Evaluation of Structurally Distorted Split GFP Fluorescent Sensors for Cell-Based Detection of Viral Proteolytic Activity

Miguel R Guerreiro,Ana S Coroadinha,Ana R Fernandes

doi:10.3390/s21010024

Miguel R Guerreiro, Ana S Coroadinha + Show 1 more

Open Access

https://doi.org/10.3390/s21010024

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Abstract

Cell-based assays are essential for virus functional characterization in fundamental and applied research. Overcoming the limitations of virus-labelling strategies while allowing functional assessment of critical viral enzymes, virus-induced cell-based biosensors constitute a powerful approach. Herein, we designed and characterized different cell-based switch-on split GFP sensors reporting viral proteolytic activity and virus infection. Crucial to these sensors is the effective—yet reversible—fluorescence off-state, through protein distortion. For that, single (protein embedment or intein-mediated cyclization) or dual (coiled-coils) distortion schemes prevent split GFP self-assembly, until virus-promoted proteolysis of a cleavable sequence. All strategies showed their applicability in detecting viral proteolysis, although with different efficiencies depending on the protease. While for tobacco etch virus protease the best performing sensor was based on coiled-coils (signal-to-noise ratio, SNR, 97), for adenovirus and lentivirus proteases it was based on GFP11 cyclization (SNR 3.5) or GFP11 embedment distortion (SNR 6.0), respectively. When stably expressed, the sensors allowed live cell biosensing of adenovirus infection, with sensor fluorescence activation 24 h post-infection. The structural distortions herein studied are highly valuable in the development of cellular biosensing platforms. Additionally highlighted, selection of the best performing strategy is highly dependent on the unique properties of each viral protease.

Highlights

Received: 9 October 2020Accepted: 21 December 2020Published: 23 December 2020Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.license
As shown in transient screenings, for tobacco etch virus protease the best performing sensor was based on coiled-coils, while for adenovirus and lentivirus proteases it was based on GFP11 cyclization (SNR 3.5) or GFP11 embedment distortion (SNR 6.0), respectively
For the establishment of split fluorescent sensors whose self-assembly is controlled by proteolytic activity, three strategies of structural distortion of split Green fluorescent protein (GFP) fragments were developed, characterized, and evaluated (Scheme 1)

Summary

Introduction

Received: 9 October 2020Accepted: 21 December 2020Published: 23 December 2020Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.license Antiviral drugs targeted to block virus entry in host cells or to block viral enzymes crucial to virus replication—such as the proteases—constitute the only therapeutic approach to reduce virus load in infected patients and decrease the development of chronic disease [2]. The last years witnessed an explosion of new virus-based biopharmaceuticals. To support fundamental and applied research in virology, development of virusbased biopharmaceuticals, and screening of antivirals, cell-based assays are of outmost importance for functional characterization of viral enzymes and viruses. Regarding the latter, despite viral plaque and colony forming assays [6,7] being considered the “gold standards” for virus detection and quantification, they are time-consuming and lack highthroughput. Faster and easier to perform methods often rely on viral genome coupled licenses/by/4.0/)

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