Visualizing single-cell dynamics of picornavirus replication and virus-host interactions

Abstract

Picornaviruses are a family of positive-sense single-stranded RNA viruses that infect both humans and animals and impose a large socio-economical and health burden. Early replication dynamics and virus-host interactions are increasingly recognized as critical determinants of infection outcome. Thus, to develop effective therapeutic options for picornaviruses requires a good understanding of early infection dynamics and virus-host interactions at the single-cell level. However, conventional experimental approaches are often not sufficiently sensitive, fail to provide temporal or spatial information, or measure thousands of cells simultaneously - averaging out any subtle differences between different individual infected cells. In my PhD I set out to overcome these limitations. To this end, we aimed to develop new microscopy assays to visualize different aspects of early picornavirus infection and virus-host interactions with high sensitivity at the single-cell level. Previously, the Kuppeveld and Tanenbaum labs collaborated to develop the VIrus Infection Real-time IMaging (VIRIM) assay, which allows visualization of translation of the viral RNA with single-molecule sensitivity in single infected cells. Expanding our imaging toolbox, we set up live cell viral RNA imaging assay using the RhoBAST fluorescence light-up aptamer system. Furthermore, we developed a live cell imaging reporter system for double-strand RNA (dsRNA) - a byproduct of viral replication which can be recognized by sensors of the cellular innate immune system such as MDA5 and PKR. This allowed us to examine the role which virus-induced membrane modifications and the ensuing formation of replication organelles play in shielding viral dsRNA from such sensors. Finally, we employed both single-molecule imaging assays and classical virology approaches to study the role that the enterovirus 2A protease plays throughout the entire infection cycle. In particular, we studied how early 2Apro-mediated cleavage events reshape the host cell environment to render it favorable for viral replication and to evade host antiviral response mechanisms.