Reovirus uses macropinocytosis-mediated entry and fast axonal transport to infect neurons

Several barriers protect the central nervous system (CNS) from pathogen invasion. Yet viral infections of the CNS are common and often debilitating. Understanding how neurotropic viruses co-opt host machinery to overcome challenges to neuronal entry and transmission is important to combat these infections. Neurotropic reovirus disseminates through neural routes and invades the CNS to cause lethal encephalitis in newborn animals. To define mechanisms of reovirus neuronal entry and directional transport, we used primary neuron cultures, which reproduce in vivo infection patterns displayed by different reovirus serotypes. Treatment of neurons with small-molecule inhibitors of different endocytic uptake pathways allowed us to discover that the cellular machinery mediating macropinocytosis is required for reovirus neuronal entry. This mechanism of reovirus entry differs from clathrin-mediated endocytosis, which is used by reovirus to invade non-neuronal cells. Analysis of reovirus transport and release from isolated soma or axonal termini of neurons cultivated in microfluidic devices indicates that reovirus is capable of retrograde but only limited anterograde neuronal transmission. The dynamics of retrograde reovirus movement are consistent with fast axonal transport coordinated by dynein along microtubules. Further analysis of viral transport revealed that multiple virions are transported together in axons within non-acidified vesicles. Reovirus-containing vesicles acidify after reaching the soma, where disassembly of virions and release of the viral core into the cytoplasm initiates replication. These results define mechanisms of reovirus neuronal entry and transport and establish a foundation to identify common host factors used by neuroinvasive viruses. Furthermore, our findings emphasize consideration of cell type-specific entry mechanisms in the tailored design of neurotropic viruses as tracers, oncolytic agents, and delivery vectors. Author summary Viral infections of the central nervous system (CNS) cause a significant health burden globally and compel a better mechanistic understanding of neural invasion by viruses to develop effective interventions. Neurotropic reovirus disseminates through neural routes to infect the CNS and serves as a tractable model to study neural invasion by viruses. Despite knowledge of reovirus neurotropism for decades, mechanisms mediating reovirus neuronal infection remain undefined. We used primary neurons cultured in microfluidic devices to study entry and directional transport of reovirus. We discovered that reovirus uses macropinocytosis for neuronal entry as opposed to the use of a clathrin-mediated pathway in non-neuronal cells. We are unaware of another virus using macropinocytosis to enter neurons. Following internalization, reovirus spreads in the retrograde direction using dynein-mediated fast axonal transport but exhibits limited anterograde spread. We further demonstrate that reovirus disassembly and replication occur in the neuronal soma subsequent to axonal transport. Remarkably, these entry and transport mechanisms mirror those used by misfolded proteins implicated in neurodegenerative diseases. Our findings establish the mechanics of reovirus neuronal uptake and spread and provide clues about therapeutic targets to limit neuropathology inflicted by pathogens and misfolded proteins.