The Pennsylvania State University The Graduate School College of Agricultural Science HENIPAVIRUS ASSEMBLY AND BUDDING A Dissertation in Pathobiology by Weina Sun © 2015 Weina Sun Submitted in Partial Fulfillments of the Requirements for the Degree of Doctor of Philosophy May 2015 The dissertation of Weina Sun was reviewed and approved* by the following: Anthony P. Schmitt Associate Professor of Molecular Virology Dissertation Advisor Chair of the Committee Director of Pathobiology Graduate Program K. Sandeep Prabhu Professor of Immunology and Molecular Toxicology Pamela A. Hankey Giblin Professor of Immunology Immunology and Infectious Disease Undergraduate Advisor Robert F. Paulson Professor of Veterinary and Biomedical Science Craig E. Cameron Professor of Biochemistry and Molecular Biology Eberly Chair in Biochemistry and Molecular Biology *Signatures are on file in the Graduate School ii ABSTRACT The emergence of a group of paramyxoviruses called henipavirus has caused fatal illness such as severe vasculitis and encephalitis which resulted in high fatality rate in both humans and animals since 1990’s in south Asia and Australia. Like other paramyxoviruses, the essential viral protein that organizes the process of henipavirus assembly and budding is the matrix protein (M), where it functions to link together the viral glycoproteins and ribonucleoprotein complex (RNPs). Current studies have sought out to investigate the host factors that are involved in virus assembly and budding by interacting with M protein to facilitate M functions. In an effort to identify host factors that are important for henipavirus assembly and budding, proteomics-based approach was performed to identify host proteins that interact with viral M protein. Here, we affinity purified viral M proteins by FPLC and identified co-purifying host proteins by mass spectrometry. Co- immunoprecipitation was used as a secondary screening of protein candidates identified from mass spec results. One of the host proteins candidates, AP3B1, the beta subunit of AP-3 complex, was selected for further investigations. Importantly, a 29 amino acids polypeptide derived from AP3B1 hinge domain was identified as the minimal fragment to bind M protein as well as effectively inhibit henipavirus-like particles (VLPs) production. This inhibitory effect is due to disruption of M protein association with membrane. Additionally, in AP3B1 depleted cells by siRNA knockdown, Nipah VLP production is significantly reduced. By immunofluorescence iii microscopy, M protein colocalization with endogenous AP3B1 was also observed in mammalian cells. Our results suggested that AP-3 might play an important role in M protein functions. Henipaviruses have two types of surface glycoproteins, the attachment protein (G) and fusion protein (F). They were responsible for virus entry by mediating virus attachment to cell receptors and fusion with cell membrane. Numerous studies have illustrated how the virus entry occurs under the coordination of G and F proteins. However, little is known about how glycoproteins assemble into virions or VLPs. We know that M protein is the main driving force of particles formation, so we wondered how M protein coordinates with G protein and F protein to facilitate their trafficking and assembly into virions or VLPs. Here, under the unique pathway of HeV F protein identified by our collaborator Dr. Rebecca Dutch at University of Kentucky, we have found that HeV M protein, F protein and G protein all partially colocalized with Rab11a-REs, and overexpression of a DN Rab11a could significantly inhibit Hendra M-VLPs as well as F-VLPs formation in the cells. Interestingly, in cells that express inhibitory AP3B1 Hinge domain, M colocalization with Rab11-REs was disrupted. Unlike M protein or F protein, G proteins releases poorly into particles when expressed alone in cells. We observed that G incorporation into VLPs could be facilitated by co-expression of M protein but not F protein, likely through G-M interaction at the plasma membrane. Moreover, we have found that F protein incorporation into VLPs depended on its endocytic trafficking event regardless its cleavage status, as an endocytosis defective mutant F S490A that was retained on iv the cell surface was shown to be significantly defective on particles assembly. On the other hand, HeV F protein still well incorporated into VLPs when its cleavage was prevented by a cathepsin L inhibitor, E-64d. Therefore, we hypothesized that henipavirus M and F proteins have separate mechanisms for trafficking to Rab11-REs, with the M protein trafficking facilitated by its interaction with AP3B1. M and cathepsin-cleaved F proteins must then assemble together within these compartments prior to their delivery to the cell surface for particle budding and assemble with G protein through G-M interaction at the plasma membrane. v TABLE OF CONTENTS LIST OF FIGURES ................................................................................................... ix LIST OF TABLES ..................................................................................................... xi LIST OF ABBREVIATIONS ...................................................................................... xii CHAPTER 1 ............................................................................................................ 1 Introduction .......................................................................................................... 1 1.1 Paramyxovirus classification and significance ............................................... 1 1.2 Paramyxovirus genome and replication ........................................................ 6 1.3 Paramyxovirus matrix (M) protein and virus assembly ............................... 10 1.4 Virus-like particles production ..................................................................... 15 1.5 Trafficking of paramyxovirus glycoproteins ................................................. 17 1.6 Adaptor protein (AP) complexes ................................................................. 20 1.7 Adaptor protein 3 (AP-3) complex ............................................................... 23 1.8 AP-3 complex and viral trafficking ............................................................... 24 1.9 Rab11 GTPase in negative-strand RNA virus assembly ................................ 26 1.10 Identification of protein-protein interactions between virus and host ......... 29 1.11 Preview ........................................................................................................... 31 CHAPTER 2 .......................................................................................................... 34 Materials and Methods ....................................................................................... 34 Plasmids .................................................................................................................. 34 Henipavirus M protein affinity purification and mass spectrometry ..................... 37 Coimmunoprecipitation .......................................................................................... 38 Measurements of VLP production .......................................................................... 39 Membrane flotation assays to measure M protein membrane association .......... 41 RNA interference (RNAi) ......................................................................................... 41 Immunofluorescence microscopy ........................................................................... 42 Brefeldin A (BFA) treatment ................................................................................... 44 Cathepsin L inhibition VLP assay ............................................................................. 45 Phosphoinositide 3-kinase (PI3K) inhibition VLP assay .......................................... 46 Sedimentation gradient analysis of Hendra VLPs density ...................................... 46 vi Subcellular fractionation ......................................................................................... 47 CHAPTER 3 .......................................................................................................... 49 Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes .......................................................................................................... 49 3.1 Introduction ...................................................................................................... 49 3.2 Results ............................................................................................................... 51 Identification of henipavirus M-associating host proteins by affinity purification and mass spectrometry ...................................................................................... 51 AP3B1 interacts with Henipavirus M proteins via its hinge domain .................. 55 Overexpression of M-binding, AP3B1-derived polypeptides blocks production of henipavirus VLPs ................................................................................................. 61 Depletion of AP3B1 from cells impairs Nipah VLP production ........................... 66 Partial co-localization of Nipah virus M protein with endogenous AP3B1 in transfected cells .................................................................................................. 68 3.3 Discussion .........................................................................................................