University of Nevada, Reno Coils, Loops, and Fingers: Functional Motifs in Hantavirus Proteins A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cellular and Molecular Biology by Daniel M. Boudreaux Dr. Stephen C. St Jeor / Dissertation Advisor December 2009 THE GRADUATE SCHOOL We recommend that the dissertation prepared under our supervision by DANIEL M. BOUDREAUX entitled Coils, Loops, And Fingers: Functional Motifs In Hantavirus Proteins be accepted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Stephen C. St Jeor, Ph.D., Advisor Gregory S. Pari, Ph.D., Committee Member David P. AuCoin, Ph.D., Committee Member William H. Welch, Ph.D., Committee Member Ellen J. Baker, Ph.D., Graduate School Representative Marsha H. Read, Ph. D., Associate Dean, Graduate School December, 2009 i ABSTRACT New world hantaviruses are a threat as an emerging pathogen with the potential to cause outbreaks in rural regions remote from medical facilities. Medical authorities emphasize the need for preventative education programs and the development of antiviral drug treatments based on an understanding of the viral replication process. The virus is composed of three negative sense RNA genomic segments and four proteins; the RNA-dependent RNA polymerase (RdRP), glycoprotein Gn, glycoprotein Gc, and the nucleocapsid protein (Npro). This dissertation presents the structures of three motifs within hantavirus proteins as part of an effort to identify potential targets for antiviral drugs. 1) The NMR structure of the N-terminal region of Npro is described here to form a helical coiled-coil motif. Three acidic amino acids in this structure significantly contribute to the ability of Npro to self associate, an important process in viral replication. 2) The middle region of Npro is proposed at a low resolution to contain a helix-loop- helix motif which interacts with the cytoskeletal filament, vimentin. The Npro-vimentin interaction contributes to the proper trafficking of Npro through the cytoplasm. 3) The glycoprotein Gn contains an unusually long cytoplasmic tail with a conserved dual CCHC sequence. NMR resolution revealed that this sequence forms a zinc finger motif. The potential for this zinc finger to bind viral RNA was predicted by molecular modeling and molecular dynamic simulations. This interaction is thought to coordinate the packaging of each of the three viral genome segments. The presentation of these three structures contributes a better understanding of the physical properties of proteins involved in viral infections. ii TABLE OF CONTENTS Abstract……………………………………………………………………………………………i Table of Contents…………………………………………………………..………………….….ii List of Tables………………………………………………………………………………….….iii List of Figures………………………………………………………………………...………….iii Introduction…………………………………………………………………………...…………..1 Prologue: Identification of Hantavirus Nucleocapsid Binding partners suggests essential interactions in the assembly process……………...………………………………………..…..19 Chapter 1: NMR structure of the N-terminal coiled coil domain of the Andes hantavirus nucleocapsid protein.....................................................................................................................29 Abstract…………………………………………………………………………………..29 Introduction…………………………………………………………………………...….30 Methods…………………………………………………………………………………..31 Results……………………………………………………………………………………35 Discussion………………………………………………………………………………..43 Chapter 1 Addendum: The N-Terminal Coiled Coil Domain of the Andes Nucleocapsid Protein is associated with Trimerization………………………………………………………48 Introduction………………………………………………………………………………48 Materials and Methods…………………………………………………………………...49 Results…………………………………………………………………………………....50 Discussion……………………………………………………………………………..…51 Chapter 2: The Middle Region of Hantavirus nucleocapsid binds Vimentin………….……54 Introduction………………………………………………………………………………54 Methods………………………………………………………………………………..…57 Results…………………………………………………………………………..………..62 Discussion……………………………………………………………………………..…74 Chapter 3: The Hantavirus Glycoprotein G1 Tail Contains Dual CCHC-type Classical Zinc Fingers............................................................................................................................................79 Abstract…………………………………………………………………………………..79 Introduction………………………………………………………………………………80 iii Materials and Methods…………………………………………………………….……..81 Results……………………………………………………………………………………86 Discussion………………………………………………………………………………..94 Chapter 3 Addendum: The Zinc Finger Motif of Andes Hantavirus Gn has the potential to bind to the viral RNA panhandle………………………………………………………………98 Introduction……………………………………………………………………………....98 Materials and Methods………………………………………………………..