Genome Packaging and Host-‐Pathogen Interactions In
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The Pennsylvania State University The Graduate School Department of Veterinary and Biomedical Sciences GENOME PACKAGING AND HOST-PATHOGEN INTERACTIONS IN PARAMYXOVIRUS ASSEMBLY AND BUDDING A Dissertation in Pathobiology by Greeshma Ray © 2016 Greeshma Ray Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2016 ii The dissertation of Greeshma Ray was reviewed and approved* by the following: Anthony P. Schmitt Associate Professor of Molecular Virology Dissertation Advisor Chair of Committee Director of Pathobiology Graduate Program K. Sandeep Prabhu Professor of Immunology and Molecular Toxicology Robert F. Paulson Professor of Veterinary and Biomedical Sciences Richard T. Frisque Professor of Molecular Virology Associate Department Head for Equity and Diversity *Signatures are on file in the Graduate School iii ABSTRACT Paramyxoviruses possess non-segmented, negative-sense RNA genomes that are encapsidated by viral nucleocapsid protein (NP), and are packaged into budding virus particles via NP protein interaction with viral matrix (M) proteins, thereby creating infectious viruses. We had previously identified a DLD sequence near the C- terminal end of parainfluenza virus 5 (PIV5) NP protein that was important for interaction with PIV5 M protein, and for enhancing PIV5 virus-like particle (VLP) production. We have shown here that a DLD-containing 15 amino acid sequence derived from PIV5 NP or Nipah virus N proteins is sufficient to direct a foreign protein, Renilla luciferase, into virus-like particles. This short DLD-containing sequence was also able to replace the requirement for NP protein in PIV5 VLP production. Mumps virus NP protein contains a DWD sequence instead of DLD, and consequently, PIV5 NP protein cannot interact efficiently with mumps virus M protein, in spite of the two viruses being very closely related. Altering PIV5 NP DLD sequence to mumps virus NP DWD sequence creates a new PIV5 NP protein that is now compatible with M proteins from both PIV5 and mumps virus. We hypothesize that DLD-like sequences define compatibilities between paramyxovirus M and NP proteins that can be manipulated to drive production of virus-like particles containing therapeutics for delivery to cells. A hallmark of enveloped virus infection is the hijacking of host cellular pathways during viral egress. To define host factors involved in paramyxovirus budding, a yeast two-hybrid screening approach was used previously, which identified iv angiomotin-like 1 (AmotL1) as a host factor that binds to the matrix (M) proteins of parainfluenza virus 5 (PIV5) and mumps virus. AmotL1 belongs to a family of proteins that also includes angiomotin (Amot) and angiomotin-like 2 (AmotL2). All three angiomotins harbor PPXY motifs and interact with WW-domain containing proteins such as Nedd4 and YAP. We found that the PIV5 and mumps virus M proteins bind to AmotL1, but not to Amot or AmotL2. Binding was mapped to a 83- amino acid region from the C-terminal portion of AmotL1. Overexpression of M- binding AmotL1-derived polypeptides inhibited production of PIV5 and mumps virus-like particles (VLPs), while overexpression of the corresponding regions of Amot and AmotL2 had little effect on VLP production. Co-immunoprecipitation studies support the presence of a three-way interaction between Nedd4, AmotL1, and M. Efficient pulldown of M with Nedd4 was observed only when AmotL1 was expressed to bridge the gap. Our findings support a model in which paramyxoviruses indirectly recruit the same WW domain-containing proteins to virus assembly sites through AmotL1 that other enveloped viruses recruit directly via PPXY late domains. v TABLE OF CONTENTS LIST OF FIGURES…………………………………………………………………………………………vii LIST OF TABLES……………………………………………………………………………………………ix LIST OF ABBREVIATIONS………………………………………………………………………………x ACKNOWLEDGMENTS…………………………………………………………………………………xii CHAPTER 1……………………………………………………………………………………………………1 LITERATURE REVIEW……………………………………………………………………………………1 1.1 Significance and classification of paramyxoviruses………………………………………...1 1.2 Structure and composition……………………………………………………………………………7 1.3 Life cycle of paramyxoviruses……………………………………………………………………..14 1.4 Paramyxovirus virus-like particles……………………………………………………………...17 1.5 Paramyxovirus matrix-nucleocapsid interactions………………………………………..18 1.6 ESCRT pathway and late domains……………………………………………………………….19 1.7 Angiomotins………………………………………………………………………………………………23 1.8 Preview...