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Functional characterization of a small nonstructural protein of the parvovirus B19 Fan, Mannie Man Yee 2001

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FUNCTIONAL CHARACTERIZATION OF A SMALL NONSTRUCTURAL PROTEIN OF THE PARVOVIRUS B19. by MANNIE MAN YEE FAN B.Sc. (Honours), McGill University, 1998  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Biochemistry and Molecular Biology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 2001 © Mannie Man Yee Fan, 2001  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by his or  her representatives.  It  is understood  that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  The University of British Columbia Vancouver, Canada  DE-6  (2/88)  ABSTRACT The pathogenic human parvovirus B19 produces a class of small, l l k D a proteins whose function is as yet undetermined. In the absence of a continuous permissive cell line in which B19 can be readily propagated, in vitro studies have been used to assess the functional role of the 11 kDa proteins. In previous studies, the l l k D a proteins have not been shown to be targets of phosphorylation or binding partners for other viral proteins, nor have they demonstrated Zn -binding or transactivation activities. However, there is ++  preliminary evidence that the l l k D a proteins are capable of interacting with the cellular growth factor receptor-binding protein 2 (Grb2) in vitro, and this thesis aims to investigate this interaction in greater detail. In studies reported in this thesis, far western experiments confirm in vitro interactions between Grb2 and the l l k D a proteins. Moreover, a fusion construct of the l l k D a protein with glutathione S-transferase (GST) is able to specifically pull down endogenous Grb2 from H E K 293 cell lysates, providing further support for our hypothesis that the two proteins interact. Grb2 is an important adaptor molecule in the M A P K signal transduction pathway leading to defined cellular responses. The protein is composed of a series of modular domains each specialized for a different function, including two SH3 domains that are capable of binding proline-rich sequences.  Such sequences  correspond to consensus "ligand" motifs found in binding partners for Grb2 such as son of sevenless (Sos), a guanine nucleotide exchange factor for the Ras small GTPbinding protein that is implicated in M A P K activation. Sos-like proline-rich regions are also present in the l l k D a proteins. Further studies have focused on the requirement for these regions in the G r b 2 / l l k D a interaction. Far western studies employing l l k D a proteins mutated at proline residues suggest reduced interactions with Grb2. Furthermore, GST-11 mutant constructs do not precipitate Grb2 from mammalian cell lysates as readily as does the wild-type. These results indicate possible involvement of the proline-rich  ii  region in mediating interactions of the 11 kDa proteins with Grb2, in an SH3dependent manner. The findings from this thesis strongly support an interaction of the 11 kDa proteins with the host protein Grb2 to influence host cell environment during the viral replicative cycle.  iii  TABLE OF CONTENTS Abstract  ii  Table of Contents  iv  List of Tables  ix  List of Figures  x  List of Abbreviations  xii  Acknowledgments  xix  1. INTRODUCTION  1  1.1. Taxonomy  1  1.1.1. Dependoviruses  1  1.1.2. Parvoviruses  3  . 1.1.3. Erythroviruses  3  1.2. The Erythrovirus B19  4  1.2.1. B19 Infection and Transient Aplastic Crisis  6  1.2.2. Persistent B19 Infection  6  1.2.3. B19 Infection In Utero  7  1.2A. B19 Infection in the Immunocompetent Host  7  1.2.5. B19 and Other Disorders  11  1.3. B19 in Cell Culture  11  1.4. The B19 Genome  12  1.4.1. The B19 Nonstructural Proteins  15  1.4.2. The B19 Structural Proteins  15  1.4.3. The B19 l l k D a Proteins  16  1.5. Review of Relevant Signal Transduction Concepts and Pathways  17  1.6. Review of Functions of Other Viral Nonstructural Proteins  19  1.6.1. Virus Entry  19  1.6.2. Nuclear Targeting and Replication of Viral Genome  19  1.6.3. Viral Gene Expression  21  1.6.4. Processing and Nuclear Export of mRNA Transcripts  22  iv  1.6.4.1. Processing and Nuclear Export of Viral Transcripts  22  1.6.4.2. Processing and Nuclear Export of Host Transcripts  23  1.6.5. Processing of Viral Proteins  24  1.6.6. Host Cell Protein Trafficking and Degradation  24  1.6.7. Virion Packaging, Assembly, and Egress  24  1.6.8. Cell Cycle Arrest  25  1.6.9. Immune Evasion  25  1.6.9.1. Inhibition of Humoral Immune Responses  25  1.6.9.2. Inhibition of Interferon Responses  26  1.6.9.3. Modulation of Cytokine and Chemokine Responses  27  1.6.9.4. Modulation of Apoptosis  28  1.6.9.4.1. Inhibition of Apoptosis  28  1.6.9.4.2. Induction of Apoptosis  29  1.6.9.5. Modulation of Major Histocompatibility Complex (MHC) Function  30  1.7. Other Proline-Rich Viral Nonstructural Proteins  31  1.8. Previous Studies on the llkDa Proteins and the Present Study  32  2. MATERIALS AND METHODS  34  2.1. Chemicals and Supplies  34  2.2. Bacterial Culture  35  2.2.1. Growth and Maintenance  35  2.2.2. Preparation of Competent Bacteria for Heat Shock Transformation  35  2.2.3. Heat Shock Transformation of £. coli  36  2.3. Tissue Culture  36  2.3.1. Growth and Maintenance  36  2.3.2. Transfection of Plasmid DNA into COS-7 Cells  37  2.3.3. Lysis of Tissue Culture Cells for GST Protein Pull Down and Immunoprecipitation Experiments  37  2.4. Molecular Cloning Techniques  38  v  2.4.1. Small-Scale Plasmid D N A Isolation  38  2.4.2. Large-Scale Plasmid D N A Isolation  39  2.4.3. D N A Quantitation and Purity Check  39  2.4.4. Plasmid D N A Digestions  39  2.4.5. Plasmids  40  2.4.5.1. pGEX2T  40  2.4.5.2. pGEX-11 2.4.5.3. pGEX-11 P42,45A/P42 45 46A/P42,46A/P43A/P43 45A /P43,45,46A/P45A/P45,46A  40  2.4.5.4. pGEX-11 P42A  42  2.4.5.5. pGEX-11 P46A  42  2.4.5.6. pGEX2T-tag  42  2.4.5.7. pGEX2T-tag-Grb2  42  2.4.5.8. pEGFP-C2  42  2.4.5.9. pEGFP-11  44  /  /  2.4.6. Oligonucleotides  /  4 2  44  2.4.6.1. HkDPro2  44  2.4.6.2. 11COMPL  44  2.4.6.3. P42A1  44  2.4.6.4. P42A2  45  2.4.6.5. P46A1  45  2.4.6.6. P46A2  45  2.4.6.7. pGEX3'  45  2.4.6.8. HSeql  45  2.4.7. Polymerase Chain Reaction (PCR)-Based Site-Directed Mutagenesis  46  2.4.8. G-Ladder Screen for Mutant Plasmid D N A  46  2.4.9. Automated Sequencing of Plasmid D N A  48  2.5. Protein Techniques  48  2.5.1. GST/GST Fusion Protein Purification  48  2.5.2. Protein Quantitation  49  vi  2.5.3. Antibodies  50  2.5.3.1. Primary Antibodies  50  2.5.3.1.1. Rabbit anti-B19 l l k D a  50  2.5.3.1.2. Mouse anti-GFP  50  2.5.3.1.3. Rabbit anti-Grb2  50  2.5.3.1.4. Mouse anti-GST  50  2.5.3.1.5. Mouse anti-HA  50  2.5.3.1.6. Mouse anti-Sosl  51  2.5.3.2. Secondary Antibodies  51  2.5.3.2.1. HRP-Conjugated Goat anti-Mouse  51  2.5.3.2.2. Super X-Absorbed HRP-Conjugated Donkey antiRabbit 2.5.4. Western Blotting  51  2.5.5. Far Western Blotting  52  2.5.6. GST Protein Pull Down Assay  52  2.5.7. Immunoprecipitation  53  51  55  3. RESULTS 3.1. Expression of Glutathione S-Transferase (GST) and GST-Tagged Fusion Proteins  55  3.2. GST-HA-Grb2 Interacts with GST-11 but not GST on a Far Western Blot  55  3.3. GST-HA-Grb2, but not GST-HA, Interacts with GST-11 on a Far Western Blot  58  3.4. Anti-Hemagglutinin (HA) Antibody Recognizes Both GST-HA and GST-HA-Grb2  58  3.5. GST-11 but not GST Interacts with Cellular Grb2 in a GST Protein Pull Down Assay  61  3.6. Exogenously Added GST and GST-11 Do Not Appear to Interact with Cellular Grb2 in Co-Immunoprecipitation Studies  61  3.7. Addition of Exogenous GST and GST-11 Proteins Do Not Affect Grb2/Sos Interaction  65  vii  3.8. Neither the Enhanced Green Fluorescent Protein (EGFP) Nor the EGFP-llkDa Fusion Protein (EGFP-11) Interact with Cellular Grb2 in Co-Immunoprecipitation Studies In Vivo  65  3.9. Levels of Sos Protein Co-Immunoprecipitated by Anti-Grb2 Antibody Are Comparable in COS-7 Cells Transiently Expressing EGFP or EGFP-11  69  3.10. The GST-HA-Grb2/GST-11 Interaction on a Far Western Blot Requires 11 kDa Proline-Rich Region  69  3.11. The Grb2/GST-ll Interaction in a GST Protein Pull Down Assay Requires 11 kDa Proline-Rich Region  73  4. DISCUSSION  75  4.1. Characterization of the B19 HkDa/Grb2 Interaction  75  4.1.1. Far Western Studies  75  4.1.2. Pull Down Assays  75  4.1.3. Immunoprecipitation Studies  76  4.2. Src Homology 3 (SH3)-Mediated Interactions and B19 HkDa Proteins  77  4.3. Effects of Proline-to-Alanine Mutations on B19 HkDa/Grb2 Interactions  78  4.3.1. Far Western Studies  78  4.3.2. Pull Down Assays  78  4.4. Implications of Present Findings on Possible Functions of B19 11 kDa Proteins  79  4.5. Future Directions  79  4.6. Conclusions  80  5. REFERENCES  81  viii  LIST OF TABLES Table 1.1.  Selected Parvoviridae and their target host species.  Table 1.2.5.  Other disorders associated with parvovirus B19 infection.  11  Table 2.4.5.  Plasmid vectors and the proteins they express.  40  ix  2  LIST OF FIGURES Figure 1.2.4.1.  Development of B19-specific IgM and IgG after experimental B19 inoculation of seronegative volunteers and a volunteer with trace amounts of IgG to B19.  Figure 1.2.4.2.  Time course of experimental inoculation of Parvovirus  9  10  B19 in a seronegative host. Figure 1.4.  Transcription and coding map of Parvovirus B19.  14  Figure 1.8.  Model of B19 llkDa protein-mediated disruption of the  33  Grb2 / Sos / Ras signaling pathway. Figure 2.4.5.1.  pGEX2T plasmid map.  41  Figure 2.4.5.8.  pEGFP-C2 plasmid map.  43  Figure 3.1.  Coomassie-stained polyacrylamide gel of purified GST/GST-fusion proteins. Western blot and far western blot demonstrating interaction of GST-11 but not GST with GST-HA-Grb2. Western blot and far western blots demonstrating interaction of GST-HA-Grb2 but not GST-HA with GST11.  56  Figure 3.2. Figure 3.3.  57 59  Figure 3.4.  Western blots demonstrating ability of anti-HA antibody to recognize HA epitope in both GST-HA and GST-HA-Grb2.  60  Figure 3.5.  Western blots demonstrating interaction of exogenously added GST-11 but not GST with cellular Grb2 in the GST protein pull down assays.  62  Figure 3.6.  Western blots of anti-Grb2-immunoprecipitated samples in the presence of exogenously added GST or GST-11.  63  Figure 3.7.  Western blots of anti-Grb2-immunoprecipitated samples in the absence or presence of exogenously added GST or GST-11.  66  x  Figure 3.8.  Western blots of anti-Grb2-immunoprecipitated samples in COS-7 cells transfected with pEGFP-C2 or pEGFP-11.  67  Figure 3.9.  Western blots of anti-Grb2-immunoprecipitated samples in COS-7 cells transfected with pEGFP-C2 or pEGFP-11.  70  Figure 3.10.  Far western blot and western blots demonstrating differential strength of interaction of GST-11 wt/mutant proteins with GST-HA-Grb2.  71  Figure 3.11.  Western blots demonstrating differential strength of interaction of exogenously added GST-11 wt/mutant proteins with cellular Grb2 in a GST protein pull down assay.  74  xi  LIST OF ABBREVIATIONS A  adenine  1  A-260/ A-280/ O ^ A-5g2  absorbance at 260,280, or 562 nm  aa  amino acid  AAV  adeno-associated virus  Ab  antibody  Ad  adenovirus  Ala  alanine  Amp  ampicillin  ASFV  African swine fever virus  ATCC  American Type Culture Collection  ATP  adenosine 5'-triphosphate  BCA  bicmchroninic acid  pME  R-mercaptoethanol  BPB  bromophenol blue  BPV  bovine papilloma virus  BSA  bovine serum albumin  C  cytosine  CaCl  2  calcium chloride  cdc  cell division cycle mutant  cDNA  complementary DNA  CH COOK  potassium acetate  Ck  chemokine  CkR  chemokine receptor  CMV  cytomegalovirus  CNS  central nervous system  co  carbon dioxide  3  2  CPSF  cleavage and polyadenylation specificity factor  CPV  canine parvovirus  crm  cytokine response modifier  xii  CTL  cytotoxic T-lymphocyte  dATP  deoxy-ATP  ddGTP  dideoxy-GTP  DEAE  diethylaminoethyl  dH 0  distilled water  DMEM  Dulbecco's modified Eagle's medium  DMSO  dimethylsulfoxide  DNA  deoxyribonucleic acid  dNTPs  deoxy-NTPs  ds  double-stranded  EBNA  EBV nuclear antigen  EBV  Epstein-Barr virus  ECL  enhanced chemiluminescence  EDTA  ethylene-diamine tetra-acetic acid  EGF  epidermal growth factor  EGFP  enhanced green fluorescent protein  EGFP-11  enhanced green fluorescent protein-llkDa fusion  EI  erythema infectiosum  ER  endoplasmic reticulum  ERK  extracellular signal-regulated kinase  FasL  Fas ligand  FBS  fetal bovine serum  FLICE  Fas-associated death domain-like ICE  FLIP  FLICE-inhibitory protein  FPV  feline parvovirus  FW  far western blot  g  gram  G  guanine  GDP  guanosine diphosphate  GEB  glutathione elution buffer  2  xiii  GEF  guanine nucleotide exchange factor  GFP  green fluorescent protein  gP Grb2  glycoprotein  GS or HS  goat or horse serum  GSH  glutathione  GST  glutathione S-transferase  GST-11  glutathione S-transferase-11 kDa fusion  GTP  guanosine 5'-triphosphate  HIV  human immunodeficiency virus  H 0  water  HA  hemagglutinin  HCOOH  formic acid  HCV  hepatitis C virus  