Imaging Poliovirus Entry in Live Cells

Abstract

Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to–plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubule-independent, but tyrosine kinase- and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100–200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses. During travel between hosts, the genome of a virus is well protected by the viral capsid and/or envelope. After binding specifically to target cells, the virus particles enter cells by hijacking cell trafficking pathways and then deliver the viral genome into the appropriate compartment of the cell where it directs the production of progeny virus particles. How nonenveloped viruses, such as poliovirus, enter target cells is not well understood. Here, we produced fully infectious poliovirus with both genome and capsid specifically labeled by fluorescent dyes. We could then use real-time fluorescent microscopy to follow single virus particles during infection, to define how they enter cells and to determine when and where in the cell the genome gets released. We have complemented the microscopic studies with virological assays, which demonstrate that the pathways observed by microscopy are productive. We show that poliovirus enters live cells in a process that requires energy, an intact actin cytoskeleton, and cell signaling pathways, but does not depend on the well-known markers of endocytic pathways. We show that after internalization, the genome release is surprisingly efficient and occurs from vesicles that are very close to the cell surface. Our experiments offer new insights into the early steps of poliovirus infection, and describe methods that can be used for a wide variety of other viruses.