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

Abstract

Enteroviruses (EVs) are globally important human and animal pathogens which cause a diverse spectrum of disease, ranging from febrile illness to paralysis. Despite decades of research, parts of the EV lifecycle remain poorly understood. Replicons, in which reporter genes replace the structural protein coding region, have proved useful for the study of EV biology. However, it is not possible to study the molecular mechanism(s) of entry, capsid uncoating and genome release without the production of virus particles. To utilise the benefits provided by replicons for the study of viral cell entry, it would be necessary to supply the structural proteins in trans. Here, we present an EV trans-encapsidation (TE) system in which reporter replicons are transfected into cells modified to express the viral structural proteins. The nascent replicons are packaged in trans to form virus particles containing fluorescent or luminescent replicon genomes. This enables the real-time assessment of EV entry and replication through quantification of fluorescence using live-cell imaging. We demonstrate that these TE particles are biologically accurate proxies to EVA71 virions and show utility for the study of EV entry, uncoating and replication. Additionally, we demonstrate the use of TE particles as platforms for drug discovery and immunological screening, applicable to the development of antiviral therapeutics and assessment of immunisation outcomes.

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.

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.

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.

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.

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.