HEK  human embryonal kidney  HEPES  (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid])  HHV  human herpesvirus  hnRNP  heteronuclear ribonucleoprotein  HPV-2  human parvovirus  HRP  horse radish peroxidase  HSV  herpes simplex virus  hSos  human Sos  HTLV  human T-lymphotropic virus  IAP  inhibitor of apoptosis protein  ICE  inter leukin-ip-converting enzyme  ICP  infected cell protein  IE  immediate early  IFN  interferon  Ig IL  immunoglobulin  2  growth factor receptor-binding protein 2  interleukin  xiv  IMP  inflammation modulatory protein  IN  integrase  IP  immunoprecipitation  IPTG  isopropylthio-(3-D-galactoside  ITR  inverted terminal repeat  Kan  kanamycin  kb  kilobase  kDa  kilodalton  KLH  keyhole limpet hemacyanin  KSHV  Kaposi's Sarcoma-associated herpesvirus  LB  Luria-Bertani  LMP  latent membrane protein  MA  matrix  MAC  membrane attack complex  MAPK  mitogen-activated protein kinase  MCS  multiple cloning site  MCV  molluscum contagiosum virus  MEK  MAPK/ERK kinase  mg  milligram  US  microgram  MgCl  magnesium chloride  2  MgS0  4  magnesium sulfate  MHC  major histocompatibility complex  MHV  murine y-herpesvirus  MIP  macrophage inflammatory protein  ml  milliliter  III  microliter  mM  millimolar  mm  millimeter  mRNA  messenger RNA  XV  mSos  murine Sos  mu  map unit  MVM  minute virus of mice  MW  molecular weight  NaCl  sodium chloride  NAK  nef-associated kinase  NaOH  sodium hydroxide  NES  nuclear export signal  g NK  nanogram  NLS  nuclear localization signal  nm  nanometer  NS  nonstructural  nt  nucleotide  NTP  nucleoside triphosphate  OD  optical density  ORF  open reading frame  OS  oligoadenylate synthetase  P(number)A  proline-to-alanine mutation at specified amino acid position  n  P  6  natural killer  promoter at map unit 6  PABII  poly(A)-binding protein II  PAGE  polyacrylamide gel electrophoresis  PAK  p21-activated protein kinase  PAP  poly(A) polymerase  PBS  phosphate-buffered saline  PBS-T  PBS supplemented with 0.05% Tween-20  PCR  polymerase chain reaction  PEG  polyethylene glycol  PIC  pre-integration complex  PKR  dsRNA-dependent protein kinase  xvi  pmol  picomole  PMSF  phenylmethylsulfonyl fluoride  PNK  polynucleotide kinase  poly(A)  polyadenylated  PP-II  polyproline type II  PrCl  preclear  pRB  retinoblastoma protein  Pro  proline  PSB  protein sample buffer  PTMV  pig-tailed macaque virus  PVDF  polyvinylidene fluoride  RF  replicative form  RMV  rhesus macaque virus  RNA  ribonucleic acid  rpm  revolutions per minute  RRE  rev-responsive element  sdH 0  sterilized distilled water  SDM  site-directed mutagenesis  SDS  sodium dodecylsulfate  SFV  shope fibroma virus  SH  Src homology  SMP  skim milk powder  snRNA  small nuclear R N A  Sos  son of sevenless  SPV  simian parvovirus  ss  single-stranded  STET  sucrose-Triton-EDTA-Tris  SV40  simian virus 40  T  thymine  TAC  transient aplastic crisis  2  xvii  TAE  tris-acetate-EDTA  TBE  tris-borate-EDTA  TNF  tumor necrosis factor  TNFR  TNF receptor  tris  tris[hydroxymethyl]aminomethane  U  unit  UTR  untranslated region  UV  ultraviolet  vEGF  viral EGF homolog  VETF  vaccinia virus early transcription factor  v-FLIP  viral FLIP homolog  VGF  vaccinia growth factor  Vif  virion infectivity protein factor  vIL  viral IL homolog  vMIP  viral MIP homolog  VP  viral protein (structural)  Vpr  viral protein R  Vpu  viral protein U  VSV  vesicular stomatitis virus  vTNFR  viral TNFR homolog  WB  western blot  wt  wild-type  Zn  ++  zinc ion  xviii  ACKNOWLEDGMENTS I would like to express my gratitude to the people who have helped me through my studies and who have been providing support and encouragement through my family crisis. I am forever indebted to Dr. Caroline Astell, who gave me this opportunity to train, and to Dr. Cynthia Shippam-Brett, who taught me my first lessons in molecular biology at the bench. Special thanks go to members of the Astell lab, past and present, for being wonderful friends and for being there when I need you (to answer a question, to listen to my nonsensical babbles, and so on). Many thanks to members of my committee Dr. Roger Brownsey, Dr. Shoukat Dedhar, and Dr. Alice Mui, for their continual guidance and useful suggestions. Also thank you to all my other friends in the department—without you my life would have been that much less enjoyable... I would also like to thank my family for the love and support they've shown. Last but not least, I'd like to thank the Almighty for placing me on this path and for giving me love, hope, and strength. I have not been your best pupil but I try. May I live to see the day my father will recover and be able to appreciate the contents of this thesis. Amen.  xix  Introduction  INTRODUCTION  1.1. Taxonomy Parvoviruses are small eukaryotic viruses (diameter ranges from 18-25nm) with linear, single-stranded DNA (ssDNA) genomes roughly 5kb long. These nonenveloped viruses have a T=l icosahedral protein capsid coat which protects against inactivation by moderate pH changes, high temperatures, high salt concentrations and lipid solvent extraction. The members of the family Parvoviridae are classified according to sequence similarity, genome organization, host range, and requirement for co-infection by a helper virus (Pringle, 1993). The family consists of two subgroups, the Densovirinae and the Parvovirinae. While the former infects arthropods, members of Parvovirinae infect vertebrate organisms and can in turn be subdivided into three genera:  the Dependoviruses, Parvoviruses, and  Erythroviruses (reviewed in Berns, 1996; Young, 1996; Astell, 1999a; Astell, 1999b).  1.1.1. Dependoviruses While all members of Parvoviridae require cellular functions associated with S phase of the cell cycle to replicate successfully, Dependoviruses such as adenoassociated virus (AAV) further require co-infection by a helper virus (adenovirus or herpesvirus) in order to establish a productive infection. The requirement appears to be more for an altered cellular environment than for specific functions associated with the helper virus, as it has been shown that treatment of cells by various chemicals or UV irradiation can replace the need for a helper virus (Yakobson et al, 1987; Yakobson et al, 1989). In the absence of a helper virus, infection by AAV results in site-specific integration of the AAV genome into the host genome (Kotin et al, 1990). AAV replication is initiated upon subsequent infection by adenovirus or herpesvirus. (An exception is AAV5, a recently discovered member of the family that can replicate in the absence of a helper virus.)  1  Introduction  Table 1.1. Selected Parvoviridae and their target host species. Parvoviridae  Densovirinae (invertebrate) Contravirus Densovirus Iteravirus  Parvovirinae (vertebrate) Dependovirus  Adeno-Associated Virus (most animals) Avian Adeno-Associated Virus (bird) Bovine Adeno-Associated Virus (cattle)  Parvovirus  Aleutian Mink Disease Virus (mink) Bovine Parvovirus (cattle) Canine Parvovirus (dog) Feline Panleukopenia Virus (cat) Goose Parvovirus (goose) HI Virus (rodent) Kilham Rat Virus (rat) LuIII (unknown) Minute Virus of Canines (dog) Minute Virus of Mice (mouse) Mouse Parvovirus (mouse) Hamster Parvovirus (hamster) Mink Enteritis Virus (mink) Porcine Parvovirus (pig)  Erythrovirus  B19, Human Parvovirus-2 (human) V9 Parvovirus (human) Pig-Tailed Macaque Virus (monkey) Rhesus Macaque Virus (monkey) Simian Parvovirus (monkey) Chipmunk Parvovirus (chipmunk)  2  Introduction  Infection by Dependoviruses produces no known pathological symptoms. Hence viruses such as AAV are attractive for use as vectors in gene therapy. AAV not only integrates site-specifically into the host genome; it does so in both dividing and senescent cells, without causing cellular transformation in the infected cell.  1.1.2. Parvoviruses In contrast to the Dependoviruses, members of the Parvoviruses and Erythroviruses are capable of replicating autonomously, in the absence of any helper viruses. Parvoviruses infect a wide range of domestic animals as well as several murine species. Because successful replication of Parvoviruses requires progression of the host cell into the S phase, cells that are rapidly dividing are the most susceptible to infection. These include cells of the hematopoietic system, epithelial cells lining the gastrointestinal tract, as well as fetal or neonatal cells. Correspondingly, Parvovirus infection in newborn domestic animals and livestock are frequently lethal, leading to significant financial consequences.  1.1.3. Erythroviruses While members of Erythroviruses share some common characteristics with the Parvoviruses, they differ from each other in genome organization and in specific host range. These viruses appear to require certain transcription factors or other conditions unique to progenitor cells of the erythroid lineage in mammals and therefore display a tropism towards red cell precursors in humans and other primate and rodent species. Among the Parvovirinae, Erythrovirus is the most recently discovered genus. Currently, the genus is comprised of six members: simian parvovirus (SPV) (O'Sullivan et al, 1994; Brown et al, 1995a), rhesus macaque virus (RMV) (Green et al, 2000), pig-tailed macaque virus (PTMV) (Green et al, 2000), chipmunk parvovirus (Yoo et al, 1999), V9 (Nguyen et al, 1999), and B19 (also known as human parvovirus or HPV-2) (Cossart et al, 1975).  3  Introduction  1.2. The Erythrovirus B19 The B19 parvovirus was first discovered by Yvonne Cossart in a screen for hepatitis in blood donors (Cossart et al, 1975; structure reported in Agbandje et al, 1994) and is the only member of the Parvoviridae known to cause disease in humans (reviewed in Young, 1995; Rogers, 1999; molecular aspects reviewed in Astell et al, 1997). Roughly 45% of children test seropositive for antibodies against the virus, and the antibody prevalence rises in an age-dependent manner up to about 70% in the adult population (Gay et al, 1994), indicating infection during childhood. There are no known animal or insect vectors for B19, and viral transmission can take place through respiratory (foamites and aerosol droplets), oral-fecal, blood-borne, and transplacental routes. The tropism of B19 parvovirus for human erythroid progenitor cells appears to be regulated at multiple levels. One determinant is the presence of the blood group P antigen, which has been identified as the cell surface receptor for B19 parvovirus (Brown et al, 1993). The virus binds with high affinity and specificity to the carbohydrate moiety of the P antigen, also known as globoside or tetrahexose ceramide. Cells lacking the P antigen are resistant to B19 infection (Brown et al, 1994a). While the P antigen is expressed in a variety of cell types including mature erythrocytes, erythroid progenitors, megakaryocytes and platelets, endothelial cells, and fibroblasts (von dem Borne et al, 1986), only bone marrow cells expressing the erythrocyte P antigen are permissive to B19 infection (Kerr et al, 1995). In nonpermissive cells, it has been proposed that the block in replication may be mediated in part by an inhibition of transcript maturation (Liu et al, 1992). Other studies suggest that the 3' untranslated region (3' UTR) of the mRNAs encoding B19 structural proteins may block their own translation, thereby inhibiting viral replication (Pallier et al, 1997). Moreover, Brunstein and co-workers (Brunstein et al, 2000) have identified a novel splicing pattern for B19 transcripts in semipermissive cells which may lead to a translational block of structural protein expression. Their findings implicate differences in host cell splicing machinery as one determinant of B19 tropism.  In other studies, the presence of as yet  4  Introduction  uncharacterized transcription factors in erythroid progenitor cells has been implicated in the restriction of B19 replication to these cells. The B19 p6 promoter cannot function in non-erythroid cells (Kurpad et al, 1999) but is able to direct erythroid cell-specific expression in infected cells when cloned into a recombinant AAV-2 virus (Wang et al, 1995). In permissive erythroid progenitor cells, entry of the virus is believed to be mediated by endocytosis via clathrin-coated pits, independent of cell cycle progression. B19 DNA is detected in the nucleus two days following infection (Morey et al, 1993), and the virus proceeds to induce apoptosis in the infected cell (Moffatt et al, 1998). The cessation of erythropoiesis leads to a deficiency in reticulocytosis and a decrease in red cell count (hematological consequences of B19 infection reviewed in Brown, 2000). Despite similar pathological alterations, clinical manifestations of B19 infection are highly variable and dependent on the hematological and immunological state of the infected host. In some cases, infection is asymptomatic or the symptoms may be sub-clinical. In the majority of cases, however, B19 infection is typically associated with mild pyrexia and flu-like illness within the first week, concurrent with viremia and a depression in red cell count. This occurrence is usually transient and not problematic in healthy individuals. However, B19 infection becomes clinically significant and may even be life-threatening in certain groups of patients, namely in patients with an abnormality in red cell function or with a weakened immune system.  