Rabies Glycoprotein-Mediated Uptake Into Epithelial Cells and Compartmentalized Primary Neuronal Culture The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Piccinotti, Silvia. 2015. Rabies Glycoprotein-Mediated Uptake Into Epithelial Cells and Compartmentalized Primary Neuronal Culture. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:23845493 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA RABIES GLYCOPROTEIN-MEDIATED UPTAKE INTO EPITHELIAL CELLS AND COMPARTMENTALIZED PRIMARY NEURONAL CULTURE A dissertation presented by SILVIA PICCINOTTI to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the subject of VIROLOGY Harvard University Cambridge, Massachusetts August 2015 © 2015 Silvia Piccinotti All rights reserved. Dissertation Advisor: Sean P. Whelan Silvia Piccinotti 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. iii TABLE OF CONTENTS Abstract ....................................................................................................................................................... iii Table of Contents ......................................................................................................................................... iv List of Figures ................................................................................................................................................ v Introduction .................................................................................................................................................. 1 Chapter 1: Uptake of rabies virus into epithelial cells by clathrin-mediated endocytosis depends upon actin ............................................................................................................................................................ 24 Abstract ...................................................................................................................................... 25 Introduction ............................................................................................................................... 26 Materials and Methods .............................................................................................................. 28 Results ........................................................................................................................................ 35 Discussion .................................................................................................................................. 49 Acknowledgments...................................................................................................................... 54 Author Contributions ................................................................................................................. 54 Chapter 2: Clathrin-mediated uptake and long range axoplasmic transport of virions incorporating rabies glycoprotein ..................................................................................................................................... 55 Abstract ...................................................................................................................................... 56 Introduction ............................................................................................................................... 57 Material and Methods ............................................................................................................... 60 Results ........................................................................................................................................ 68 Discussion .................................................................................................................................. 85 Acknowledgments...................................................................................................................... 90 Author Contributions ................................................................................................................. 90 Discussion ................................................................................................................................................... 91 Acknowledgments .................................................................................................................................... 112 References ................................................................................................................................................ 113 Appendix ................................................................................................................................................... 127 Co-uptake of rVSV SAD B19 G virus with exogenously expressed receptor p75NTR into BS-C-1 cells ..................................................................................................................................... 127 Movie Captions for Chapter 1 .................................................................................................. 142 Movie Captions for Chapter 2 .................................................................................................. 143 Movie Captions for Appendix .................................................................................................. 144 iv LIST OF FIGURES Introduction Figure I.1. Global distribution of the risk of contracting rabies. ................................................................... 2 Figure I.2. Rhabdoviral particle structure and morphology. ........................................................................ 4 Figure I.3. Putative rabies receptors and known binding sites. ................................................................... 6 Figure I.4. Model of VSV uptake into actin-dependent, partially coated pits. ........................................... 14 Figure I.5. Model of microtubule engagement for RABV axoplasmic transport. ....................................... 17 Chapter 1 Figure 1.1 Characterization of recombinant VSV expressing RABV G. ....................................................... 33 Figure 1.3. Absence of M protein release and expression in cells treated with BAF A1 or dynasore. ....... 39 Figure 1.4. Dynasore blocks internalization at the plasma membrane. ..................................................... 41 Figure 1.5. Visualization of clathrin-dependent uptake of rVSV RABV G by transmission electron microscopy. ................................................................................................................................................. 43 Figure 1.6. Live-cell imaging and kinetics analysis of rVSV RABV G internalization. .................................. 44 Figure 1.7. Impact of actin depolymerization on rVSV RABV G internalization. ........................................ 46 Chapter 2 Figure 2.1. Production of a recombinant VSV expressing rabies CVS G. .................................................... 66 Figure 2.2. Compartmentalized cultures of dorsal root ganglion and ventral spinal cord neurons. ......... 69 Figure 2.3. Dynasore and EIPA inhibit rVSV CVS G infection in compartmentalized neuronal culture. ..... 71 Figure 2.4. rVSV SAD B19 G infection of compartmentalized DRG neurons is dynamin-dependent. ........ 74 Figure