Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization

Abstract

Targeting protein–protein interactions (PPI) is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV)-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of HIV-1 proteins with many host cellular components. In this study, a bimolecular fluorescence complementation (BiFC)-based screening assay was developed that can quantify changes in dimerization, using HIV-1 viral protein R (Vpr) dimerization as a “proof of concept.” Results demonstrated that Venus Vpr (generated by BiFC Vpr constructs) could be competed off in a dose-dependent manner using untagged, full-length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of small molecules for an effect on Vpr dimerization. Among the tested molecules, a few of the small molecules demonstrate an effect on Vpr dimerization in a dose-dependent manner.

Keywords:

• BiFC
• protein-protein interaction
• HIV-1 Vpr
• dimerization
• drug targets

Acknowledgments

This work was supported in part by R01 MH087247 to VA from the NIMH, NIH. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 consensus B Vpr (15-mer); peptides – complete set. This project used the UPCI cytometry facility that is supported in part by award P30CA047904.

Disclosure

The authors report no conflicts of interest in this work. The authors have no financial conflicts of interest.