5  Introduction  1.2.1. B19 Infection and Transient Aplastic Crisis B19 infection can become life-threatening in patients with an underlying hematological disorder (eg. sickle cell anemia) (Pattison et al, 1981; Rao et al, 1992; Serjeant et al, 1993), with a pre-existing hemolytic condition (eg. (3-thalassemia, hereditary spherocytosis) (Kelleher et al, 1983; Davidson et al, 1984; Goss and Szer, 1997), or under erythroid stress (eg. iron deficiency, hemorrhage) (Kojima et al, 1988; Kamoshita et al, 1993). In these individuals, because they have a low red cell count or shortened red cell half-life to begin with, a cessation in red blood cell production resulting from B19 infection may lead to a life-threatening transient aplastic crisis (TAC) (Young et al, 1984; Chorba et al, 1986).  While this  hematological condition is ultimately self-limiting, the resulting anemia may be so severe as to not only cause fatigue but possibly precipitate congestive heart failure and/or cerebrovascular accidents as well. The anemic condition arising from TAC may be resolved by blood transfusion.  1.2.2. Persistent B19 Infection In individuals with congenital or acquired immunodeficiencies (eg. from HIV infection, bone marrow or solid organ transplant procedure, chemotherapy treatment, etc.), B19 establishes a chronic infection due to the inability of the host to clear the virus, thus leading to chronic red cell aplasia (Neild et al, 1986; Kurtzman et al, 1988; Coulombel et al, 1989; Frickhofen and Young, 1989; Graeve et al, 1989; Weiland et al, 1989; Frickhofen et al, 1990; Bertoni et al, 1995). Analysis of B19specific antibodies in these individuals indicates a persistence of the IgM antibody and a lack of seroconversion of the IgG antibody from the anti-major capsid species to the anti-minor capsid species. Immunocompromised individuals with chronic hypoplastic anemia resulting from persistent B19 infection may be treated by immune globulin therapy (Kurtzman et al, 1989).  6  Introduction  1.2.3. B19 Infection In Utero The B19 parvovirus is able to cross the placenta. Frequency of maternal infection with B19 is estimated at 1 in roughly every 400 pregnancies (Gay et al, 1994), of which 9% of the cases result in fetal death (Gay et al, 1994; Public Health Laboratory Service Working Party of Fifth Disease, 1990). Incidence of fetal loss is comparable in normal pregnancies and in pregnancies complicated by B19 infection during the first trimester, while the risk for spontaneous abortion increases substantially in cases of infection during the second trimester. Many more cells in the fetus are rapidly dividing and hence susceptible to B19 infection, and the severe anemia resulting from intrauterine infection with B19 could cause death of the fetus in a condition called non-immune hydrops fetalis (Brown et al, 1984; Anand et al, 1987; Anderson L.J., 1990; Wright et al, 1996). The anemia could also cause hypoxia leading to severe developmental defects in the central nervous system (CNS) if the fetus survives to term (Torok, 1995). Surviving neonates would also be expected to develop immune tolerance to B19 capsid proteins as a result of exposure of the fetal immune system to the virus early in pregnancy.  1.2.4. B19 Infection i n the Immunocompetent Host Following infection, B19 propagates itself in infected erythroid progenitor cells leading to the transient depression in red cell count at the peak of viremia during the first week of infection. In the immunocompetent host who has not had a previous B19 infection, virus can be detected from days 5-6 post-infection onwards, reaching maximum levels at days 8-9 (serum titer can reach as high as 10 viral 11  particles per ml). Viremia is accompanied by non-specific flu-like symptoms such as headache, myalgia, and chills resulting from the action of various inflammatory cytokines. The host then launches an IgM-based immune response leading to the formation of immune complexes which are responsible for the secondary symptoms of B19 infection observed by about 15 days post-infection. In children and adults alike, the neutralizing IgM response may coincide with the onset of a rubella-like rash, characterized by maculopapular cutaneous  7  Introduction  eruptions on the cheeks, trunks, and extremities. This condition is known as "fifth disease" (as it is the 5 of 6 classical childhood exanthema) or erythema infectiosm th  (E.I.; Anderson M.J. et al, 1983; Chorba et al, 1986). E.I. symptoms typically resolve within a few days or could recur over a period of several weeks. About half of B19-infected adults (especially women) may experience arthralgias and develop arthritis-like complications in the small joints of the hands and feet (Reid et al, 1985; White et al, 1985; Ueno et al, 1993), which normally resolve within several weeks but have been documented to last as long as two years. Both types of inflammatory responses are manifested secondary to immune complex formation, such that by the time the patients present to medical attention the period of viremia has usually passed. B19-specific IgG expression is also increased following viremia, at around 2 weeks following infection, and is essential for neutralization of the virus (Anderson M.J. et al, 1985; Anderson L.J. et al, 1986). The IgG antibody, which persists for life, is a convenient marker of past exposure to B19 and confers a strong and durable protective effect against recurrent infection by B19. The first wave of seroconversion usually occurs among school-age children; a second wave is observed among parents of school-age children if they had failed to seroconvert beforehand.  8  Introduction  Figure 1.2.4.1. Development of B19-specific IgM (left) and IgG (right) after experimental B19 inoculation of seronegative volunteers (closed circles) and a volunteer with trace amounts of IgG to B19 (open circles). (Figure taken from Anderson M.J. et al, 1985.)  9  Introduction  Symptoms Haemoglobin (g/d I) Itching Headache — Myalgia Chill — Rash — Arthralgia — ArthritisI  I  I  I  I  I  I  • •  I  I  I  I  I  I  0-  I  I  I  I  I  I  I  I  I  I  I  II  9  1  Immune complexes  ••••  <log )5-, 4 • 321• 0-  I  1  1 Neutrophils (xlO" /!)  -I  Virus excretion  10  -r  i l i i t i i t i i i i i i i Reticulocytes ('•)  to0.S' 9.6 • 0.4' 0.2-  I  -2  2  I  I  6  I  I  10  I  I  14  I  I  13  I  I  22  I I  26  -2 Days after P e c u l a t i o n  "i  2  i  i  6  i  i  10  i  i rn  14  18  i  i  22  n  26  Figure 1.2.4.2. Time course of experimental inoculation of Parvovirus B19 in seronegative host. (Figure taken from Anderson M.J. et al, 1985.)  10  Introduction  1.2.5. B19 and Other Disorders A number of disorders not described above have also been associated with or attributed to B19 infection. These are summarized in Table 1.2.5 below. Table 1.2.5. Other disorders associated with parvovirus B19 infection. Cardiovascular manifestations Acute congestive heart failure Myocarditis Pericarditis Cutaneous manifestations Peripheral edema Vascular purpura Vesicular lesions Hematological disorders Aplastic anemia Autoimmune hemolytic anemia Chronic neutropenia Idiopathic thrombocytopenic purpura Hepatobiliary tract disorders Acute hepatic sequestration Fulminant liver failure Neurological diseases Coma Seizure Sensorineural abnormalities Renal diseases Acute renal failure Nephrotic syndrome Rheumatic diseases Juvenile rheumatoid arthritis Rheumatoid arthritis Vasculitis  1.3. B19 i n Cell Culture The species tropism of B19 parvovirus is retained in cell culture. B19 infects and replicates in burst forming erythroid progenitors (BFU-E) as well as in late erythroid progenitor cells (Mortimer et al, 1983). When cultured in the presence of erythropoietin, B19 will also replicate in erythroid progenitor cells from human bone marrow (Ozawa et al, 1986; Ozawa et al, 1987b; Srivastava and Lu, 1988),  11  Introduction  human fetal liver (Yaegashi et al, 1989; Brown et al, 1991; Westmoreland and Cohen, 1991), and normal peripheral blood (Serke et al, 1991), as well as in cells from patients with chronic myelogenous leukemia (CML) or erythroleukemia (Takahashi et al, 1989). Additionally, human megakaryoblastoid cells will also support low levels of B19 replication after being adapted to growth in the presence of erythropoietin (Shimomura et al, 1992; Munshi et al, 1993). The B19-infected primary culture systems described thus far are not readily adaptable to laboratory studies, and to date no known continuous permissive cell line for propagating the virus has been identified. The use of transfected cell systems has aided in the elucidation of various molecular and cellular aspects of B19 replication and transcription. Viral transcripts and proteins are generated in HeLa cells transfected with plasmids containing part or almost all of the B19 genome (Doerig et al, 1987; Ozawa et al, 1988a; Doerig et aZ.,1990) and in COS-7 cells transfected with B19/SV40 hybrid vectors (Beard et al, 1989). A full-length clone containing the entire B19 genome has also been generated (Deiss et al, 1990), though it has not been shown to be infectious.  1.4. The B19 Genome In contrast to other parvoviruses which package predominantly negativesense ssDNA, B19 and AAV package their genome as both positive- and negativesense ssDNA in separate particles in a one-to-one ratio. The 5112nt genome of B19 parvovirus is arranged as a single-stranded coding region flanked on either end by a palindromic inverted terminal repeat (ITR) sequence. The ITR consists of 383nt of which 365nt are folded into a duplex hairpin structure (Deiss et al, 1990), providing a free 3'-hydroxyl group that is essential for initiation of DNA synthesis and replication of the viral genome (Astell, 1990) which take place in the nucleus. The currently accepted model for parvoviral replication (reviewed in Berns, 1990; Berns, 1996) stipulates that the viral ssDNA genome monomer is first converted into a double-stranded DNA (dsDNA) monomer, referred to as the replicative form (RF). This conversion into the RF requires a functional host DNA  12  Introduction  polymerase, while the viral nonstructural (NS) protein becomes essential in subsequent stages of the replicative cycle. The dsDNA RF monomer is converted into a dimer intermediate which is then resolved into two monomer RF molecules. From these RF monomers, viral progeny ssDNA strands (both positive- and negative-sense) are generated and packaged into capsids in the nucleus, and assembled virions are released from the infected cell upon lysis. Unlike some insect densoviruses which encode proteins from opposite strands of the genome, B19 produces proteins from one strand only. The coding strand, by definition the plus strand, contains two large and one smaller open reading frames (ORFs). In contrast to other parvoviruses, B19 produces all of its transcripts from a single promoter located at map unit 6 (p6) (Blundell et al, 1987; Doerig et al, 1987). At least nine different transcripts are known to be generated from the three open reading frames (Figure 1.4), through the extensive use of alternate splicing and end processing mechanisms (Ozawa et al, 1987a; Beard et al, 1989; St. Amand et al, 1991; Luo and Astell, 1993). All transcripts in permissive cell lines have a 56nt leader sequence and are 3'-end processed (polyadenylated) either in the middle or at the far right end of the genome. The open reading frame on the left gives rise to the nonstructural (NS) proteins, while the structural capsid proteins (viral proteins VP1 and VP2) are encoded by the open reading frame spanning the right half of the genome (Ozawa et al, 1988b). ). Differential levels of transcript production are observed in non-permissive and permissive cells; VP transcripts predominate in B19-infected cells (Brown et al, 1994b), while NS transcripts accumulate in HeLa cells transfected with a B19-based plasmid due to a functional block in transcription towards the middle of the genome(Liu et al, 1992). In addition to the transcripts encoding the NS and VP proteins, B19 also produces two classes of abundant small mRNAs encoding proteins with molecular weights of 7.5kDa and llkDa (St. Amand et al, 1991; St. Amand and Astell, 1993; Luo and Astell, 1993).  13  Introduction  open reading frames 20 1000  40 2000  60  100  80  m a p units (m.u.)  L—  4000  3000  5000  nucleotides (nt) Transcript Length 2309nt 807nt 687nt  NS 7.5kDa  ? 7.5kDa,VP1  5010 nt  3156nt  5010 nt  2980nt  5010 nt  2282/2288nt  7.5kDa,VP2  5010 nt  2162/2168nt  VP2  5010 nt  638nt  7.5kDa,11kDa  5010 nt  518nt  11 kDa  Figure 1.4. Transcription and coding map of Parvovirus B19.  