title: Development and validation of a high-content bimolecular fluorescence complementation assay for small-molecule inhibitors of HIV-1 Nef dimerization.
abstract: Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.
authors.affiliation: 1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Jerrod A
authors.initials: JA
authors.lastname: Poe
authors.forename: Laura
authors.initials: L
authors.lastname: Vollmer
authors.forename: Andreas
authors.initials: A
authors.lastname: Vogt
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D055503:Protein Multimerization / Q000187:drug effects*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors*
chemical_list: D019380:Anti-HIV Agents; D008164:Luminescent Proteins; D054852:Small Molecule Libraries; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1
keywords: BiFC; HIV-1; HIV-1 Nef; YFP; fluorescence complementation; high-content screening