Real-Time Imaging of Polioviral RNA Translocation across a Membrane

Genome transfer from a virus into a cell is a critical early step in viral replication. Enveloped viruses achieve the delivery of their genomes into the cytoplasm by merging the viral membrane with the cellular membrane via a conceptually simple mechanism called membrane fusion. In contrast, genome translocation mechanisms in nonenveloped viruses, which lack viral membranes, remain poorly understood. Although cellular assays provide useful information about cell entry and genome release, it is difficult to obtain detailed mechanistic insights due both to the inherent technical difficulties associated with direct visualization of these processes and to the prevalence of nonproductive events in cellular assays performed at a very high multiplicity of infection. To overcome these issues, we developed an in vitro single-particle fluorescence assay to characterize genome release from a nonenveloped virus (poliovirus) in real time using a tethered receptor-decorated liposome system. Our results suggest that poliovirus genome release is a complex process that consists of multiple rate-limiting steps. Interestingly, we found that the addition of exogenous wild-type capsid protein VP4, but not mutant VP4, enhanced the efficiency of genome translocation. These results, together with prior structural analysis, suggest that VP4 interacts with RNA directly and forms a protective, membrane-spanning channel during genome translocation. Furthermore, our data indicate that VP4 dynamically interacts with RNA, rather than forming a static tube for RNA translocation. This study provides new insights into poliovirus genome translocation and offers a cell-free assay that can be utilized broadly to investigate genome release processes in other nonenveloped viruses.