Rabies Glycoprotein-Mediated Uptake Into Epithelial Cells and Compartmentalized Primary Neuronal Culture

Abstract

Rabies virus (RABV) subverts host neuronal circuitry to gain access to the brain where it causes generally incurable, lethal encephalitis. The single glycoprotein (G) dictates two defining steps for infection and neuroinvasion: receptor-mediated endocytosis and transport of virus. We generate two recombinant VSV (rVSV) clones that genetically incorporate G (rVSV RABV G) from the fixed RABV strains, SAD B19 and CVS, to study internalization into epithelial cells and compartmentalized primary cultures of peripheral neurons. Through the use of chemical inhibitors and markers for specific endocytic routes, we demonstrate that the predominant RABV entry route in both epithelial and neuronal cells is dynamin- and clathrin- dependent. Viral endocytosis is mediated by actin-dependent, partially coated clathrin pits as evidenced by live high resolution confocal microscopy of envelopment in epithelial cells and transmission electron micrographs in neuronal and non-neuronal cells. Thus, we corroborate the hypothesis that particle size is the sole viral determinant of actin-dependence of coated pits. Through a combination of high resolution microscopy and infectivity-based approaches, we link molecular mechanisms of viral uptake at the single particle level to productive infection. Targeted pharmacological disruption of endosomal acidification at the neurites or cell bodies of peripheral neurons demonstrates that fusion and viral genome release at the cell body, the site of replication, is a prerequisite for infection. This work extends the current understanding of RABV entry by providing a detailed characterization of endocytosis from the plasma membrane to the site of fusion and correlating it with establishment of infection into neuronal populations relevant for pathogenesis in vivo.