14  Protein  VP1  Introduction  1.4.1. The B19 Nonstructural Proteins The full-length NS protein translated from the 2309nt transcript has a molecular weight (MW) of ~70kDa (Cotmore et al, 1986; Ozawa and Young, 1987). There are, however, several lower molecular weight species, and it is not clear whether these represent degradation products or whether they arise from posttranslational modifications of the full-length protein. The functions of NS proteins are highly conserved among the different members of Parvoviridae. In a manner analogous to the NS1 protein of the minute virus of mice (MVM, a prototype member of the genus Parvovirus) and the Rep proteins of AAV2, the NS protein of B19 erythrovirus effects transactivation of the p6 promoter (Doerig et al, 1990) and is likely indispensable for initiation of viral replication through its associated helicase/ATPase and presumed site-specific endonuclease activities.  Recent  evidence also suggests that the B19 NS protein mediates cytotoxicity by induction of apoptosis in infected erythroid cells (Moffatt et al, 1998).  1.4.2. The B19 Structural Proteins Capsid proteins VP1 and VP2 are translated from overlapping open reading frames. They share an identical amino acid sequence except for 227 additional amino acid residues at the amino-terminus of VP1 which are absent from the VP2 sequence. The capsid coat of a native B19 particle is composed of 60 capsomers of VP1 and VP2, of which VP2 is the predominant species. The shorter VP2 protein (58kDa) comprises about 95% of the capsid coat and is termed the "major" structural protein, while the longer VP1 protein (83kDa) accounts for about 5% and is referred to as the "minor" capsid species.  It has been shown by other  investigators that VP2 protein produced from a baculovirus expression system in insect cells is capable of self-assembly into viral particles, with or without VP1, while VP1 does not self-assemble in the absence of VP2 (Kajigaya et al, 1991). In addition to forming the capsid to house the viral genome, structural proteins may also be important in determination of host range and tissue specificity of parvoviruses. Recent evidence points to VP1 and VP2 as being at least partly  15  Introduction  responsible for the tissue tropism of different strains of MVM (Spalholz & Tattersall, 1983) as well as for host range differences between canine and feline parvoviruses (CPV and FPV, respectively; Parrish, 1991). Immunodominance studies suggest that the major neutralization epitopes in the viral capsid reside in the unique aminoterminal portion of the VP1 protein (Saikawa et al, 1993; Brown et al, 1995b), which is believed to protrude from the surface of the viral particle (Rosenfeld et al, 1992).  1.4.3. The B19 l l k D a Proteins The 11 kDa proteins are produced from the third, smaller open reading frame in the B19 genome. Due to the use of alternate start codons, three related proteins of similar molecular weight are translated from both the 638nt and 518nt mRNAs (see Figure 1.4) (St. Amand et al, 1991; St. Amand and Astell, 1993). A functional role for these small proteins in the B19 life cycle has yet to be identified. Other viruses have been shown to encode gene products which modulate the host immune response and evade the immune surveillance system (reviewed in Spriggs, 1994; McFadden, 1995; Spriggs, 1996; Barry and McFadden, 1997; Ploegh, 1998; Spriggs, 1999; Lalani and McFadden, 1999; Lalani et al, 2000), to prevent premature apoptosis of the infected cell, or to induce apoptosis after a productive infection has been established (reviewed in Shen and Shenk, 1995; Teodoro and Branton, 1997; Roulston et al, 1999). The B19 llkDa proteins do not appear to be related in sequence to any other known protein, nor do other members of the Parvoviridae produce them, though a comparable ORF with three regions of homology exists in the SPV genome. Absence of llkDa expression has not been shown to affect either viral capsid assembly (Cohen et al, 1995) or expression of other viral proteins (St. Amand, 1992). The llkDa proteins do not appear to have any region corresponding to transmembrane domains, DNA-binding domains, or Zn -binding domains, and ++  immunoprecipitation experiments have not shown them to interact with other virally encoded proteins. A region has been identified as a putative transactivation motif, though previous experiments have failed to demonstrate any such activity  16  Introduction  from the llkDa proteins (W. Luo and CR. Astell, unpublished results). Although several potential target phosphorylation sites for protein kinase C and casein kinase II have been identified, there is no evidence to suggest that the llkDa proteins are phosphorylated in vivo. Analysis of the amino acid sequence of the llkDa proteins indicates an unusually high proportion of proline residues; 14 out of 94 residues or 15%. The proline residues can be grouped into three regions which share similarity with certain sequences involved in mediating protein-protein interactions, thus leading to the speculation that the llkDa proteins may be involved in binding to other proteins via their proline-rich regions.  1.5. Review of Relevant Signal Transduction Concepts and Pathways In order for an organism to be viable, each cell must perform specific functions at specific times. Co-ordination of behavior in different cells must be exact and occurs through a concerted series of actions cued by various signals from outside or inside the cell. A large number of signals are transduced to the interior of a cell by a relatively small number of signaling molecules, and specificity of signaling is achieved through the assembly of selected signaling molecules at specific times and locations. Signals are propagated through a series of molecules, with each level or "node" subject to signaling divergence or convergence to or from other signaling networks. Proteins interact with each other through various means, including the use of specialized modular domains which were first identified as regions of high sequence homology in signaling molecules. Subsequently, these domains were found to belong to a growing assortment of non-catalytic folding protein modules whose primary role is to mediate moderate to high affinity binding with other proteins. Prototypical protein binding domains include the Src homology (SH) domains, first identified within the signaling molecule and non-receptor protein tyrosine kinase Src. SH2 domains mediate inducible, specific high affinity binding to phosphotyrosine residues on target proteins, while SH3 domains are involved in  17  Introduction  largely constitutive, moderate affinity binding to proline-rich sequences. The latter acts more as a general recruitment type of mechanism than as a molecular on/off switch. One example of a signaling protein which contains these types of domains is the growth factor receptor binding protein (Grb2; Downward, 1994), with two SH3 domains flanking a single SH2 domain. This protein belongs to a class of "adaptor" molecules which possess no intrinsic enzymatic activity and are believed to function solely to bring one protein into close proximity with another protein so that the signal can be propagated. In the case of the signaling network involving Grb2, a paradigm pathway involving receptor tyrosine kinases has been well characterized.  A signal  originating from outside the cell in the form of a growth factor (for example epidermal growth factor or platelet-derived growth factor) is transmitted to its cell surface receptor. The receptor typically traverses the plasma membrane and either possesses intrinsic tyrosine phosphorylation activity or is associated with a nonreceptor tyrosine kinase which mediates self-phosphorylation on critical tyrosine residues on the cytoplasmic tail upon ligand stimulation. The phospho-tyrosine residues then serve as docking sites for SH2 domain-containing proteins such as Grb2, which then recruits another protein, son of sevenless (Sos), to the plasma membrane through SH3 interactions with proline-rich regions or "ligands" on Sos. Sos is a guanine nucleotide exchange factor (GEF) which, when brought into close vicinity to membrane-bound GTPase Ras, is able to exchange GDP for GTP on Ras to activate it. Activated Ras is responsible for propagating the signal via a series of phosphorylation events through downstream kinase effectors Rafl, MAPK/ERK kinase (MEK), and mitogen-activated protein kinase (MAPK) (reviewed in Guan, 1994; Margolis and Skolnik, 1994; Garrington and Johnson, 1999). Phosphorylated MAPK is translocated from the cytoplasm to the nucleus, where it exerts its effects by phosphorylating target proteins on serine residues. A number of target proteins have been identified, including the transcription factor Elk-1 which controls expression of c-fos. C-fos is known to form the API complex in conjunction with c-  18  Introduction  jun, which in turn regulates expression of various genes involved in cell cycle progression and proliferation. In this way, a signal initiated outside the cell is transmitted with high fidelity to the interior of the cell and is capable of eliciting a highly specific response within the cell.  1.6. Review of Functions of Other Viral Nonstructural Proteins In order to maximize chances of survival and propagation, viruses have evolved numerous mechanisms to manipulate the host environment for their own benefit, through interactions with viral or host cell components and alterations in host cell signaling events. At this point, it is useful to review the functions of other viral nonstructural proteins in order to better understand the possible functional roles that the B19 llkDa proteins may play in the viral replicative cycle.  1.6.1. Virus Entry Viral entry into the host cell is typically mediated by interactions of viral capsid or envelope proteins with receptors on the host cell membrane. Recent findings point towards a role for nonstructural proteins in enhancing the process of intracellular delivery of virions into the infected cell. In the case of retrovirus human immunodeficiency virus type 1 (HIV-1), it has been well established that viral entry takes place by fusion, though increasing evidence suggests endocytosis may also play a role. This process is CD4- and chemokine receptor-dependent and appears to be enhanced by the HIV-1 accessory protein Nef, which is believed to carry out a number of other additional functions that contribute to increased viral infectivity (to be discussed further) (Schaeffer et al, 2001).  1.6.2. Nuclear Targeting and Replication of Viral Genome The Rhabdoviridae vesicular stomatitis virus (VSV) expresses a small, basic C protein which functions to enhance viral replication. The C protein increases production  of  full-length  genomic  RNAs  genome/transcriptase complex (Peluso et al, 1996).  19  by  stabilizing the  viral  Introduction  Poliovirus encodes a nonstructural polyprotein, 2BC, which is also involved in viral replication. Sites of genome replication have been localized to membrane vesicles whose formation is coupled to phospholipid synthesis (Guinea and Carrasco, 1990). The 2BC polyprotein enhances viral replication through the induction of membrane vesicle proliferation. HIV-1 also expresses a variety of proteins (the so-called accessory proteins) which are involved in multiple aspects of nuclear targeting and replication of the genome. Up to 100 molecules of the 23kDa virion infectivity protein factor (Vif) are packaged into a single viral particle. These proteins co-localize with and are required for proper packaging of the nucleoprotein core structures (Liu et al, 1995; Hoglund et al., 1994). Vif is required for the synthesis of pro viral D N A in infected cells (von Schwedler et al., 1993) and has been suggested to be involved in nuclear targeting of the genome (Strebel and Bour, 1999) once the viral particle has been uncoated to release the genome in the form of a ribonucleoprotein complex known as the pre-integration complex (PIC).  