Institute for Frontier Life and Medical Sciences, Kyoto University

The development of an antiviral drug screening platform with a FRET biosensor for measurement of arenavirus Z assembly

Tatsuaki Mizutani1,2, Yusuke Ohba3, Satoshi Mizuta4, Jiro Yasuda5,6, Shuzo Urata5,6
(1 Laboratory of Cell Regulation, Institute for Frontier Life and Medical Sciences Kyoto University, 2 Laboratory of Cell Regulation and Molecular Network, Graduate School of Biostudies, Kyoto University 3 Department of Cell Physiology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University 4 Graduate School of Biomedical Sciences, Nagasaki University 5 Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University 6 National Research Center for the Control and Prevention of Infectious Diseases (CCPID), Nagasaki University)

An antiviral drug screening platform with a FRET biosensor for measurement of arenavirus Z assembly

Cell Structure and Function (2020) https://doi.org/10.1247/csf.20030

Abstract

The smallest arenavirus gene product, Z protein, plays critical roles in the virus life cycle. Z is the major driving force of budding and particle production because of a unique property that defines self-assembly. In addition to the roles in budding, Z also participates in the suppression of type I interferon production to evade host antiviral immunity. Therefore, Z and its assembled form are an attractive drug target for arenaviral hemorrhagic fever, such as Lassa fever. Here, we developed a biosensor that enabled the evaluation of the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), Z assembly using the principle of Förster resonance energy transfer (FRET). This FRET biosensor consisted of three tandem Z that were sandwiched between super-enhanced cyan-emitting fluorescent protein and variant of a yellow-emitting mutant of green fluorescent protein so that Z-Z intermolecular binding via the really interesting new gene finger domain increased the emission ratio. To identify novel anti-arenavirus compounds, the FRET biosensor was employed to screen the PathogenBox400 for inhibitors of Z assembly in a 96-well plate format. The assay performed well, with a Z’-factor of 0.89, and identified two compounds that decreased the emission ratio of the FRET biosensor in a dose-dependent manner. Of them, the compound, 5,6,7,8-tetrahydro-7-(benzyl) -pyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-2,4-diamine, was found to significantly inhibit LCMV propagation in infected cells. Thereby, the present study demonstrated that a novel FRET biosensor incorporating Z assembly built on FRET and named Zabton, was a valuable screening tool to identify anti-arenavirus compounds in the context of inhibition of Z assembly.