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.
Highlights
As obligatory intracellular parasites with limited genetic capacity, viruses have evolved to hijack intrinsic cellular pathways to enter the cell and deliver their genomes to specific cellular locations for replication
After binding to target cells, the virus particles enter cells by hijacking cell trafficking pathways and deliver the viral genome into the appropriate compartment of the cell where it directs the production of progeny virus particles
We show that after internalization, the genome release is surprisingly efficient and occurs from vesicles that are very close to the cell surface
Summary
As obligatory intracellular parasites with limited genetic capacity, viruses have evolved to hijack intrinsic cellular pathways to enter the cell and deliver their genomes to specific cellular locations for replication. Mechanistic understandings of viral entry may lead to new therapies for combating viral infection, and provide new insights into fundamental cellular functions [1]. A number of distinct strategies have been exploited for viral entry and gene delivery. Protein-assisted fusion of viral and cellular membranes provides a conceptually simple mechanism for capsid or genome release into the cytoplasm [2]. The mechanism is less well understood, but appears to rely upon viral capsid proteins (VPs) to disrupt cellular membranes or to form pores through them [3]. The cellular sites where genome release occurs are unknown for most nonenveloped viruses
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