………...100 Results…………………………………………………………………………………..103 Discussion………………………………………………………………………………111 Conclusion………………………………………………………………………………….…..114 References………………………………………………………………………………………119 Appendix I Supplementary Figures for Chapter 1…………………………………………..146 Appendix II Supplementary Figures for Chapter 3………………………………………....150 LIST OF TABLES Introduction Table 1. Distribution, Vectors, and Diseases of Hantavirus Genotype Representatives………………………………………………………………………………….....5 Prologue Table 1. Proteins identified as potential binding partners for SNV-Npro using co- immunoprecipitation and MALDI-TOF peptide analysis………………………………..…….....23 Chapter 1 Table 1. Melting temperatures (Tm) and ellipticity ratio at 222 and 208 nm of N1-74……………………………………………………………………………...………………..41 Chapter 2 Table 1. Interaction energies of wild type and mutant SNV-N-Mid docked to VimH- 1A…………………………………………………………………………...………………...…..74 Chapter 3 Table 1. Restraints and structural statistics for 20 NMR structures…………...……..89 Appendix I Table 1. Structural statistics for 20 NMR structures of Andes virus N1-74 coiled coil domain………………………………………………………………………………………...…148 LIST OF FIGURES Introduction Figure 1. The Life Cycle of Hantaviruses…………………………………...……12 Introduction Figure 2. Properties of Hantavirus Proteins………………………………………16 iv Prologue Figure 1. Outline for the Interactions of Hantavirus proteins described in this dissertation ………………………………………………………………………………………28 Chapter 1 Figure 1. Assigned 1H – 15N HSQC spectrum of Andes virus N1-74 domain ……..…36 Chapter 1 Figure 2. Heptad repeats of conserved hydrophobic residues form the interface of the helix alpha1 and alpha2 that stabilize the coiled coil domain ………………………................…39 Chapter 1 Figure 3. CD spectra of wild type N1-74 (WT) and point mutants (K26E, (R22F)...…40 Chapter 1 Figure 4 Immunocytochemistry of full-length n protein with point mutations in the N1-74 coiled coil domain…………………………………………………………………...………43 Chapter 1 Addendum Figure 1.Granular versus Globular distribution of N in transfected Cos-7 cells …………………………………………………………………………………………...….51 Chapter 1 Addendum Figure 2. Cross linking of ANDV N proteins individually expressed….52 Chapter 2 Figure 1. Structure and Assembly of Intermediate filaments…………..…...…….…55 Chapter 2 Figure 2. Immunocytochemistry of Hantavirus genotypes…………………...…….. 63 Chapter 2 Figure 3. Co-immunoprecipitation of Hantavirus Genotypes and SNV-N- Mid………………………………………………………………………………………………..65 Chapter 2 Figure 4. Model for the SNV-Npro middle region …………………...…………..…66 Chapter 2 Figure 5. Primary Sequence of SNV-N-Mid…………………………………...……67 Chapter 2 Figure 6.Co-immunoprecipitation of Vimentin with Mutant Nucleocapsids.…….…68 Chapter 2 Figure 7. Helix-Loop-Helix motif………………………………………...…………69 Chapter 2 Figure 8. Primary sequence of the Vimentin Head…………………………..………71 Chapter 2 Figure 9. Model for VimH-1A……..……………………………………………..….72 Chapter 2 Figure 10. Docking of the SNV-Npro Middle Region to VimH-1A……………...…73 Chapter 3 Figure 1. The G1 tail of Hantaviruses, Nairoviruses, and Orthobunyaviruses (genera of Bunyaviridae) contains a cysteine/histidine-rich region with two CCHC arrays………..…….85 Chapter 3 Figure 2. CD spectroscopy and titration with EDTA and ZnSO4 for recombinant Andes virus G1 tail CCHC-region………………………………………………………………..87 Chapter 3 Figure 3. The Andes virus G1 tail zinc finger domain 9residues 543-599) shows a well dispersed two-dimensional 1H – 15N HSQC spectrum……………………………………...90 Chapter 3 Figure 4. The NMR structure of the Andes virus G1 tail zinc-binding domain reveals two classical beta beta alpha fold zinc fingers that are joined together…………..………………93 Chapter 3 Addendum Figure 1. ChIP assay of pAD-ANDV-M transfected cells……………104 Chapter 3 Addendum Figure 2. Primary sequence of Gn-CT-Arms protein……….…...……105 v Chapter 3 Addendum Figure 3. Addition of N- and C-terminal arms to ANDV-Gn-ZF is predicted to form a dsRNA binding motif………………………………………………………106 Chapter 3 Addendum Figure 4. Molecular Docking of Gn-ZF-Arms to dsRNA………….…108 Chapter 3 Addendum Figure 5. The ANDV-S viral panhandle……………………...………109 Chapter 3 Addendum Figure 6. Interaction energies and percentage of pretone residues within 3A of the RNA for 11 docking attempts of Gn-ZF-Arms with the ANDV-S-panhandles …..…110 Chapter 3 Addendum Figure 7. The Gn-ZF-Arms protein initially docked with the ZF region on top of the second bulge (g18) ………………………………………………...…………...…111 Appendix I Supplementary Figures for Chapter 1 Figure 1. Sequence alignment of hantavirus nucleocapsid N1-74 coiled coil domain with the conserved hydrophobic and polar heptads highlighted……………...………………………………………………………………146 Appendix I Supplementary Figures for Chapter 1 Figure 2. Secondary C, H, C’, and