…………………………………………………………………………………………………...27 CHAPTER 2………………………………………………………………………………………………….30 MATERIALS AND METHODS……………………………………………………………………......30 Plasmids………………………………………………………………………………………………………….30 Antibodies……………………………………………………………………………………………………….32 Membrane co-flotation assays measuring M-NP interactions……………………………..33 Measurements of VLP production……………………………………………………………………..34 Measurement of luciferase activity……………………………………………………………………36 Co-immunoprecipitation assays………………………………………………………………………..36 Immunofluorescence microscopy……………………………………………………………………..37 Amino acid sequence comparisons…………………………………………………………………...38 CHAPTER 3………………………………………………………………………………………………….39 C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles…………………………………………………………………..39 3.1 Introduction……………………………………………………………………………………………….39 3.2 Results……………………………………………………………………………………………………….42 Manipulation of genome packaging interactions to direct a foreign protein into PIV5 VLPs…………………………………………………………………….………………………………………….42 PIV5 NP protein and mumps virus M protein can be engineered for compatibility…………………………………………………………………………………………………...46 Alterations affecting the C-terminal end of mumps virus NP protein impair interaction with M protein and block VLP production………………………………………..52 Manipulation of genome packaging interactions to direct a foreign protein into Nipah VLPs………………………………………………………………………………………………………54 vi Amino acid residues 523 to 528 of N protein are important for RLuc packaging into Nipah VLPs………………………………………………………………………………………………………55 3.3 Discussion………………………………………………………………………………………………….59 CHAPTER 4………………………………………………………………………………………………….63 Role of angiomotin-like 1 in paramyxovirus budding…………………………………63 4.1 Introduction. …………………………………………………………………………………………......63 ESCRT and viral late domains…………………………………………………………………………...63 Angiomotins and virus budding……………………………………………………………………......67 4.2 Results……………………………………………………………………………………………………….69 PIV5 M binds selectively to AmotL1………………………………………………………………….69 The C-terminal fragment of AmotL1, and not that of AmotL2 or Amotp130, can inhibit PIV5 VLP production..…………………………………………………………………………...70 AmotL1 can bind to different members of the Nedd4 family of E3 ubiquitin ligases…………………………………………………………………….……………………………………….74 PIV5 M, AmotL1 and Nedd4-like proteins interact and form a complex……………...76 PIV5 M, Nedd4L and AmotL1 co-localization within cells………………………………......79 Mumps virus matrix protein binds only AmotL1, not AmotL2 or Amotp130…….....85 4.3 Discussion.………………………………………………………………………………………………....86 CHAPTER 5………………………………………………………………………………………………....91 SUMMARY AND FUTURE DIRECTIONS…………………………………………………………91 5.1 Summary…………………………………………………………………………………………………...91 5.2 Future directions…………………………………………………………………………………….....94 Manipulating genome packaging interactions for therapeutic uses……………………94 Examining compatibilities between M-NP proteins from different paramyxoviruses…………………………………………………………………………………………….96 Studying the role of angiomotins and Nedd4 ligases in paramxovirus budding.....97 APPENDICES…………………………………………………………………………………………….100 Appendix A: Packaging of foreign proteins into PIV5 virus-like particles and delivery into cells…………………………………………………………………………………..100 Appendix B: Packaging foreign proteins into mumps virus-like particles………..105 Appendix C: PIV5 M proteins with PPXY late domains can bind directly to Nedd4L………………………………………………………………………………………………………..108 BIBLIOGRAPHY………………………………………………………………………………………..110 vii LIST OF FIGURES Figure 1.1. Phylogenetic tree of Paramyxoviridae- subfamily Paramyxovirinae……………………………………………………………………………………………4 Figure 1.2. Gene orders among different paramyxoviruses………………………………8 Figure 1.3. Structure of a paramyxovirus virion………………………………………………8 Figure 1.4. Paramyxovirus life cycle……………………………………………………………...16 Figure 1.5. The ESCRT pathway in viral budding……………………………………………23 Figure 3.1. A total of 15 C-terminal residues of PIV5 NP protein are sufficient to trigger VLP production and to direct a foreign protein into budding particles...44 Figure 3.2. C-terminal ends of paramyxovirus N/NP proteins………………………...45 Figure 3.3. DLD and DWD sequences define compatibilities between PIV5 and mumps virus M/NP protein pairs………………………………………………………………….48 Figure 3.4. Compatibilities of PIV5 and mumps virus M/NP protein pairs measured by membrane coflotation analysis…………………………………………………………………50 Figure 3.5. PIV5 NP L507W protein retains its compatibilities with PIV5 and Nipah virus M proteins…………………………………………………………………………………………...51 Figure 3.6. The DWD-containing C-terminal region of mumps virus NP protein is important for its VLP production function……………………………………………………..53 Figure 3.7. A total of 15 C-terminal residues of Nipah virus N protein are sufficient to direct a foreign protein into budding particles………………………………………………56 Figure 3.8. Amino acid residues 523-NDLDFV-528 within Nipah virus N protein are important for the ability to direct a foreign protein