Inducible APOBEC3G-Vif double stable cell line as a high-throughput screening platform to identify antiviral compounds.
Inhibition of the interaction of the human cytidine-deaminase APOBEC3G (A3G) with the human immunodeficiency virus (HIV) type 1-specific viral infectivity factor (Vif) represents a novel therapeutic approach in which a cellular factor with potent antiviral activity (A3G) plays a key role. In HIV-infected cells, the interaction of Vif with A3G leads to the subsequent degradation of A3G by the 26S proteasome via the ubiquitin pathway and to the loss of antiviral activity. To establish a stable and convenient cellular testing platform for the high-throughput screening of potential antiviral compound libraries, we engineered a double transgenic cell line constitutively expressing an enhanced yellow fluorescent protein expressor (EYFP-A3G) fusion as well as a Tet-Off controllable Vif protein. With this cell line, we were able to measure precisely the Vif-induced degradation of A3G in the presence of potential antiviral compounds in an easy-to-handle, robust, and practical high-throughput multiwell plate format with an excellent screening window coefficient (Z factor) of 0.67.
University of Würzburg, Institute for Virology and Immunobiology, Versbacher Str. 7, Würzburg 97078, Germany.
Boris
B
Nowotny
Thomas
T
Schneider
Gabriele
G
Pradel
Tanja
T
Schirmeister
Axel
A
Rethwilm
Marc
M
Kirschner
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019380:Anti-HIV Agents / Q000138:chemical synthesis / Q000191:economics / Q000494:pharmacology*; D000998:Antiviral Agents / Q000138:chemical synthesis / Q000191:economics / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003362:Cost-Benefit Analysis; D003470:Culture Media; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000191:economics / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000187:drug effects; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015203:Reproducibility of Results; D014162:Transfection
D000900:Anti-Bacterial Agents; D019380:Anti-HIV Agents; D000998:Antiviral Agents; D003470:Culture Media; D004279:DNA, Viral; D005456:Fluorescent Dyes; D003564:Cytidine Deaminase; D004318:Doxycycline