Development of enterovirus trans-encapsidation assays as tools to understand viral entry

Figures

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  Figure 1.. Generation of GFP-containing TE particles Replication kinetics of A) EVA71-GFP and B) EVA71-mCherry replicons over 24 hours 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 on the Incucyte S3. Assays performed in triplicate, graphed mean and ± SEM n = 3 in triplicate.

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  Figure 2.. Generation of luciferase-containing TE particles A) Luciferase signal generated by EVA71 fLuc replicon 24 hours post transfection. Luciferase activity was measured graphed mean ± SEM, n = 3 in triplicate. B) Cells infected with fLuc-TE particles were assessed for the presence of fLuc after 24 hours. Graphed mean ± SEM n = 3 in triplicate.

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  Figure 3.. Characterisation 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. RTqPCR was used to determine the presence of viral RNA in A) WT virus and B) P1-GFP, graphed mean and ± SEM, n = 3 in duplicate. C) Infectious titre of WT virus and D) P1-GFP particles, graphed mean and ± SEM, n = 3.

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  Figure 4.. Entry inhibition A) Virus neutralisation assay. GFP TE particles were incubated with rat anti-EVA71 immune serum for 1 hour at indicated dilutions. The mixtures were added to pre-seeded HeLa cells, and the fluorescent counts quantified across 24 hours post inoculation. 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 calculated in GraphPad. D) Cell viability with MTS assay after incubation with a concentration series of Bafilomycin A1, normalised to DMSO control at 24-hours. Assays performed in triplicate, graphed mean ± SEM, n = 3 in triplicate.

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  Figure 5.. Replication inhibition HeLa cells were treated with A) enviroxime B) rupintrivir or DMSO and infected with P1-GFP TE particles. Fluorescence measures were normalised to a DMSO control at 24 hours. C) EC50 curves of enviroxime and rupintrivir. EC50 values calculated in GraphPad. D) MTS assay measuring cell viability at 24 hours in the presence of compound, normalised to DMSO. Assays performed in triplicate, graphed mean ± SEM, n = 3 in triplicate.