Development of enterovirus transencapsidation assays as tools to understand viral entry

Figures

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  Fig. 1.. Generation of fluorescent reporter-containing TE particles. Replication kinetics of (a) EVA71-GFP and (b) EVA71-mCherry replicons over 24 h after transfection into HeLa cells, alongside GNN controls for input translation. (c) Diagrammatic representation of the protocol used to generate and assess TE particles (BioRender). (d) Kinetics of P1-GFP and (e) P1-mCherry TE infection assays. HeLa cells were infected with TE particles alongside mock controls. The production of fluorescence was monitored across 24 h using an Incucyte S3. Total green or red object integrated intensity is presented as green or red fluorescence. Assays were performed in triplicate, graphed as mean ± sem (n=3 in triplicate).

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  Fig. 2.. Generation of luciferase-containing TE particles. (a) Luciferase signal generated by the EVA71 fLuc replicon 24 h post-transfection. Luciferase activity was measured and graphed as mean ± sem (n=3 in triplicate). (b) Cells infected with fLuc-TE particles were assessed for the presence of fLuc after 24 h. Graphed as mean ± sem (n=3 in triplicate).

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  Fig. 3.. Characterization of TE particles and virus. Samples of WT EVA71 and P1-GFP TE particles were recovered directly from in vitro-transcribed RNA electroporated into HeLa cells and separated on 15–45% sucrose gradients. RT-qPCR was used to determine the presence of viral RNA in (a) WT EVA71 virus and (b) P1-GFP, with total genome content presented as the percentage of genome detected within the gradient, graphed as mean ± sem (n=3 in duplicate). (c) Infectious titre of WT virus and (d) P1-GFP particles (24 h timepoint), with total green object integrated intensity presented as green fluorescence, graphed as mean ± sem (n=3).

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  Fig. 4.. Entry inhibition. (a) Virus neutralization assay. GFP TE particles were incubated with rat anti-EVA71 immune serum for 1 h at the indicated dilutions. The mixtures were added to pre-seeded HeLa cells, and the fluorescent counts quantified 24 h post-inoculation using an Incucyte S3. (b) HeLa cells were treated with bafilomycin A1 or DMSO and infected with P1-GFP TE particles. (c) EC50 curve of bafilomycin A1. EC50 values were calculated in GraphPad. Total green object integrated intensity is presented as green fluorescence, normalized to the DMSO control at 24 h. (d) Cell viability with an MTS assay after incubation with a concentration series of bafilomycin A1, normalized to the DMSO control at 24 h. Assays were performed in triplicate, graphed as mean ± sem (n=3 in triplicate). MTS is the standard nomenclature for this assay, the chemical used is: (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

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  Fig. 5.. Replication inhibition. HeLa cells were treated with (a) enviroxime, (b) rupintrivir or DMSO and infected with P1-GFP TE particles. Fluorescence measurements were normalized to a DMSO control at 24 h. (c) EC50 curves of enviroxime and rupintrivir. EC50 values were calculated in GraphPad. Total green object integrated intensity is presented as green fluorescence, normalized to the DMSO control at 24 h. (d) MTS assay measuring cell viability at 24 h in the presence of compound, normalized to DMSO. Assays were performed in triplicate, graphed as mean ± sem (n=3 in triplicate). MTS is the standard nomenclature for this assay, the chemical used is: (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).