IDENTIFICATION OF BOTH STRUCTURAL AND FUNCTIONAL DOMAINS OF THE SENDAI VIRUS NUCLEOCAPSID PROTEIN REQUIRED FOR VIRAL RNA REPLICATION By TINA MARIE MYERS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1996 . ACKNOWLEDGMENTS I would like to thank Dr. Sue Moyer for giving me the opportunity to work on such an interesting virus and for her unerring guidance and support during my graduate training. I would also like to thank the members of my committee, Drs Thomas O'Brien, Richard Condit, and James B. Flanegan, for their participation and helpful advice. I would like to express my appreciation to the Flanegan group for the many useful suggestions I received during our combined laboratory meetings. Also, I want to thank the other faculty members and graduate students for the suggestions I received during research presentations. I would like to acknowledge both Sandra Horikami and Sherin Smallwood for making me feel welcome and for their expert advice. I am grateful to all members of the laboratory, past and present, for making the lab an enjoyable and exciting place to work. For her valuable technical assistance in cloning and sequencing the central conserved region (CCR) NP mutants, I want to thank Cheryl Zack. I would also like to thank Ami Pieters for continuing the cloning, sequencing, and initial characterization of the CCR mutants during her laboratory rotation. I would particularly like to thank Sherin Smallwood for her superb technical assistance in the characterization of the CCR NP mutants, including protein expression, GST-P binding, and in vitro replication assays. Special thanks go to my mother, Shirley, for her love, encouragement, and most importantly, friendship, throughout my entire life. Likewise, I thank my brother, Scott, for being a wonderful friend and always willing to lend an ear. Their unending love and support has made this possible. TABLE OF CONTENTS ACKNOWLEDGMENTS ii LIST OF FIGURES vi LIST OF TABLES ix KEY TO SYMBOLS X ABSTRACT xiii CHAPTERS 1 INTRODUCTION AND BACKGROUND 1 Viral Taxonomy 1 Genomic Structure 3 The Virion and the Viral Lifecycle 4 Deletion Mutants of the Paramyxoviruses ... 7 RNA Synthesis 9 The NP Protein 17 2 MATERIALS AND METHODS 24 Cells and Viruses 24 Plasmids, Antibodies, and Probes 26 Construction of NP Mutants 28 Infection and Transfection 40 RNA Replication 40 Protein Analysis 45 3 TEMPLATE FUNCTION 56 Introduction 56 Results 58 Discussion 79 4 IDENTIFICATION OF A PUTATIVE RNA BINDING SITE . 88 Introduction 88 Results 89 Discussion 107 CHAPTERS 5 IDENTIFICATION OF THE NP SELF-ASSEMBLY DOMAIN . 114 Introduction 114 Results 115 Discussion 140 6 CONCLUDING REMARKS 147 LIST OF REFERENCES 150 BIOGRAPHICAL SKETCH 163 .. .. LIST OF FIGURES Figure Page 1. A schematic of the NP protein and the amino acid sequences (aa) from 107 to 130 of the charge-to-alanine NP mutants 59 2. Pulse-chase analysis and in vitro replication with the mutant NP107 62 3. In vitro DI-H RNA synthesis with the charge-to-alanine mutant NP proteins 64 4 Cobinding of the NP protein with the GST-P protein to glutathione Sepharose beads 68 5. CsCl step gradient centrifugation of the self-assembled wt and mutant nucleocapsidlike particles 72 6. Template function of the (+JDI-H RNA-NPs assembled in vivo with wt or mutant NP proteins 75 7. Northern analysis of DI-H RNA replication in vivo 77 . 8. Binding of the viral polymerase to the wt and mutant self-assembled nucleocapsidlike particles 81 9 A summary of the protein-protein interactions and in vitro DI-H RNA replication data of the wt and mutant NP proteins 83 10. A schematic of the NP protein and the amino acid sequences (aa) from 361 to 377 of the alanine-scanning NP mutants 90 11 Pulse-chase analysis of the alanine-scanning mutants described in Fig. 10 92 12. In vitro DI-H RNA replication with the mutant NP362, NP370, and NP373 proteins 95 13 Cobinding of the mutant NP proteins with the GST-P protein to glutathione Sepharose beads 97 vi . Figure Page 14. Electron microscopy of the wt NP and NP370 proteins and CsCl gradient analysis of the wt NP, NP370, and NP362 proteins 101 15. CsCl step gradient centrifugation of the self-assembled wt and mutant nucleocapsidlike particles 105 16. A summary of the wt and mutant NP protein-protein interactions and in vitro DI-H RNA replication data 108 17. Amino acid alignment of selected NP sequences Ill . . . 18. Amino acid alignment of selected NP sequences and a .... schematic representation of the NP protein 117 19. Glycerol gradient analysis of the wt NP protein, the MBP protein, the MBP.NP1 fusion protein, and a DI-H nucleocapsid 120 20. Sedimentation analysis of the MBP fusion proteins containing region 1 (CCR) of both the MV N protein (MBP.N1) and the SV NP protein (MBP.NP1) and their derivatives 124 21. Sedimentation analysis of the in vitro synthesized MBP fusion protein containing region 2 (N-terminus) of the NP protein (MBP.NP2) and truncations of the MBP.NP2 fusion protein 127 22. Sedimentation analysis of the in vitro synthesized MBP fusion proteins containing regions 3A, 3B, and 4 of the NP protein (MBP.NP3A, MBP.NP3B, and MBP.NP4, respectively) 129 23 A summary of the sedimentation data of the MBP fusion proteins 131 24. Inhibition of in vitro DI-H RNA replication by the MBP.NP1 fusion protein 133 25. Amino acid sequence of the CCR (aa 258-357) of the NP protein and the random-primed site-directed NP mutants 135 26. Analysis of the wt NP and mutant NP proteins by CsCl step gradient centrifugation 137 27. In vitro DI-H RNA synthesis with the CCR mutant NP proteins 139 Figure Page 28. A combined summary of the wt and mutant NP protein-protein interactions and in vitro DI-H RNA replication data from the CCR NP mutants Fig. 16 142 . . 29. A schematic of the NP protein and the domains required for NP-NP interaction, template function, and NP-RNA binding 145 . LIST OF TABLES Table £age 1. Oligonucleotide primers 29 2 Site-directed mutations in the central conserved region 37 3. MBP-NP fusion protein primers and plasmids 39 ix KEY TO SYMBOLS aa amino acid ATP adenosine 5 -triphosphate ' bp base pair C- carboxy 'C degrees centigrade CCR central conserved region cfu colony forming units Ci curie cm centimeter cpm counts per minute cs cold sensitive CTP cytidine 5'-triphosphate dCTP deoxycytidine 5 -triphosphate ' DI defective interfering particle DNA deoxyribonucleic acid ds double stranded GTP guanosine 5'-triphosphate h hour I.U. international unit kb kilobase kDa kilodalton