Another protein implicated in nuclear  targeting of the PIC is the above-mentioned Nef protein, which has been shown to stimulate replication in quiescent CD4 lymphocytes (Spina et ah, 1994) and reverse transcription (Aiken and Trono, 1995; Schwartz et al., 1995) in association with cellular serine/threonine kinases (Sawai et al., 1994). At least three other HIV-1-encoded proteins are involved in the actual translocation and import of the PIC into the nucleus of the infected cell. These include the viral protein R (Vpr) (reviewed in Bukrinsky and Adzhubei, 1999), which connects the PIC with cellular nuclear import machinery even in the absence of active cell cycling (Heinzinger et al, 1994; Popov et al, 1998; Vodicka et al, 1998), thereby facilitating subsequent proviral D N A synthesis and integration into the host genome without the need for cell division. The integrase (IN) and matrix (MA) proteins both contain nuclear localization signals and are also involved in mediating nuclear import and translocation of the PIC (Bukrinsky et al, 1993; von Schwedler et al, 1994; Gallay et al, 1997)  20  Introduction  1.6.3. Viral Gene Expression Viruses encode numerous gene products to regulate expression at the level of transcription (reviewed in Flint and Shenk, 1997). These include the HIV-1 Tat protein (reviewed in Kingsman and Kingsman, 1996), which transactivates cellular and viral genes (Mavankal et al, 1996; Parada and Roeder, 1996), and the bovine papilloma virus type 1 (BPV-1) E2 protein (reviewed in Ham et al, 1991; McBride et al, 1991), which stimulates transcription through the binding of enhancers and transcription factors (Rank and Lambert, 1995; Grossel et al, 1996). Another member of the Papovaviridae family, simian virus 40 (SV40), expresses the large Tantigen which both stimulates late viral gene transcription as well as autorepresses early transcription (Reed et al, 1976; Casaz et al, 1991; Gilinger and Alwine, 1993; Gruda et al, 1993; Rice and Cole, 1993). The small DNA viruses Ad2 and Ad5 from Adenoviridae also encodes several transcriptional regulators. The E1A protein (reviewed in Hagmeyer et al, 1995; Pruzan and Flint, 1995; Shenk, 1996) binds a cellular co-factor p300 and stimulates E2F-dependent transcription by sequestering the cellular retinoblastoma protein (pRB). El A has been shown to activate early viral transcription (Nevins, 1981) and to regulate transcription of genes by either RNA polymerase II or III. Ad2 and Ad5 also encode the IVa2 protein which binds the major late promoter to stimulate late viral gene transcription (Tribouley et al, 1994; Lutz and Kedinger, 1996). Examples of transcriptional regulators from large DNA viruses include the virus early transcription factor (VETF) from vaccinia virus of the Poxviridae family (reviewed in Moss et al, 1991). VETF is a DNA-dependent ATPase which binds the early promoter and is essential for early gene transcription by the vaccinia virus RNA polymerase (Li and Broyles, 1993a; Li and Broyles, 1993b; Cassetti and Moss, 1996). Herpes simplex virus type 1 (HSV-1) of the Herpesviridae encodes the tegument viral protein VP16, also known as ICP25 or a-TIF (reviewed in O'Hare, 1993; Roizman and Sears, 1996), which activates immediate early (IE) gene transcription from the HSV-1 genome (Batterson and Roizman, 1983; Campbell et al, 1984).  21  Introduction  1.6.4. Processing and Nuclear Export of m R N A Transcripts Following transcription of the cellular genes, nascent pre-mRNA species normally undergo a series of post-transcriptional processing steps to become mature mRNA ready for nucleocytoplasmic  export  as  heteronuclear  ribonucleoprotein (hnRNP) complexes. The export process is tightly regulated to prevent escape of any improperly processed or potentially deleterious transcripts, with one of the requirements being the complete removal of all introns within the transcript before it will be exported. This "spliceosome retention" hypothesis stipulates that the mRNA export machinery recognizes only fully spliced mRNAs, and that partially spliced or unspliced pre-mRNA species are retained in the nucleus.  1.6.4.1 Processing and Nuclear Export of Viral Transcripts Viruses have evolved a number of strategies to ensure export of viral transcripts, be they fully spliced or not. One of the best characterized modes of viral transcript escape involves the HTV-1 accessory protein Rev (reviewed in Pollard and Malim, 1998). This 19kDa protein has both nuclear localization and export signals (NLS and NES, respectively) (Fischer et al, 1995) and binds to a sequence known as the Rev-responsive element (RRE) on partially spliced or unspliced viral transcripts. When Rev is bound to the RRE, the NLS becomes masked and the NES associates with the nuclear export machinery to mediate nucleocytoplasmic transport of the viral transcript (Malim et al, 1989; Cullen, 1995). This NES-dependent export is dependent on the cellular protein Crml (Fornerod et al, 1997; Fukuda et al, 1997) and may also require the U l small nuclear RNA (snRNA) from the splicing machinery (Lu et al, 1993). In a similar manner, the adenovirus 55kDa E1B and 34kDa E4 proteins form a complex to mediate export of late viral transcripts (Krug, 1993; Dobbelstein et al, 1997), possibly by sequestration of nuclear export factors away from spliced host mRNAs to sites of viral replication. Other viral proteins involved in viral transcript processing or export include the influenza NS1 protein, which regulates transport of  22  Introduction  spliced NS2 mRNA and its precursor NS1 mRNA (Alonso-Caplen et al, 1992), and the 63kDa HSV-1 IE protein ICP27, which stabilizes the 3' ends of late viral premRNAs and enhances viral transcript export (Brown et al, 1995; Phelan et al, 1996; Sandri-Goldin, 1998).  1.6.4.2. Processing and Nuclear Export of Host Transcripts A number of viral proteins mentioned above in the processing of viral transcripts also play a role in limiting host transcript processing or export. In addition to its positive effects in mediating viral transcript export, the HSV-1 ICP27 protein causes a decrease in host protein synthesis by blocking the splicing of host pre-mRNA species (Hardy and Sandri-Goldin, 1994). A variation of this strategy to block host transcript export is exemplified by the influenza NS1 protein, which binds the U6 snRNA to inhibit host pre-mRNA splicing (Fortes et al, 1994; Qiu et al, 1995).  The NS1 protein also safeguards against host mRNA export a step further  by selectively blocking the export of fully spliced mRNAs containing poly(A) tails that were generated by the cellular two-step cleavage/polyadenylation system, which most host transcripts employ. In this way, fully spliced host mRNAs are retained in the nucleus while export of viral transcripts (whose poly(A) tails are produced differently) is unaffected (Fortes et al, 1994; Qiu and Krug, 1994). The influenza NS1 protein also mediates its negative effects on cellular mRNA export by inhibiting the host cellular 3'-end processing machinery. NS1 launches a double attack to accomplish this: it binds to and blocks the function of the 30kDa subunit of the cleavage and polyadenylation specificity factor (CPSF) (Nemeroff et al, 1998), which is required for 3'-end cleavage and subsequent polyadenylation of the transcript; it also binds to the poly(A)-binding protein II (PABII) (Chen et al, 1999). Binding to PABII prevents the latter's interaction with poly(A) polymerase (PAP), thereby disrupting the processive elongation of poly(A) chains. It is also possible that NS1 may inhibit PABII's ability to directly shuttle cellular mRNAs into the cytoplasm (Chen and Krug, 2000).  23  Introduction  1.6.5. Processing of Viral Proteins Viruses also encode nonstructural proteins that function to process viral gene products. For example, the flavivirus hepatitis C virus (HCV) produces a 70kDa NS3 protein which is required for processing of the single HCV polyprotein. NS3 has an amino-terminal serine protease domain as well as a carboxy-terminal ATPdependent RNA helicase domain. In association with its co-factor NS4A and several host cellular enzymes, NS3 mediates self cleavage from the polyprotein and then proceeds to cleave the rest of the peptide into distinct viral proteins (Tomei et al, 1993; Bartenschlager et al, 1995; Landro et al, 1997).  1.6.6. Host Cell Protein Trafficking and Degradation Some viral proteins function to alter host cell protein trafficking or degradation. HIV-1 alone encodes several of these, including gpl60, viral protein U (Vpu), and Nef. The Env precursor gpl60 protein retains CD4 in the endoplasmic reticulum (ER) to block its trafficking to the cell surface (Crise et al, 1990; Jabbar and Nayak, 1990), and the ER-bound CD4 is then induced by Vpu to undergo degradation via the proteasomal pathway (Willey et al, 1992; Jabbar, 1995). Nef also downregulates CD4 but does this through the enhancement of the rate of cell surface CD4 internalization into endosomes (Rhee and Marsh, 1994). The poliovirus 2BC polyprotein alters protein trafficking in a different manner; it induces membrane vesicle proliferation as discussed earlier and functions to block the exocytic pathway (Barco and Carrasco, 1995).  1.6.7. Virion Packaging, Assembly, and Egress As mentioned above in the context of nuclear targeting of the viral genome, the HIV-1 Vif protein co-localizes with and is required for proper packaging of nucleoprotein core structures (Liu et al, 1995). Additionally, the proper maturation, targeting and release of virions require the function of the 16kDa viral protein U (Vpu) (Subbramanian and Cohen, 1994; Jabbar, 1995; Schubert et al, 1995a; Schubert  24  Introduction  et al, 1995b) and the co-ordinated expression and processing of the Gag, Pol, Env, Vif and Nef proteins (Strebel and Bour, 1999).  1.6.8. Cell Cycle Arrest In addition to its role in facilitating translocation of the HIV-1 PIC into the nucleus, Vpr (reviewed in Bukrinsky and Adzhubei, 1999) also causes cell cycle arrest at the G 2 / M checkpoint via inhibition of Cdc2 kinase activity (He et al, 1995; Jowett et al, 1995; Re et al, 1995; Bartz et al, 1996).  1.6.9. Immune Evasion In order to successfully establish a productive infection in the host, viruses need to escape surveillance or at least delay detection by the host immune system (reviewed in Alcami and Koszinowski, 2000) and avoid any associated anti-viral response.  To accomplish this, different viruses have evolved widely ranging  strategies to interfere with the host humoral immune responses, inhibit interferon responses (reviewed in Goodbourn et al, 2000), regulate cytokine and chemokine function (reviewed in Barry and McFadden, 1997; Spriggs, 1999; Lalani and McFadden, 1999; Lalani et al, 2000), modulate apoptosis signaling (reviewed in Roulston et al, 1999), as well as regulate function of the major histocompatibility complex classes I and II molecules (reviewed in Ploegh, 1998).  1.6.9.1. Inhibition of Humoral Immune Responses The complement cascade forms part of the innate immune response and consists of a series of enzymatic events and formation of complexes involving -20 proteins. The cascade is activated by the binding of a recognition component to an antibody bound to antigen and functions to mediate local inflammation and lysis of infected host cells by the membrane attack complex (MAC). The cowpox virus (member of the Poxviridae family) expresses an inflammation modulatory protein (IMP) which is believed to inhibit production of macrophage  chemoattractant  factors C3a and C5a to limit tissue inflammation/damage and to block M A C -  25  Introduction  mediated lysis (Kotwal, 2000).  The HSV-1 glycoprotein C (gC) also inhibits  complement-mediated lysis of infected cells, by binding C3b, thereby blocking the stablizing effects of properdin on the C3b/Bb convertase complex (Harris et al, 1990; Hung et al, 1994). Several members of the Coronaviridae and the Herpesviridae families (including HSV-1, HSV-2, and murine cytomegalovirus (mCMV)) encode functional IgG Fc receptors. Sequestration of host antibodies thus prevents IgG Fcdependent activation of the complement cascade (Alcami and Koszinowski, 2000).  1.6.9.2. Inhibition of Interferon Responses Host interferon (IFN) expression is typically upregulated upon viral infection.  Effects of the I F N response include (1) upregulation of the 2'-5'  oligoadenylate synthetase (2'-5' OS) which activates RNase L to degrade viral m R N A , (2) activation of the double-stranded R N A (dsRNA)-dependent protein kinase (PKR) which phosphorylates and inactivates the translation factor eIF-2oc to block translation of viral proteins, and (3) induction of apoptosis to prevent viral propagation (Samuel et al, 1997; Clemens, 1997; Clemens and Elia, 1997; DiazGuerra et al, 1997; Castelli et al, 1997; Rivas et al, 1998). Again, viruses have evolved numerous ways to counter host-mediated IFN responses (Goodbourn et al, 2000). The vaccinia virus from the Poxviridae family encodes a viral homolog of the I F N - a / P receptor, which prevents binding and activation of the cellular receptor and thereby blocks IFN function (Smith et al, 1998).  