Inducible APOBEC3G-Vif Double Stable Cell Line as a High-Throughput Screening Platform To Identify Antiviral Compounds

Abstract

Inhibition of the interaction of the human cytidine-deaminase APOBEC3G (A3G) with the human immunodeficiency virus (HIV) type 1-specific viral infectivity factor (Vif) represents a novel therapeutic approach in which a cellular factor with potent antiviral activity (A3G) plays a key role. In HIV-infected cells, the interaction of Vif with A3G leads to the subsequent degradation of A3G by the 26S proteasome via the ubiquitin pathway and to the loss of antiviral activity. To establish a stable and convenient cellular testing platform for the high-throughput screening of potential antiviral compound libraries, we engineered a double transgenic cell line constitutively expressing an enhanced yellow fluorescent protein expressor (EYFP-A3G) fusion as well as a Tet-Off controllable Vif protein. With this cell line, we were able to measure precisely the Vif-induced degradation of A3G in the presence of potential antiviral compounds in an easy-to-handle, robust, and practical high-throughput multiwell plate format with an excellent screening window coefficient (Z factor) of 0.67.

Suppression of APOBEC3-mediated restriction of HIV-1 by Vif

“…The A3-HIV host and pathogen relationship creates the possibility of developing novel therapeutics ( Greene et al, 2008 ; Albin and Harris, 2010 ) and high-throughput screening approaches for small-molecule inhibitors have uncovered positive results. There are strategies to induce either A3G-mediated viral hypermutation by disrupting the Vif-A3G interaction ( Nathans et al, 2008 ; Cen et al, 2010 ; Nowotny et al, 2010 ; Ejima et al, 2011 ; Ali et al, 2012 ; Mohammed et al, 2012 ; Matsui et al, 2014a ) or viral hypomutation by blocking A3G catalytic activity ( Li et al, 2012 ; Olson et al, 2013 ). For the “therapy by hypermutation” strategy, the rationale is to find small-molecule inhibitors that antagonize Vif function and increase the cellular level of A3G available for virus restriction.…”

“…For the “therapy by hypermutation” strategy, the rationale is to find small-molecule inhibitors that antagonize Vif function and increase the cellular level of A3G available for virus restriction. A few candidate molecules that recover A3G expression levels and enable HIV restriction in the presence of Vif have been discovered ( Nathans et al, 2008 ; Cen et al, 2010 ; Nowotny et al, 2010 ; Ejima et al, 2011 ; Ali et al, 2012 ; Mohammed et al, 2012 ; Matsui et al, 2014a ), although there are little biochemical data to understand the mechanism of action. For the “therapy by hypomutation” strategy, small-molecule inhibitors have been designed that target a key residue in A3G (C321) that inhibits the catalytic activity of A3G ( Li et al, 2012 ; Olson et al, 2013 ).…”

HIV-1 Vif versus the APOBEC3 cytidine deaminases: An intracellular duel between pathogen and host restriction factors

“…We and others have used different approaches to find an inhibitor of the interactions of Vif with A3G, cullin5, and elonginC (Cao et al, 2005; Nowotny et al, 2010)(W. Yonomoto, and W. C. Greene, unpublished data). Using YFP-tagged A3G and HIV-1 vectors with and without Vif, Rana and colleagues screened a library of 30,000 small molecules and identified 66 compounds, which were also scored in a secondary screen.…”