University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Defining A Regulatory Role For The Hsv Glycoprotein B Membrane Proximal Region In Membrane Association Spencer Shelly University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biology Commons, and the Virology Commons Recommended Citation Shelly, Spencer, "Defining A Regulatory Role For The Hsv Glycoprotein B Membrane Proximal Region In Membrane Association" (2013). Publicly Accessible Penn Dissertations. 799. https://repository.upenn.edu/edissertations/799 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/799 For more information, please contact [email protected]. Defining A Regulatory Role For The Hsv Glycoprotein B Membrane Proximal Region In Membrane Association Abstract Herpes simplex virus (HSV) entry requires four essential glycoproteins (gD, gH/gL, and gB) to enable fusion between the virion envelope and the cellular membrane. The fusion cascade is activated by gD binding to one of its receptors, nectin-1 or HVEM. Glycoprotein B (gB), a class III viral fusion protein, mediates the fusion reaction, while data indicates that gH/gL acts as a regulator of gB. gB is trimeric and has a 773 amino acid ectodomain with a highly hydrophobic membrane proximal region (MPR) (residues 731-773) and two fusion loops (FL) per protomer. The post-fusion structure of gB was solved from the gB(730t) construct, which is truncated to remove the hydrophobic MPR residues. In this dissertation I investigated the MPRs influence on gBs ability ot interact with membranes. I hypothesize that the MPR regulates fusion loop exposure by interacting with the fusion loops and masks them until fusion begins. To investigate this process I constructed a series of MPR deletion, truncation, and point mutations using both full-length mammalian expression vectors and purified baculovirus expressed protein. I found that deletions in the MPR from full-length gB resulted in a disruption in cell surface expression in transfected cells. This suggests the MPR is necessary for proper folding or transport of gB. Soluble gB MPR truncations [gB(759t), gB(749t), gB(739t)] were expressed and purified using the baculovirus expression system, and compared to MPR-less gB(730t) and full MPR containing gB(773t). I found that gB containing an MPR segment were all compromised in their ability to bind liposomes in comparison to gB(730t), which lacks any MPR residues. Supporting our hypothesis we found that residues 731 to 739 were sufficient epr vent liposome association and mutation of two aromatic residues, F732 and F739, to alanine in gB(739t) restored gBs ability to bind liposomes. Together, my data suggests the MPR does indeed regulate gBs ability to associate with liposomes, and that aromatic residues in the MPR are important for this function. This supports our model that the MPR masks the gB FLs to prevent premature membrane association and adds another layer of regulation to the HSV entry cascade. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Roselyn J. Eisenberg Second Advisor Gary H. Cohen Keywords entry, fusion, herpesvirus, hsv Subject Categories Biology | Virology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/799 DEFINING A REGULATORY ROLE FOR THE HSV GLYCOPROTEIN B MEMBRANE PROXIMAL REGION IN MEMBRANE ASSOCIATION Spencer S. Shelly A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2013 Supervisor of Dissertation Co-Supervisor of Dissertation ___________________________ _________________________ Roselyn J. Eisenberg Gary H. Cohen Professor of Microbiology Professor of Microbiology Graduate Group Chairperson ___________________________ Daniel S. Kessler, Associate Professor of Cell and Developmental Biology Dissertation Committee Paul F. Bates, Professor of Microbiology Jeffrey M. Bergelson, Professor of Pediatrics Harvey M. Friedman, Professor of Medicine Yan Yuan, Professor of Microbiology DEDICATION For my wife and daughter ii ACKNOWLEDGMENTS The work presented in this thesis could not have been completed without the tremendous support provided by many individuals. I would like to express my gratitude to my advisors, Gary Cohen and Roselyn Eisenberg, for allowing me to join the laboratory and for the tremendous support and guidance they provided in all aspects of this work. Their continuous enthusiasm and dedication is inspiring. Also, I need to thank Michael Atchison for his personal support and his commitment to the VMD/PhD program. I must also thank the members of my thesis committee Paul Bates, Jeff Bergelson, Harvey Freidman, and Yan Yuan for helping me throughout this process. My thanks to Sara Cherry and the members of the Cherry laboratory where I learned a great deal. I also thank the fantastic group of people that makes up the Cohen/Eisenberg laboratory. A big thanks to the entire lab past and present, including Doina Atanasiu, Chwan Hong Foo, John Gallagher, Huan Lou, Manuel Ponce-de-Leon, Wan Ting Saw, Katie Stiles, and Chuck Whitbeck. I’d especially like to thank Tina Cairns who helped with many aspects of this project, and made completion of this work possible. I’m incredibly grateful for my wonderful wife and daughter, and their constant support and encouragement. Lastly, I would like to thank my parents for their unconditional support. iii ABSTRACT DEFINING A REGULATORY ROLE FOR THE HSV GLYCOPROTEIN B MEMBRANE PROXIMAL REGION IN MEMBRANE ASSOCIATION Spencer S. Shelly Roselyn J. Eisenberg Gary H. Cohen Herpes simplex virus (HSV) entry requires four essential glycoproteins (gD, gH/gL, and gB) to enable fusion between the virion envelope and the cellular membrane. The fusion cascade is activated by gD binding to one of its receptors, nectin-1 or HVEM. Glycoprotein B (gB), a class III viral fusion protein, mediates the fusion reaction, while data indicates that gH/gL acts as a regulator of gB. gB is trimeric and has a 773 amino acid ectodomain with a highly hydrophobic membrane proximal region (MPR) (residues 731-773) and two fusion loops (FL) per protomer. The post-fusion structure of gB was solved from the gB(730t) construct, which is truncated to remove the hydrophobic MPR residues. In this dissertation I investigated the MPRs influence on gBs ability to interact with membranes. I hypothesize that the MPR regulates fusion loop exposure by interacting with the fusion loops and masks them until fusion begins. To investigate this process I constructed a series of MPR deletion, truncation, and point mutations using iv both full-length mammalian expression vectors and purified baculovirus expressed protein. I found that deletions in the MPR from full-length gB resulted in a disruption in cell surface expression in transfected cells. This suggests the MPR is necessary for proper folding or transport of gB. Soluble gB MPR truncations [gB(759t), gB(749t), gB(739t)] were expressed and purified using the baculovirus expression system, and compared to MPR-less gB(730t) and full MPR containing gB(773t). I found that gB containing an MPR segment were all compromised in their ability to bind liposomes in comparison to gB(730t), which lacks any MPR residues. Supporting our hypothesis we found that residues 731 to 739 were sufficient prevent liposome association and mutation of two aromatic residues, F732 and F739, to alanine in gB(739t) restored gBs ability to bind liposomes. Together, my data suggests the MPR does indeed regulate gBs ability to associate with liposomes, and that aromatic residues in the MPR are important for this function. This supports our model that the MPR masks the gB FLs to prevent premature membrane association and adds another layer of regulation to the HSV entry cascade. v TABLE OF CONTENTS Page DEDICATION ............................................................................................................... ii ACKNOWLEDGEMENTS ......................................................................................... iii ABSTRACT ................................................................................................................... iv TABLE OF CONTENTS ............................................................................................. vi LIST OF TABLES ...................................................................................................... viii LIST OF ILLUSTRATIONS ....................................................................................... ix CHAPTER 1: GENERAL INTRODUCTION ............................................................1 A. Herpesvirus overview ...........................................................................................1 B. Virion structure .....................................................................................................4 C. HSV replication cycle ...........................................................................................5 D. Viral entry strategies .............................................................................................9 E. Viral fusion proteins ............................................................................................11 a. Class I .......................................................................................................12