Other viral nonstructural  proteins intercept the IFN response further downstream; the H C V proteins NS5A and E2, and the H I V Tat protein, bind to and inhibit P K R activity. The multifunctional influenza NS1 protein, on the other hand, binds the dsRNA directly to inhibit IFN-induced activation of PKR, so that protein synthesis continues unaffected (Lu et al, 1995). NS1 binding to the dsRNA also inhibits activation of the 2'-5' OS, hence viral mRNA does not become degraded by RNase L.  26  Introduction  1.6.9.3. Modulation of Cytokine and Chemokine Responses Cytokines are soluble proteins that mediate immune and inflammatory responses in cases of infection or injury, and chemokines in particular are chemoattractant cytokines that mediate chemotaxis and trafficking of leukocytes to the affected site. HIV-1 as well as many large DNA viruses have evolved a number of gene products derived from host counterparts to block or stimulate the function of various cytokine, cytokine receptors, chemokines (Ck's), or chemokine receptors (CkR's) in order to best serve the virus' needs for its survival and propagation. The HIV-1 transcriptional activator Tat protein shares partial sequence similarity with a known cellular Ck and has been shown to function as a chemoattractant for monocytes (Albini et al, 1998). Tat also causes changes in host cytokine expression (Brady et al, 1995) by upregulating interleukin-4 receptor (IL4R) expression in a human B cell line (Puri and Aggarwal, 1992) and by increasing IL-2 secretion from activated T cells (Westendorp et al, 1994). The Herpesviridae Epstein-Barr virus (EBV) expresses the latent membrane protein 1 (LMP1), which recruits components of the tumor necrosis factor (TNF) receptor (TNFR)-mediated signal transduction machinery to mimic cytokine responses beneficial to the virus, such as proliferation (Farrell, 1998). Towards the same end, EBV also expresses a viral IL-10 homolog (vIL-10) (Hsu et al, 1990), which stimulates B cell growth and differentiation by inhibiting the synthesis of antiviral cytokines.  The Kaposi's Sarcoma-associated herpesvirus (KSHV, also  known as human herpesvirus 8 or HHV-8) encodes a viral homolog of the macrophage inflammatory protein-la (vMIP-la) from its K6 ORF (Moore et al, 1996) which acts as a functional chemokine to inappropriately induce chemotaxis to favor viral survival. In contrast, the murine y-herpesvirus 68 (MHV-68) secretes a Ck binding protein, vCkBP-III, which functions to sequester a broad range of Ck's (Parry et al, 2000). Members of the Poxviridae have also expended a great deal of effort, genetically speaking, to mimic and modulate the activities of various cellular cytokines, growth factors, and receptors. Vaccinia encodes a viral homolog of the  27  Introduction  epidermal growth factor (vEGF) from its 19K ORF, known as the vaccinia growth factor (VGF), to inappropriately stimulate cell growth, while the molluscum contagiosum virus (MCV) produces from its MC148R ORF a viral MIP-ip homolog which lacks the amino-terminal domain required for activation of the CkR. The vMIP-ip is therefore believed to act as an inhibitor of chemotactic signaling by competing with cellular Ck's for binding to CkR and blocking subsequent leukocyte mobilization (Senkevich et al, 19g6). Other poxvirus-encoded proteins are secreted and function as cytokinebinding proteins to inhibit cytokine signaling. Shope fibroma virus (SFV) and myxoma virus each encodes a T2 protein (Smith et al, 1991; Upton et al, 1991), and cowpox virus encodes a crmB protein (Hu et al, 1994), which are secreted, soluble binding proteins for tumor necrosis factor (vTNFR). These soluble receptors act as a molecular sink to soak up TNF to prevent TNF-mediated cell lysis. In addition to M-T2, myxoma virus also produces M-T7, a secreted IFN-y binding protein (vIFNyR) which correspondingly inhibits IFN function (Nash et al, 1999).  1.6.9.4. Modulation of Apoptosis 1.6.9.4.1. Inhibition of Apoptosis Anti-viral and anti-proliferative responses often involve induction of apoptosis to block viral replication and propagation, though viruses have developed ways to overcome these hindrances. The Papovaviridae SV40 large Tantigen not only transactivates viral gene expression but also binds and inactivates p53, an important mediator of the apoptotic response.  Further downstream,  expression of the anti-apoptotic factor Bcl-X is increased in the presence of the Tax L  protein from human T-lymphotropic virus type 2 (HTLV-2) and is believed to mediate the viral protein's inhibitory effects on Fas-mediated apoptosis (Zehender et al, 2001). The human CMV (hCMV) also encodes two immediate early proteins, IE-1 and IE-2, which have been shown to inhibit TNF-induced apoptosis (Hengel et al, 1998).  28  Introduction  At a level downstream of Fas and TNFR activation, caspase activity is also targeted by virally encoded proteins. Cowpox crmA is a serpin-like protein which inhibits initiator caspase (caspase 1/8) activity to protect against apoptosis mediated by cytotoxic T-lymphocytes (CTLs), TNF, or Fas (Ploegh, 1998; Alcami and Koszinowski, 2000), and KSHV produces a viral homolog of caspase 8/FLICEinhibitory proteins (v-FLIP) that interfere with the recruitment and activation of caspase 8 to block TNF-induced apoptosis (Irmler et al, 1997). African swine fever virus (ASFV), on the other hand, encodes a member of the inhibitor of apoptosis protein (IAP) family named A224L which interacts with the proteolytic fragment of caspase 3 to inhibit its activity. This inhibitory interaction protects the host cell against apoptosis induced by a variety of means including TNF-oc, cycloheximide, and staurosporine in a transfected cell model (Nogal et al, 2001).  1.6.9.4.2. Induction of Apoptosis Occasionally, it may be to the viral pathogen's advantage to promote apoptosis, such as when cell lysis is desirable at the end of the replicative cycle for dissemination of viral progeny. Despite the wealth of knowledge on viral proteins that block apoptosis, those virally encoded proteins which stimulate the process are much less well characterized. Among the well-established examples are the adenovirus E1A protein which induces sensitivity to TNF (Duerksen-Hughes et al, 1989) and upregulates p53 (Debbas and White, 1993; Lowe and Ruley, 1993), and the HIV-1 Tat protein which upregulates the FasL promoter to sensitize cells to Fasmediated apoptosis (Westendorp et al, 1995). And of course, the NS1 proteins from Parvoviridae members M V M and B19 have been shown to cause D N A strand breaks (Op de Beeck and Caillet-Fauquet, 1997), induce cell cycle arrest at the G 2 / M checkpoint (Op de Beeck et al, 1995), and mediate apoptosis of the infected cell (Moffatt et al, 1998).  29  Introduction  1.6.9.5.  Modulation of Major Histocompatibility Complex  ( M H O Function Major histocompatibility complex (MHC) molecules are important vehicles for antigen presentation and for host detection of foreign substance. Helper T cells express CD4 co-receptors for MHC class II product recognition (sampling endosomal compartments), while cytotoxic T-lymphocytes (CTLs) express CD8 coreceptors for MHC class I product recognition (sampling the proteasomal pathways). MHC products are typically formed from a pair of ER-associated heavy and light subunits stabilized by a short antigenic peptide derived from cleavage of proteins from the proteasomal or endosomal degradation pathways. The assembled products are then trafficked to the plasma membrane for cell surface presentation of the antigen. Virtually every component of the MHC-mediated antigen presentation system is subject to viral counterattack, as are most elements of other immune response pathways. The EBV nuclear antigen 1 (EBNA-1) contains a glycine-alanine repeat domain that interferes with recognition of its own proteasomal cleavage site. By escaping proteolysis, EBNA-1 is able to avoid MHC class I presentation of its fragments and subsequent recognition by CTLs (Levitskaya et al, 1997). Other proteins, such as ICP47 from HSV-1 and HSV-2, act to inhibit the peptide transporter protein TAP by competing with peptides for binding to the cytosolic surface of the TAP complex, thereby blocking transport of antigenic peptides to complete assembly of the MHC molecules (York et al, 1994; Hill et al, 1995; Tomazin et al, 1996). Assembled MHC molecules are by no means immune to viral countermeasures. For instance, the human CMV encodes a protein named US3 which binds and retains MHC class I molecules in the ER (Ahn et al, 1996); HIV-1 Nef and KSHV K3 and K5 gene products also downregulate MHC class I products (Schwartz et al, 1996; Ishido et al, 2000; Coscoy and Ganem, 2000). HIV-1 Nef and EBV vIL-10 homolog also interfere with processing of MHC class II molecules, thus delaying recognition by T helper cells (Koppelman et al, 1997).  30  Introduction  Cells with downregulated display of MHC products are susceptible to killing by natural killer (NK) cells, as cells bearing surface MHC molecules normally send NK cells signals to prevent killing of themselves. The human CMV has come up with expression of a MHC class I homolog, UL18 (homologs also produced by murine mCMV and rat rCMV), to function as a decoy to fool NK cells and inhibit NK-mediated cell lysis (Farrell et al, 1997).  1.7. Other Proline-Rich Viral Nonstructural Proteins It is difficult to predict what the function of the llkDa proteins may be, given the widely ranging roles that nonstructural proteins from other viruses play. In the characterization of unknown proteins, it has been instructive to look for similarities with known proteins in terms of sequence and structure in order to delineate function. In the case of the llkDa proteins, there are very few clues upon which to base these types of comparisons, though other virally encoded, nonstructural proteins containing proline-rich regions or SH3 recognition sites have been catalogued. These include the aforementioned HCV NS5A protein, which has been shown to interact with the SH3 domain-containing protein Grb2 to disrupt mitogenic signaling in addition to its role in inhibition of IFN-induced PKR antiviral activity (Tan et al, 1999). In addition, the Nef protein from HIV-1 contains a highly conserved sequence with repeated proline residues in PxxP motifs which was shown to mediate binding to the SH3 domain in various Src-family nonreceptor tyrosine kinases (Saksela et al, 1995). This SH3 domain-binding function is also important in mediating interaction of Nef with a p21-activated protein kinase (PAK)-like Nef-associated kinase (NAK), which implicates the HIV-1 Nef protein in modulation of host cell signaling cascades that may be of importance in HIV-1 pathogenesis (Manninen et al, 1998).  31  Introduction  1.8. Previous Studies on the l l k D a Proteins and the Present Study The llkDa proteins, like the signaling protein Sos, contain proline-rich regions which conform to consensus SH3 recognition sites with the sequence xxxPPxPxx. Because such sequences have been identified in numerous signaling molecules and are known to mediate binding to SH3 domains on other proteins, we speculate that the llkDa proteins, like Sos, may be involved in protein-protein interactions. To investigate this possibility, affinity chromatography experiments have been carried out to identify putative cellular interactors of the llkDa proteins. An ~83kDa and a ~26kDa protein from an erythroleukemic cell lysate were shown to bind to an llkDa protein fused carboxy-terminal to a glutathione S-transferase (GST) epitope tag but not to GST alone. Based on the assumption that the llkDa proline-rich region may be mediating protein-protein interactions, a computer search was done to identify proteins with these molecular weights containing one or more SH3 domains. The adaptor protein Grb2 fits these criteria and was therefore tested for its ability to interact with the llkDa proteins. Preliminary far western blotting studies suggested that a Grb2 fusion protein can interact specifically with the GST-llkDa fusion protein but not with GST, demonstrating interactions between llkDa and Grb2, at least in vitro (Zagrodney, 1998). The present study attempts to characterize the HkDa/Grb2 interaction further, through the use of a mutagenic approach coupled with far western blotting studies and GST fusion protein pull down assays. Our working hypothesis is that the B19 llkDa proteins interact with Grb2, attenuating the normal cellular Grb2/Sos/Ras signaling pathway and affecting downstream signaling (see Figure 1.8).  32  Introduction  Growth Factor  Receptor  Figure 1.8. Model of B19 l l k D a protein-mediated disruption of the Grb2/Sos/Ras signaling pathway.  33  Materials and Methods  MATERIALS A N D METHODS  2.1. Chemicals and Supplies All chemicals used were purchased from Fisher Scientific Co., Sigma Chemical Co., Gibco BRL, or Roche Diagnostics (formerly Boehringer Martnheim) unless otherwise specified. Bacterial culture reagents were purchased from BDH Inc. or ABI. Tissue culture reagents were from Gibco BRL or Sigma Chemical Co. Cell Culture Reagents. Agarose and polyacrylamide gel supplies were from Gibco BRL or Bio-Rad Laboratories. DNA ladders and protein molecular weight standards were obtained from Gibco BRL and Bio-Rad Laboratories, respectively. DNA purification kits were obtained from Qiagen (QIAprep Spin Miniprep Kit) or Bio/Can Scientific (GeneClean II kit). Radiolabeled dATP was purchased from New England Nuclear (NEN) Dupont (now part of Perkin Elmer (PE) Applied Biosystems), ultrapure deoxyribonucleotide triphosphates (dNTP's) were from Amersham, and the BigDye Terminator Reaction Ready Mix for automated sequencing was supplied by PE Applied Biosystems.  