title: HIV virions as nanoscopic test tubes for probing oligomerization of the integrase enzyme.
abstract: Employing viruses as nanoscopic lipid-enveloped test tubes allows the miniaturization of protein-protein interaction (PPI) assays while preserving the physiological environment necessary for particular biological processes. Applied to the study of the human immunodeficiency virus type 1 (HIV-1), viral biology and pathology can also be investigated in novel ways, both in vitro as well as in infected cells. In this work we report on an experimental strategy that makes use of engineered HIV-1 viral particles, to allow for probing PPIs of the HIV-1 integrase (IN) inside viruses with single-molecule Förster resonance energy transfer (FRET) using fluorescent proteins (FP). We show that infectious fluorescently labeled viruses can be obtained and that the quantity of labels can be accurately measured and controlled inside individual viral particles. We demonstrate, with proper control experiments, the formation of IN oligomers in single viral particles and inside viral complexes in infected cells. Finally, we show a clear effect on IN oligomerization of small molecule inhibitors of interactions of IN with its natural human cofactor LEDGF/p75, corroborating that IN oligomer enhancing drugs are active already at the level of the virus and strongly suggesting the presence of a dynamic, enhanceable equilibrium between the IN dimer and tetramer in viral particles. Although applied to the HIV-1 IN enzyme, our methodology for utilizing HIV virions as nanoscopic test tubes for probing PPIs is generic, i.e., other PPIs targeted into the HIV-1, or PPIs targeted into other viruses, can potentially be studied with a similar strategy.
authors.affiliation: Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, KU Leuven , Celestijnenlaan 200F, 3001 Heverlee, Flanders, Belgium.
authors.forename: Doortje
authors.initials: D
authors.lastname: Borrenberghs
authors.forename: Wannes
authors.initials: W
authors.lastname: Thys
authors.forename: Susana
authors.initials: S
authors.lastname: Rocha
authors.forename: Jonas
authors.initials: J
authors.lastname: Demeulemeester
authors.forename: Caroline
authors.initials: C
authors.lastname: Weydert
authors.forename: Peter
authors.initials: P
authors.lastname: Dedecker
authors.forename: Johan
authors.initials: J
authors.lastname: Hofkens
authors.forename: Zeger
authors.initials: Z
authors.lastname: Debyser
authors.forename: Jelle
authors.initials: J
authors.lastname: Hendrix
mesh_terms: D055162:Biocatalysis; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D019427:HIV Integrase / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology; D006801:Humans; D008958:Models, Molecular; D036103:Nanotechnology / Q000379:methods*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D014771:Virion / Q000378:metabolism*
chemical_list: D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1