HIV virions as nanoscopic test tubes for probing oligomerization of the integrase enzyme

TY - JOUR

T1 - HIV virions as nanoscopic test tubes for probing oligomerization of the integrase enzyme

AU - Borrenberghs, Doortje

AU - Thys, Wannes

AU - Rocha, Susana

AU - Demeulemeester, Jonas

AU - Weydert, Caroline

AU - Dedecker, Peter

AU - Hofkens, Johan

AU - Debyser, Zeger

AU - Hendrix, Jelle

PY - 2014/4/22

Y1 - 2014/4/22

N2 - 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.

AB - 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.

U2 - 10.1021/nn406615v

DO - 10.1021/nn406615v

M3 - Journal article

C2 - 24654558

SN - 1936-0851

VL - 8

SP - 3531

EP - 3545

JO - A C S Nano

JF - A C S Nano

IS - 4

ER -