Restriction and  deoxyribonucleic acid (DNA)-modifying enzymes were obtained from New England Biolabs (NEB), Gibco BRL, or Boehringer Mannheim unless otherwise noted. Oligonucleotide primers were synthesized at the Nucleic Acid and Protein Synthesis (NAPS) unit at the University of British Columbia. GlutathioneSepharose 4B and Protein A-sepharose CL-4B resins for protein purification, protein pull down assays and immunoprecipitation studies were supplied by Amersham Pharmacia Biotech.  Polyvinylidene fluoride (PVDF) membranes and other  apparatus for protein transfer were from Bio-Rad Laboratories (Immun-Blot PVDF) or Pall Corp. Gelman Laboratory (BioTrace PVDF). Primary antibodies were from StressGen Biotechnologies, Boehringer Mannheim, or Becton Dickinson (BD) PharMingen/Transduction Laboratories unless otherwise noted. Secondary antibodies were purchased as part of the enhanced chemiluminescence (ECL) kit from Amersham Pharmacia Biotech or from  34  Materials and Methods  Jackson ImmunoResearch. Film for ECL detection was from Amersham Pharmacia Biotech.  2.2. Bacterial Culture 2.2.1. Growth and Maintenance Routine cloning, plasmid propagation, and protein expression were carried out in DHlOb E. coli [F mcrA A(mrr-hsdKMS-mcrBC) <|)80d/acZAM15 A/acX74 deoR rec A l end A l araD139 A(ara, leu)7697 gal\J galK X' rpsL nupG] from Gibco BRL.  Bacteria were routinely grown on Luria-Bertani (LB) agar plates (1% peptone, 0.5% yeast extract, 1% sodium chloride (NaCl), and 1.5% agar) or in LB media for plasmid isolation; for protein expression, bacteria were cultured in 2xYT media (16g peptone, lOg yeast extract, 5g NaCl per liter). Media or agar were supplemented with 100(ig/ml ampicillin (Amp) or 30|ig/ml kanamycin (Kan) when required for plasmid selection. Frozen stocks of bacterial cultures were routinely prepared with 25% glycerol and stored at -80°C.  2.2.2.  Preparation of Competent Bacteria for Heat Shock  Transformation Competent DHlOb E. coli bacteria were prepared from a 10ml overnight culture grown at 37°C in LB media which was used to inoculate 200ml LB media. The 200ml culture was then grown at 37°C with vigorous shaking until an optical density at 600nm (OD ) of -0.9 was reached.  Bacteria were harvested by  600  centrifugation at 5000rpm for 5 minutes at 0°C, and the supernatant was decanted. The pellet was resuspended in 50ml of ice-cold lOOmM MgCl and centrifuged 2  again at 5000rpm for 5 minutes at 0°C. After the supernatant was removed, the cells were resuspended in 10ml ice-cold lOOmM CaCl . A further 100ml of ice-cold 2  lOOmM CaCl was added, and the solution was mixed and incubated for 1 hour on 2  ice. The suspension was spun as before and the pellet was resuspended in ~12ml of ice-cold 85mM CaCl solution containing 15% glycerol. The mixture was separated 2  35  Materials and Methods  into smaller aliquots and frozen in a dry ice/ethanol bath. Frozen stocks of competent bacteria were stored at -80°C for up to a year.  2.2.3. Heat Shock Transformation of E. coli Plasmid DNA was routinely introduced into bacteria by heat shock transformation. 50ul of heat shock competent DHlOb E. coli were mixed with 0.5pg or more of plasmid DNA or with lpl of a ligation reaction. The mixture was incubated on ice for 30 minutes, heat shocked at 42°C for 1 minute, and replaced on ice for a further 2 minutes before being diluted with 950ul fresh LB media. After incubation for 1 hour at 37°C with vigorous shaking, the mixture was centrifuged for 5 minutes at 4000rpm. The supernatant was removed by aspiration, and the pellet was resuspended in lOOpl of LB media and plated in entirety onto LB agar with appropriate antibiotic selection for overnight growth at 37°C.  2.3. Tissue Cell Culture 2.3.1. Growth and Maintenance The human embryonal kidney (HEK) 293 cell line has been transformed with sheared human adenovirus type 5 (Ad 5) DNA and expresses the Ad 5 transforming gene (info from ATCC).  COS-7 cells are simian CV-1 cells  transformed with an origin-defective SV40 mutant virus which expresses the large T-antigen and therefore supports high copy number replication of plasmids containing the simian virus SV40 origin (info from ATCC). Both HEK 293 and COS7 cells were cultured as monolayers in complete media consisting of low glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), lOmM HEPES at pH 7.4, and lx antibiotic-antimycotic (Gibco BRL; lOOunits penicillin, lOOpg streptomycin, and 0.25pg amphotericin B per milliliter of media). Cells were propagated in 100mm culture dishes in a water-jacketed incubator at 37°C with 5% C0 . Frozen stocks of cells were quickly thawed at 37°C 2  and diluted 1:10 in fresh, complete media. Cells were routinely trypsinized and  36  Materials and Methods  passaged in a 1:10 dilution in fresh media when they have reached about 90% confluency, usually every 3 to 4 days. Frozen stocks of HEK 293 and COS-7 cells were routinely prepared as one milliliter aliquots of roughly 1-2 x 10 cells in 6  freezing media consisting of DMEM supplemented with 10% FBS and 15% dimethylsulfoxide (DMSO). Cells were frozen for several hours at -20°C and then overnight at -80°C before being transferred to liquid nitrogen for long term storage. Selected aliquots were thawed and checked for viability at three or more weeks after the initial date of freezing.  2.3.2. Transfection of Plasmid D N A into COS-7 Cells COS-7 cells were transfected by a modified Diethylaminoethyl (DEAE)Dextran procedure. Briefly, nearly confluent cells in 100mm culture dishes were passaged at a 1:6 dilution one day prior to transfection. Five micrograms of plasmid DNA purified by ethanol precipitation was dissolved in lOOul of filtersterilized nanopure distilled water (dH 0), mixed with 200ul of 2mg/ml DEAE2  Dextran, and diluted to 3ml final volume with serum-free DMEM. Cells were washed twice with DMEM and then overlaid with the transfection solution. After 8hr of incubation at 37°C 5% C0 , the transfection solution was removed and 2  replaced with 3ml of phosphate-buffered saline (PBS) containing 10% DMSO. Cells were subjected to the DMSO shock for exactly 5 minutes and then washed twice with PBS before the addition of 10ml of complete media. Transfected cells were incubated at 37°C 5% C 0 for 2-3 days before being analyzed for target protein 2  expression.  2.3.3. Lysis of Tissue Culture Cells for GST Protein Pull Down and Immunoprecipitation Experiments Nearly confluent cells were trypsinized, resuspended in complete media, and counted. The suspension was then centrifuged for 5 minutes at 1200rpm to pellet the cells. The supernatant was removed by aspiration and the pellet was washed once with PBS and centrifuged as above. After removal of the supernatant,  37  Materials and Methods  the washed pellet was resuspended in ice-cold hypotonic buffer (20mM Tris-Cl pH7.4) supplemented with protease inhibitors at 2pg/ml antipain, 0.5mM phenylmethylsulfonyl fluoride (PMSF), lug/ml leupeptin, and 4pg/ml aprotinin. Cells were resuspended at a concentration of roughly 3 x 10 cells per milliliter of 6  hypotonic buffer and incubated on ice for 15 minutes to allow for cell swelling. The cells were then transferred to a prechilled dounce homogenizer and lysed with 8 up and down strokes using a type-B pestle. Lysates were routinely prepared fresh and used within one hour.  2.4. Molecular Cloning Techniques 2.4.1. Small-Scale Plasmid D N A Isolation Plasmid DNA was isolated according to a modified alkaline lysis protocol based on the method described in Le Gouill et al. (1994). The bacterial pellet from 1.5ml of an overnight culture of bacterial cells was resuspended in lOOul of Solution I (50mM glucose, lOmM ethylene-diamine tetra-acetic acid (EDTA), 25mM Tris-Cl, pH8.0).  Then 200ul of Solution II (0.2N NaOH, 1% SDS; prepared fresh  immediately before use) and 200pl of chloroform were added, mixed by vortexing, and the cells were lysed for 1 minute. 150ul of Solution III (29.4g CH COOK, 5ml 3  HCOOH and H 0 to 100ml final volume) was added and mixed by vortexing for 22  3 seconds. The mixture was then centrifuged at top speed for 2 minutes to pellet cell debris. The upper, aqueous phase was transferred to a fresh microfuge tube, and DNA was precipitated with 2 volumes of ice-cold 95% ethanol and pelleted by centrifugation. After the DNA pellet was washed once with 70% ethanol and recentrifuged, it was dissolved in 50ul of sterilized distilled H 0 (sdH 0) containing 2  2  0.05pg/pl of RNase A to remove contaminating RNA fragments. The DNA was then subjected to screening or verification by restriction endonuclease digestion, Gladdering, or automated sequencing, as required.  38  Materials and Methods  2.4.2. Large-Scale Plasmid D N A Isolation Plasmid DNA was isolated according to an alkaline lysis protocol modified from the method described in Sambrook et al. (1989). Briefly, 200ml of LB media with appropriate antibiotic selection were inoculated with a single colony of bacteria carrying the plasmid DNA of interest for overnight growth at 37°C with vigorous shaking. Bacteria were harvested and lysed under alkaline conditions, and plasmid DNA was isolated and purified by polyethylene glycol (PEG) precipitation. The DNA was further purified by phenol/chloroform extraction before being sterilized by ethanol precipitation. The final DNA pellet was dissolved in sterilized distilled water (sdH 0) and quantitated by spectrophotometric 2  measurements at 260nm.  2.4.3. D N A Quantitation and Purity Check Plasmid DNA and oligonucleotides were routinely quantitated by spectrophotometric measurements at 260nm in a Pharmacia Biotech Ultrospec 3000 UV/Visible spectrophotometer. Absorbance at 260nm (A ) is related to DNA 260  concentration as follows: Concentration of plasmid DNA in ug/ml = (A )(50ug/ml)(dilution factor) 260  Concentration of oligonucleotide DNA in ug/ml = (A )(33ug/ml)(dilution factor) 260  The ratio of A  260  to A  280  gives an indication of the purity of the DNA; most pure  DNA preparations generate ratios of between 1.8 and 2.0.  2.4.4. Plasmid D N A Digestions All plasmid DNA digestions were done using restriction endonucleases, buffers, and protocols provided by NEB, Gibco BRL, or Boehringer Mannheim. In general, up to lug of plasmid DNA would be digested with restriction enzymes at a concentration of 4 units (U) of enzyme per microgram of DNA, in a final reaction volume of 20ul to 50ul and incubated at the specified temperature (usually 37°C) for  39  Materials and Methods  two or more hours. The samples were then resolved by agarose gel electrophoresis or purified for further use with a commercial D N A purification kit.  2.4.5. Plasmids Table 2.4.5. Plasmid vectors and the proteins they express. PLASMID  PROTEIN  pGEX2T  GST  pGEX-11 wt/mutant  GST-11 wt/mutant  pGEX2T-HA  GST-HA  pGEX2T-HA-Grb2  GST-HA-Grb2  pEGFP-C2  EGFP  pEGFP-11  EGFP-11  2.4.5.1. pGEX2T (figure 2.4.6.1) directs expression in E. coli of glutathione Stransferase (GST), a 26kDa protein from Schistosoma japonicum, under the control of the inducible tac promoter. A multiple cloning site (MCS) following the GSTencoding sequence permits construction of a fusion protein, with the protein of interest fused carboxy-terminal to GST. Because GST binds strongly to glutathione, GST and GST fusion proteins can be readily purified by affinity chromatograhy on glutathione-sepharose resin (Pharmacia Biotech, 1997). 2.4.5.2. pGEX-11 was constructed by Dr. Janet St. Amand by cloning the open reading frame (ORF) for the l l k D a protein from B19 parvovirus in frame into the Bam HI /Eco RI sites of pGEX2T. This vector directs the expression of a GST-llkDa fusion protein, GST-11.  40  Materials and Methods  D s a l 4848 B s u 3 6 l 4739  BspM I 264 EcoN I  N a r l 4286 Bbe I 4286  BstB I 930 935 935 935 940  H p a l 4151 E c o R V 4095 B s s H II 4058 Ban II 3854 A p a l 3854 BstEII 3828  ^  Mlu I 3647  r-  1115 Tth111 I 1123 BsaA I  U  1220 A a t l l  4948 base pairs Unique Sites  PflM I 3223 Pstl  AlwN I 2617  Figure 2.4.5.1. pGEX2T plasmid map.  41  BamH I Ava I Sma I Xmal EcoRI  Materials and Methods  2.4.5.3. pGEX-11 P42,45A/P42,45,46A/P42,46A/P43A/P43,45A/P43,45,46A/P45A /P45,46A were constructed from pGEX-11 by PCR-based site-directed mutagenesis using oligonucleotide primers HkDPro2 and 11COMPL. The resulting GST-11 proteins have proline-to-alanine mutations at amino acid residues corresponding to the specified positions within the native llkDa protein (i.e. P42,45A specifies a double amino acid substitution of Pro-42 and Pro-45 with Ala). 2.4.5.4. pGEX-11 P42A was constructed from pGEX-11 by PCR-based site-directed mutagenesis using oligonucleotide primers P42A1 and P42A2. The resulting GST11 protein has a proline-to-alanine mutation at an amino acid residue corresponding to position 42 of the native llkDa protein. 2.4.5.5. pGEX-11 P46A was constructed from pGEX-11 by PCR-based site-directed mutagenesis using oligonucleotide primers P46A1 and P46A2. The resulting GST11 protein has a proline-to-alanine mutation at an amino acid residue corresponding to position 46 of the native llkDa protein. 2.4.5.6. pGEX2T-tag was generously provided by Ken Harder and was constructed by insertion of a hemagglutinin (HA) epitope tag and polylinker into the Bam HI/Eco RI sites of pGEX2T. This vector encodes a GST-HA fusion protein. 