Development of enterovirus transencapsidation assays as tools to understand viral entry.

Enteroviruses (EVs) are globally important human and animal pathogens that cause a diverse spectrum of diseases, 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 utilize the benefits provided by replicons for the study of viral cell entry, it is necessary to supply the structural proteins separately. Here, we present an EV transencapsidation (TE) system in which reporter replicons are introduced into cells that are modified to express viral structural proteins. The nascent replicons are packaged 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 for EVA71 virions and show their 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 the assessment of immunization outcomes.