title: Hepatitis C virus expressing flag-tagged envelope protein 2 has unaltered infectivity and density, is specifically neutralized by flag antibodies and can be purified by affinity chromatography.
abstract: Hepatitis C virus (HCV) purification by ultracentrifugation is difficult because of the low and heterogeneous density of native and cultured viruses. It was recently shown that inserting flag tag into envelope protein 2 (E2) of HCV permitted virus purification by affinity chromatography. However, flag-tagged viruses had drastically altered properties, and purification yield was low. In this study, we found that insertion of flag tag at the N-terminus of E2 in HCV recombinant J6/JFH1 did not affect viability in Huh7.5 cells, and that flag-tagged virus had physiochemical properties similar to the original virus. Flag-tagged virus was susceptible to flag-specific antibody neutralization, and infected cells could be immuno-stained by anti-flag antibodies. Using affinity chromatography with anti-flag resin we repeatedly obtained ~30% recovery of infectious particles. The full viability and unaltered physiochemical properties of flag-tagged HCV is an important improvement for utilizing these viruses for imaging, virion composition analysis and possibly vaccine development.
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark.
authors.forename: Jannick
authors.initials: J
authors.lastname: Prentoe
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D050296:Microbial Viability; D016254:Mutagenesis, Insertional; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D014774:Virulence
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus