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

Authors Zych C, Domling A, Ayyavoo V

Received 18 February 2013

Accepted for publication 2 April 2013

Published 24 May 2013 Volume 2013:7 Pages 403—412

DOI https://doi.org/10.2147/DDDT.S44139

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2



Courtney Zych,1 Alexander Domling,2 Velpandi Ayyavoo1

1Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA; 2Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA, USA

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

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