Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

Abstract

dRabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin. R abies virus (RABV) is the prototypical member of the zoonotic lyssavirus genus responsible for fatal encephalitis in animals and humans. A single-stranded negative-sense RNA virus, RABV encases its RNA genome in a bullet-shaped, enveloped particle that incorporates a single surface glycoprotein (G). RABV G mediates all internalization steps from cell binding to membrane fusion. In addition, G is a major determinant of RABV neurotropism (1). Conjugation or pseudotyping with the ectodomain of pathogenic RABV G or peptides derived from receptor-binding regions allows retargeting of biologically active molecules to the central nervous system (CNS) for drug delivery or as neurotracers (2, 3). Since a large part of RABV pathogenesis is reliant on the virus garnering access to neurons and the CNS, G is also a determining factor in RABV virulence. The pathogenicity of attenuated strains can be effectively increased by replacing the glycoprotein with one from a neurotropic, virulent strain (4).Like other rhabdoviruses, RABV gains access to the cellular interior by endocytosis and subsequent low pH-dependent fusion (5-7). Electron micrographs of viral particles in vesicles with electron-dense coats suggest that clathrin-coated pits mediate the uptake of RABV in both neuronal and nonneuronal cells (8, 9). However, static images cannot inform on the fate of such particles or the relevance of these interactions for subsequent infection. High-resolution live-imaging techniques permit tracking of viral uptake into coated pits (10-15). Fluorescence tagging of coatedpit components and quantitative analysis methods have revealed differences for the pits engaging fluoresc...

Infectious Entry Pathway Mediated by the Human Endogenous Retrovirus K Envelope Protein

“…Since the morphology of the VSV particle dictates the requirement for actin to complete the process of internalization when entering via a clathrin-mediated process (40,48), the lack of a requirement for actin polymerization indicates that HERV-K ENV does not enter via the same clathrin-mediated entry pathway as VSV. Together, these data suggest that HERV-K ENV dictates an entry pathway that is dynamin dependent but clathrin and macropinocytosis independent, which is distinct from what we have observed with other recombinant VSVs (49,54). Available evidence supports the notion that MMTV also enters cells via clathrin-independent endocytic uptake; however, the requirement for dynamin or macropinocytosis is unknown (19).…”

Single-walled carbon nanotube exposure induces membrane rearrangement and suppression of receptor-mediated signalling pathways in model mast cells

“…Clathrin-mediated endocytosis is an entry mechanism for various environmental factors including bacteria, viruses and other particulates (Cureton et al, 2010; Pauly and Drubin, 2007; Pelkmans and Helenius, 2003; Piccinotti et al, 2013; Veiga and Cossart, 2006; Yamauchi and Helenius, 2013). Our data suggest that further experiments might be required to assess whether SWCNT also exploit this cellular entry mechanism.…”

G gene-deficient single-round rabies viruses for neuronal circuit analysis

“…Upon binding of RABV to the cell membrane, the virus is internalized by endocytosis, predominantly via partially clathrin-coated pits and in an actin-dependent manner (Piccinotti et al, 2013), which resembles the mechanism observed for VSV (Cureton et al, 2010). In neurons, following internalization at the axon terminal, incoming RABV virions are actively transported in a retrograde manner towards the cell body with or within vesicles containing p75NTR Klingen et al, 2008).…”