Research Article: Rabies Internalizes into Primary Peripheral Neurons via Clathrin Coated Pits and Requires Fusion at the Cell Body

Date Published: July 27, 2016

Publisher: Public Library of Science

Author(s): Silvia Piccinotti, Sean P. J. Whelan, Mark T. Heise.

http://doi.org/10.1371/journal.ppat.1005753

Abstract

The single glycoprotein (G) of rabies virus (RABV) dictates all viral entry steps from receptor engagement to membrane fusion. To study the uptake of RABV into primary neuronal cells in culture, we generated a recombinant vesicular stomatitis virus in which the G protein was replaced with that of the neurotropic RABV CVS-11 strain (rVSV CVS G). Using microfluidic compartmentalized culture, we examined the uptake of single virions into the termini of primary neurons of the dorsal root ganglion and ventral spinal cord. By pharmacologically disrupting endocytosis at the distal neurites, we demonstrate that rVSV CVS G uptake and infection are dependent on dynamin. Imaging of single virion uptake with fluorescent endocytic markers further identifies endocytosis via clathrin-coated pits as the predominant internalization mechanism. Transmission electron micrographs also reveal the presence of viral particles in vesicular structures consistent with incompletely coated clathrin pits. This work extends our previous findings of clathrin-mediated uptake of RABV into epithelial cells to two neuronal subtypes involved in rabies infection in vivo. Chemical perturbation of endosomal acidification in the neurite or somal compartment further shows that establishment of infection requires pH-dependent fusion of virions at the cell body. These findings correlate infectivity to existing single particle evidence of long-range endosomal transport of RABV and clathrin dependent uptake at the plasma membrane.

Partial Text

Rabies virus (RABV), a member of the Rhabdoviridae family, is a neurotropic pathogen that causes fatal encephalitis in animals and humans. The neurotropism of RABV is conferred by its single attachment and fusion glycoprotein (G) [1]. Virulence of specific RABV strains correlates with the neuroinvasiveness of their G proteins [2], such that exchange of G of an attenuated strain with that of a pathogenic strain and vice versa confers the corresponding level of pathogenicity [1,3–5]. Although differential glycosylation [6,7], dysregulation of G expression levels [8,9], and increased induction of apoptosis [8] all contribute to G-dependent attenuation of RABV strains, it is apparent that a predominant mechanism by which G modulates rabies virulence is by dictating affinity for and spread between neurons.

Here we provide evidence for a model of RABV entry into peripheral neurons that begins with clathrin- and dynamin-mediated uptake at the plasma membrane (Fig 10A). Endocytosed viruses are then transported intact and within endosomes from the distal neurites to the site of fusion at the cell body (Fig 10B). Furthermore, we show that somal fusion is required for efficient infection. This work extends the current understanding of RABV uptake by identifying the predominant internalization mechanism at the plasma membrane of two neuronal populations involved in early neuroinvasion in vivo. In addition, by combining single particle imaging and infectivity studies, we correlate single virion behavior with productive infection.

 

Source:

http://doi.org/10.1371/journal.ppat.1005753

 

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