Real-Time Monitoring of Human Enterovirus (HEV)-Infected Cells and Anti-HEV 3C Protease Potency by Fluorescence Resonance Energy Transfer

Abstract

A real-time assay system that allows monitoring of intracellular human enterovirus (HEV) protease activity was established using the principle of fluorescence resonance energy transfer (FRET). It was accomplished by engineering cells to constitutively express a genetically encoded FRET probe. The FRET-based probe was designed to contain an enterovirus 71 3C protease (3C pro ) cleavage motif flanked by the FRET pair composed of green fluorescent protein 2 and red fluorescent protein 2 (DsRed2). Efficient FRET from the stable line was detected in a real-time manner by fluorescence microscopy, and the disruption of FRET was readily monitored upon HEV infection. The level of the repressed FRET was proportional to the input virus titer and the infection duration as measured by the fluorometric method. The FRET biosensor cell line was also responsive to other related HEV serotypes, but not to the phylogenetically distant herpes simplex virus, which was confirmed by Western blot analysis. The FRET biosensor was then utilized to develop a format for the determination of antiviral susceptibility, as the reduced FRET appeared to reflect viral replication. Evaluations of the FRET biosensor system with representative HEV serotypes demonstrated that their susceptibilities to a 3C pro inhibitor, rupintrivir, were all accurately determined. In summary, this novel FRET-based system is a means for rapid detection, quantification, and drug susceptibility testing for HEVs, with potential for the development of a high-throughput screening assay.

Identification of 23-( S )-2-Amino-3-Phenylpropanoyl-Silybin as an Antiviral Agent for Influenza A Virus Infection In Vitro and In Vivo

“…FRET takes advantage of two fluorescent molecules; in FRET, an excited fluorescent molecule (donor) transfers its energy to a nearby light-absorbing molecule (acceptor) and excites it. This transfer is dependent on the proximity of the two molecules, and the technique is used to analyze molecular interactions quantitatively with spatial and temporal resolution (16)(17)(18)(19). In our study, we constructed three plasmids (pMC-atg5, pMN-atg12, and pEGFP-atg16) to form a BiFC-FRET system in which Atg5 and Atg12 are fused with the N and C fragments of a red fluorescent protein, respectively.…”

Idarubicin is a broad-spectrum enterovirus replication inhibitor that selectively targets the virus internal ribosomal entry site

“…The pCMV-FLAG-2A and pCMV-FLAG-3C plasmids, which encode the EV71 2A pro and 3C pro genes, respectively, have been described previously (Hsu et al, 2007;Tsai et al, 2009). The dicistronic reporter constructs used in this study were based on the pCREL plasmid, a dicistronic plasmid that encodes Renilla luciferase (RLuc) and firefly luciferase (FLuc), with the EMCV IRES in between (Créancier et al, 2000).…”

“…RD (ATCC, CCL-136), Vero (ATCC, CCL-81), HeLa-G2AwtR and HeLa-G3CwtR cells (Hsu et al, 2007;Tsai et al, 2009) were cultured in minimum essential medium (MEM) (Gibco-BRL) supplemented with 10 % FBS. EV stocks including EV71 (strain BrCr), CVA16, CVB1 and -2, Echo9 and -30 (Tsai et al, 2009) were propagated in RD or Vero cell culture with MEM supplemented with 2% FBS, and titrated on a Vero cell monolayer by using a plaque assay. IDR (I1656), DNR (D8809), EPI (E9406) and guanidium hydrochloride (GuHCl) were purchased from Sigma-Aldrich.…”

“…Because the screen with the FRET-biosensor cells measured the virus proteolytic process, we investigated if IDR acts against EV71 2A pro or 3C pro activity by an enteroviral protease assay (Hsu et al, 2007;Tsai et al, 2009). To this end, we used HeLa-G3CwtR cells, another FRET-biosensor cell line that responds specifically to EV71 3C activity (Tsai et al, 2009), in addition to HeLa-G2AwtR cells (Hsu et al, 2007).…”

“…To this end, we used HeLa-G3CwtR cells, another FRET-biosensor cell line that responds specifically to EV71 3C activity (Tsai et al, 2009), in addition to HeLa-G2AwtR cells (Hsu et al, 2007). A 2A pro or 3C pro expression plasmid was used to transfect HeLa-G2AwtR cells (Fig.…”