy of Medical Sciences, with a purity over 99.0%. The compound structure was confirmed with 1H-NMR and MS spectra. Interferon-α-2b (Intron A) was from Schering Plough (Brinny) (Kenilworth, NJ). BILN2061, a known NS3-4A protease inhibitor, was provided by Shanghai Lechen International Trading (Shanghai, China). Plasmid pcDNA3.1-Vif coding for HIV-1 full-length Vif was created by insertion of Vif (amplified Etoposide by polymerase chain reaction [PCR] from HIV-1 plasmid SVC21.BH10) into pcDNA3.1; the plasmids hA2, hA3B, hA3C, hA3F, and hA3G that express wildtype forms of hA2, hA3B, hA3C, hA3F, and hA3G, respectively, possess a fused HA tag at the C-terminus. The above-mentioned plasmids were gifts from Dr. Shan Cen at the Lady Davis Institute for Medical Research and McGill University AIDS Centre. The plasmid pFL-J6/JFH/JC1 containing the full-length chimeric

HCV cDNA was kindly provided by Vertex Pharmaceuticals (Boston, MA). Production of infectious HCV in hepatocytes was done as described.14 The plasmid pFL-J6/JFH/JC1 was restricted with XbaI and treated with Mung Bean nuclease (New England Biolabs) to generate the according HCV cDNA with T7 promotor. The cDNAs were purified and used as templates for RNA synthesis. HCV RNA was synthesized MCE公司 in vitro using a MEGAscript T7 kit (Ambion). The synthesized RNA was treated

with DNase I (New England Biolabs) and purified with Wizard GS-1101 ic50 SV Gel and PCR Clean-Up System (Promega). The synthesized HCV RNA was used to transfect naïve Huh7.5 cells with the addition of Lipofectamine 2000 (Invitrogen). The culture medium was collected and cleaned with centrifugation at 3,000 rpm for 10 minutes. The supernatants were stored at −70°C as HCV viral stock and quantified with the Diagnostic Kit for Quantification of Hepatitis C Virus RNA (Shanghai Kehua Bio-Engineering). Huh7.5 cells 24 hours after HCV infection with viral stock (45 IU per cell) were transfected with different concentrations of APOBEC- or Vif-containing plasmids in the FuGENE HD Transfection Reagent (Roche), with pcDNA3.1 as plasmid control. Then, 72 hours later the culture medium was removed and total intracellular proteins were extracted using CytoBuster Protein Extraction Reagent (Novagen) with 1 mM protease inhibitor cocktail (Roche Applied Science). HCV Core, NS3, and hA3G protein (or APOBEC proteins with HA tag) was detected with western blot. A similar procedure was used for the experiment using GS4.3 cells, except the infection step. Huh7.5 cells were planted into the 6-well plate with 3 × 105 cells per well in the complete growth medium and infected with HCV viral stock (45 IU per cell).