MAVS-Based Reporter Systems for Real-Time Imaging of EV71 Infection and Antiviral Testing

Abstract

Enterovirus consists of a variety of viruses that could cause a wide range of illness in human. The pathogenesis of these viruses remains incompletely understood and no specific treatment is available. Better methods to study enterovirus infection in live cells will help us better understand the pathogenesis of these viruses and might contribute to antiviral development. Here in this study, we developed fluorescent cell-based reporter systems that allow sensitive distinction of individual cells infected with enterovirus 71 (EV71). More importantly, these systems could be easily used for live-cell imaging by monitoring viral-induced fluorescence translocation after EV71 infection. We further demonstrated that these reporter systems could be used to study other enterovirus-mediated MAVS cleavage and they are sensitive for antiviral activity testing. Therefore, integration of these reporters with modern image-based analysis has the potential to generate new insights into enterovirus infection and facilitate antiviral development.

Keywords: antiviral; enterovirus; fluorescence; live cell imaging; reporter.

Figure 1

MAVS-based reporter systems for detection of EV71 infection. (A) Left: schematic diagram of RFP-NLS-MAVS2 reporter system. Right: schematic diagram of GFP-MAVS2 reporter system; (B) Huh 7 cells or cells stably expressing RFP-NLS-MAVS2 or GFP-MAVS2 were immunostained for MAVS expression with Hoechst nuclear counterstaining. Bar, 20 μm. The fluorescence intensity profiles along the selected lines are shown on the right; (C) Cells stably expressing RFP-NLS-MAVS2 or GFP-MAVS2 were infected with EV71 and fluorescence was monitored at 20 h post-infection. Nuclei were stained with Hoechst (blue). Asterisks indicate infected cells. Bar, 20 μm.

Figure 2

RFP-NLS-MAVS2 reporter could be cleaved by different enteroviruses. (A) Diagram of the RFP-NLS-MAVS and truncations. The locations of the CARD-like domain, proline-rich domain and transmembrane domain are indicated; (B) Huh 7 cells stably expressing the indicated reporter were infected with EV71 (MOI = 5), Echo7 (MOI = 5) or CVB5 (MOI = 2) and fluorescence was monitored at 20 h post-infection. Nuclei were stained with DAPI. Asterisks indicate infected cells. Bar, 20 μm.

Figure 3

GFP-MAVS2 reporter could be cleaved by different enteroviruses. (A) Diagram of the GFP-MAVS and truncations; (B) Huh 7 cells stably expressing the indicated reporter were infected with EV71 (MOI = 5), Echo7 (MOI = 5) or CVB5 (MOI = 2) and GFP or VP3 expression was detected with immunoblotting. Arrowheads indicate cleaved MAVS. β-actin was shown as loading control.

Figure 4

EV71, Echo7 and CVB5 cleave the RFP-NLS-MAVS reporter at the same residues. (A) Sequence alignment of 2A proteins from EV71, Echo7 and CVB5 with WebLogo 3. The letter height is proportional to the degree of amino acid conservation; (B) Huh 7 cells stably expressing the indicated reporter were infected with EV71, Echo7 or CVB5 and fluorescence was monitored at 24 h post-infection. Nuclei were stained with DAPI. Asterisks indicate infected cells. Bar, 20 μm.

Figure 5

Time-lapse live-cell imaging of EV71 infection. (A) Huh 7 cells stably expressing RFP-NLS-MAVS2 (upper and middle panel) or RFP-NLS-MAVS4 (lower panel) were infected with EV71 (MO = 5) and fluorescent images were taken every 2.5 h. Bar, 20 μm; (B) Huh 7 cells stably expressing RFP-NLS-MAVS2 were infected with EV71 and cells were harvested at indicated hours after infection. Cell lysates were immunoblotted with MAVS antibody and VP3 antibody. The arrow indicates cleaved MAVS. β-actin was shown as loading control.

Figure 6

Validation of fluorescence-based reporter system for antiviral drug screening. (A) Schematic diagram of the experiment timeline; (B) Representative images of cells treated with indicated amount of GuHCl. Bar, 20 μm; (C) A dose-response curve of GuHCl treatment on RFP translocation; (D) Cells as described in (A) were immunoblotted with RFP or VP3 antibody. β-actin was shown as loading control; (E) Dose-response curves of GuHCl treatment on cell viability or viral RNA copy numbers. Cells as described in (A) were subjected to q-RT-PCR and viral RNA copy number were plotted against GuHCl concentration. IC₅₀ were calculated with Prism. Cell viability was determined with Cell Titer Glo.