Detection and Quantification of Label-Free Infectious Adenovirus Using a Switch-On Cell-Based Fluorescent Biosensor

Abstract

Reliable and fast viral detection and quantification protocols are a requirement for the advance of basic research and clinical approaches with wild type or recombinant viruses. However, available cell-based assays are either timeconsuming or require labeled viral particles, which may alter virus biology or pose safety issues in clinical applications. Since adenoviruses constitute a major healthcare burden but also, when engineered, widely used vectors in vaccination and gene and oncolytic therapies, herein we developed a genetically encoded switch-on fluorescent biosensor consisting of a cyclized Green fluorescent proteincVisensorwith an adenoviral protease cleavable site as a switch. After initial sensor optimization (35% increase in performance), whole-cell biosensors were establishedby stably expressing cVisensor in mammalian cellsand used for live-cell monitoring of adenovirus infection as the intracellular biosensor is specifically activated by the viral protease. A rapid flow cytometry-based bioassay using cVisensor cells was established 48 h postinfection, showing an estimated limit of detection of 10 5 infectious particles/mL, in-line with previously reported flow cytometry assays requiring labeled virus, and significantly faster than standard plaque-forming assays requiring up to 14 days. cVisensor was also successfully applied in the detection of HIV-1 protease activity, validating its wider potential for the detection of other viruses. Overall, this work presents a fast and easy method for detection and quantification of label-free infectious virus, allowing the establishment of new biosensing platforms for basic research in virology and biotechnological applications of recombinant virus biopharmaceuticals.

Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells

“…To screen the GR effectors, we designed a sensing system based on the well-studied pair of Nostoc punctiforme (Npu) DnaE split inteins, which mediate fast and efficient protein trans-splicing (PTS) reactions [ 30 , 31 , 32 ]. The key to our approach is that cortisol-bound GR translocates to the nucleus, triggering CPS to reconstitute the split signal peptide to transport the fluorescent cargo ( Scheme 2 ).…”

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

“…Development of virus-induced cell-based biosensors is essential not only for functional characterization of label-free virus but also critical enzymes-e.g., viral proteases. In that regard, we previously developed cVisensor, a virus-induced biosensor where a circular permuted GFP is cyclized by Npu DnaE split intein and the structural distortion maintained until the cleavable sequence is recognized by the AVP [30]. As successive steps of proteolysis and fragment assembly are necessary to reconstitute fluorescence, split GFP-based sensors should theoretically present higher sensitivity and dynamic range than structurally distorted full-length GFP designs.…”

“…Label-free human recombinant E1-deleted adenovirus serotype 5 (AdV; provided by Dr. Geneviève Libeau, CIRAD-EMVT, Montpellier, France) were amplified, purified and stored as previously described [30]. AdV stocks were quantified by tissue culture infectious dose 50 assay in 293 cells according to the method of Spearman and Kärber, described in Darling et al [31].…”

Virus detection using nanosensors

“…They first optimized nanosensor and then established virus detection by stable expression in mammalian cells and utilized for livecell monitoring of adenovirus infection. They obtained a flow cytometry-based assay using cVisensor cells 48 hours postinfection and showed the limit of detection value as 10 5 infectious particles/mL [108].…”