2.4.5.7. pGEX2T-tag-Grb2 was generously provided by Dr. John Schrader and was constructed by cloning the cDNA encoding the human growth factor receptor binding protein (Grb2) into the Nhe l/Eco RI sites of pGEX2T-tag. This vector directs the expression of a GST-HA-Grb2 fusion protein. 2.4.5.8. pEGFP-C2 (figure 2.4.6.8) directs expression of a variant of wild-type green fluorescent protein (GFP). GFP is a protein from Aequorea victoria that fluoresces upon light excitation and is useful as a tag to localize proteins or monitor transgene expression in living cells. The present vector encodes a GFP variant referred to as enhanced GFP, or EGFP, under the control of a human cytomegalovirus (CMV) 42  Materials and Methods  immediate early promoter. EGFP has an excitation maximum at 488nm and an emission maximum at 507nm, and the protein has been optimized for brighter fluorescence and higher expression in mammalian cells. The vector contains an SV40 origin for high copy number replication in mammalian cells expressing the SV40 T-antigen (i.e. COS-7 cells). An MCS following the EGFP-encoding sequence permits construction of a fusion protein, with the protein of interest fused carboxyterminal to EGFP.  6 Ase I 233 Nde I 338 S n a B I 591 N h e l 594 E c o 4 7 l l l  HgiEII 4090  E c o O 1 0 9 l 3856  pEGFP-C2  4735 base pairs 1343 1347 1347 1350 1356 1363 1368 1373 1373 1379 1379 1383 1387 1390 1390 1394 1406 1416  R s r l l 3274  Tth111 I 2874 Fsp I 2859 M s c l 2839 N a r l 2758 Bbe I 2758 Cla I 2598 Stu I 2578 Sfi I 2527  Bgl II PaeR7l XhoI Sac I HinD III EcoR I Pstl Accl Sal I Asp718 Kpnl S a c II Apal Smal Xmal BamH I Xbal Bel I  1520 H D a I  Figure 2.4.5.8. pEGFP-C2 plasmid map.  43  Materials and Methods  2.4.5.9. pEGFP-11 was constructed by Dr. Hanson Chen by cloning the ORF for the B19 llkDa protein from pGFP-11 in frame into the Bgl Il/Eco RI sites of pEGFP-C2. This vector encodes an EGFP-llkDa fusion protein, EGFP-11, which is presumed to have similar excitation and emission properties as does wild-type EGFP.  2.4.6. Oligonucleotides Oligonucleotide primers were synthesized at the NAPS unit at UBC. Mutagenic oligonucleotides to be used in PCR-based site-directed mutagenesis experiments were first 5'-phosphorylated using T4 polynucleotide kinase (PNK) in the presence of the appropriate lx reaction buffer containing ImM ATP. Phosphorylation was carried out at 37°C for 30 minutes and terminated by heat inactivation at 70°C for 10 minutes. 2.4.6.1. HkDPro2 is a mutagenic oligonucleotide with random C or G at 4 positions corresponding to the first nucleotide in triplet codons encoding Pro-42,43, 45, or 46 in the native B19 llkDa protein, in the reverse complementary orientation. 5'-CAG GTA CAG TCT G G C / G TGC/G TAC T G C / G T G C / G GCG TTT AGT TAC-3' 2.4.6.2. 11COMPL is a mutagenic oligonucleotide with random C or G at 4 positions corresponding to the first nucleotide in triplet codons encoding Pro-42,43, 45, or 46 in the native B19 llkDa protein. 11COMPL is identical to HkDPro2 in the reverse complementary orientation. 5'-GTA ACT AAA CGC C/GCA C/GCA GTA C / G C A C / G C C AGA CTG TAC CTG-3' 2.4.6.3. P42A1 is a mutagenic oligonucleotide with a C-to-G mutation at the position corresponding to the first nucleotide in the triplet codon normally  44  Materials and Methods  encoding Pro-42 in the native B19 l l k D a protein, leading to incorporation at that position with Ala. 5-CGT A A C T A A A C G CGC A C C AGT A C C ACC-3' 2.4.6.4. P42A2 is a mutagenic oligonucleotide with the reverse complementary sequence to P42A1. 5'-GGT GGT ACT GGT GCG CGT TTA GTT ACG-3' 2.4.6.5. P46A1 is a mutagenic oligonucleotide with a C-to-G mutation at the position corresponding to the first nucleotide i n the triplet codon normally encoding Pro-46 in the native B19 l l k D a protein, leading to incorporation at that position with Ala. 5'-GCC C A C C A G TAC C A G C C A G A C TGT A C C TG-3' 2.4.6.6. P46A2 is a mutagenic oligonucleotide with the reverse complementary sequence to P46A1. 5 - C A G G T A C A G T C T G G C TGG T A C TGG TGG GC-3' 2.4.6.7. pGEX3' anneals to the pGEX-11 (wild-type and mutant) vector ~200nt downstream of the targeted l l k D a proline-to-alanine mutation site and sequences along the reverse complementary strand. 5'-CCG G G A GCT G C A TGT GTC A G A GG-3' 2.4.6.8.  USeql  anneals within the l l k D a ORF ~70nt downstream of the targeted  mutation site and sequences along the reverse complementary strand. 5'-GTT GTT CAT ATC TGG TTA AG-3'  45  Materials and Methods  2.4.7. Polymerase Chain Reaction (PCR)-Based Site-Directed Mutagenesis The B19 llkDa protein contains three proline-rich regions conforming to SH3 ligand motifs. Site-directed mutagenesis was performed to substitute selected proline residues with alanines in the second of these regions to determine if this region is involved in interaction of the llkDa protein with Grb2. The 25ul reaction mix consisted of lx Cloned Pfu Buffer (Stratagene), 5ng of template pGEX-11 plasmid DNA, 62.5ng of each of two sequence-complementary 5'-phosphorylated mutagenic oligonucleotide primers, 0.2mM dNTP's, and 1.25 units (U) of Pfu Turbo DNA polymerase (Stratagene).  After an initial denaturation step at 94°C, the  reaction was cycled 16-18 times through denaturation for 1 minute at 94°C, primer annealing for 1 minute at 56°C, and nascent strand extension at 68°C for 11 minutes (approximately 2 minutes per kb of template multiplied by ~5.2kb of pGEX-11). The PCR products were treated with Dpn I which specifically digested methylated and hemimethylated DNA and was therefore used to select for newly-synthesized mutation-containing plasmid DNA. Samples with or without Dpn I treatment were resolved by 1.0% agarose gel electrophoresis in Tris-Acetate-EDTA (TAE) buffer and visualized by ethidium bromide staining. The Dpn I-digested samples were then transformed into competent E. coli bacteria with or without being treated with T4 DNA ligase. The ligase would repair the nicks within the nascent plasmid molecule, though the nicks, being staggered, would not be expected to interfere with circularization of the plasmid DNA or the subsequent transformation into bacteria.  2.4.8. G-Ladder Screen for Mutant Plasmid D N A Site-directed mutagenesis using HkDPro2 and 11COMPL as primers and pGEX-11 as template yielded products with variable sequence where either C or G could be incorporated in up to four different nucleotide positions, due to partial degeneracy of the primers at these positions. In order to screen for and identify mutant pGEX-11 plasmid DNA containing defined C-to-G mutations, a modified  46  Materials and Methods  procedure based on the Sanger dideoxy manual sequencing method (Sanger et al, 1992) was employed. In this method, a- P dATP from NEN Dupont was used as 32  label in conjunction with the Reagent Kit for T7 Sequenase v2.0 DNA Polymerase from Amersham. The template typically consisted of 28pl of plasmid DNA from small-scale isolation and was denatured in 0.2M NaOH at 37°C for 30 minutes prior to ethanol precipitation. The washed and dried pellet of denatured DNA was annealed with lpmol of either the pGEX3' or USeql oligonucleotide in lx sequencing buffer. Instead of setting up four different reactions using a different dideoxyribonucleotide in each one, only dideoxyguanosine triphosphate (ddGTP) was employed in the sequencing reaction, hence the name "G-laddering" for this modified sequencing protocol. Radiolabeled dATP was incorporated into nascent DNA strands synthesized by T7 DNA polymerase. DNA fragments of different sizes were generated, depending on the position of incorporation of ddGTP, and these were resolved on a 6% polyacrylamide gel containing urea in Tris-BorateEDTA (TBE) buffer. Gels were run under constant power at 32W (voltage ~2000V) for 1 or 3 hours using HSeql or pGEX3' as the primer, respectively. Gels were then dried in a Bio-Rad gel dryer and exposed to Kodak or AGFA X-ray film overnight. The resulting banding pattern from a given sample would represent DNA fragments ending at all possible GTP positions. Because both primers employed here sequence along the reverse complementary strand from downstream of the llkDa stop codon, the nucleotide C at the first position of codons specifying prolines would be read as the nucleotide G from the reverse complementary direction. Hence the sequence analysis of wild-type pGEX-11 should yield bands corresponding to DNA fragments ending with ddGTP at each of the four positions to be mutated. In the case where a C-to-G mutation has occurred, this would become a G-to-C mutation on the reverse complementary strand which would be manifested as a missing band in the G-ladder. In this way, pGEX-11 mutants (other than pGEX-11 P42A and pGEX-11 P46A) were identified and subjected to sequence verification by automated sequencing.  47  Materials and Methods  2.4.9. Automated Sequencing of Plasmid D N A PE Applied Biosystems provides a comprehensive protocol for automated DNA sequence analysis which was followed with minor modifications. Briefly, double-stranded plasmid DNA template was isolated from a large or small scale preparation and was quantitated by A  260  measurements. 100-300ng of DNA was  mixed with 3.2pmol of sequencing primer and 2ul of the "BigDye Terminator Reaction Ready" PCR mix (PE Applied Biosystems) on ice. Sterilized distilled H 0 2  (sdH 0) was added to a final reaction volume of 5ul prior to amplification in a 2  Perkin Elmer GeneAmp PCR System 2400 instrument. Samples were denatured for 5 minutes at 95°C and then subjected to 25 cycles of 5 seconds at 95°C, 10 seconds at 55°C, and 4 minutes at 60°C. The DNA was then precipitated with 95% ethanol, washed with 70% ethanol, and dried in a Savant Speed Vac SCI 10 instrument. Samples were then submitted to Dr. Ivan Sadowski's laboratory here in the Department of Biochemistry and Molecular Biology at UBC for automated sequence analysis.  2.5. Protein Techniques 2.5.1. G S T / G S T Fusion Protein Purification GST and GST fusion proteins were purified according to a protocol modified from the method described by Pharmacia, supplier of the pGEX2T plasmid. Briefly, 200ml of 2xYT media was inoculated with 5ml of overnight culture and incubated at 37°C with vigorous shaking until an OD  600  of ~0.5 was reached. Isopropylthio-(3-  D-galactoside (IPTG) was added to a final concentration of 0.2mM to induce expression of GST and GST fusion proteins from the tac promoter, and the culture was grown at 37°C for 3 more hours prior to being harvested by centrifugation at 7200rpm for 10 minutes at 4°C. The supernatant was discarded and the pellet stored at -20°C until ready for protein purification. The pellet was first resuspended in 10ml of ice-cold PBS supplemented with protease inhibitors at 2ug/ml antipain, 0.5mM PMSF, lug/ml leupeptin, and 4ug/ml aprotinin. 1ml of 10mg/ml lysozyme  48  Materials and Methods  in STET buffer (8% sucrose, 0.5% Triton X-100, 50mM EDTA, lOmM Tris-Cl pH8.0) was added, and the solution was incubated at room temperature for 15 minutes with end-to-end rotation. Bacteria were lysed by sonication in 15ml Sarstedt screwcap conical tubes on ice with two 30-second bursts at 40% intensity separated by a 1-minute break on ice in between bursts. Protein was solubilized by the addition of Triton X-100 to 1% final concentration and incubation for 30 minutes at 4°C with end-to-end rotation. After centrifugation at 9400rpm for 10 minutes at 4°C, the supernatant was treated with 2mM ATP/lOmM MgSO /50mM Tris-Cl pH8 for 10 4  minutes at 37°C prior to incubation with PBS-equilibrated glutathione-sepharose 4B resin for 1 hour at 4°C with end-to-end rotation. The resin and all bound proteins were pelleted by centrifugation and washed three times with ice-cold PBS 5mM ATP supplemented with protease inhibitors. Bound proteins were eluted by resuspending the pellet in glutathione elution buffer (GEB, lOmM reduced glutathione (GSH) and 50mM Tris-Cl pH8.0) and incubating for 10 minutes at room temperature with end-to-end rotation. The resin was discarded after centrifugation and the supernatant was stored at -20°C as a resin-purified protein fraction.  2.5.2. Protein Quantitation Purified protein preparations were routinely quantitated by the bicinchroninic acid (BCA) protein determination method using BCA reagents A and B from Pierce. Briefly, lOOpl of 1:25 diluted protein samples were prepared with sdH 0 alongside serial dilutions of bovine serum albumin (BSA) protein standards 2  with known concentrations (ranging from 0-1.0mg/ml). The working reagent was prepared by mixing 1 part of reagent B with 50 parts of reagent A. 2ml of the working reagent is added to each lOOpl sample and the resulting mixtures were incubated at 37°C for 30 minutes and then allowed to cool to room temperature. Absorbance at 562nm (A ) was measured and protein concentrations were 562  calculated based on a calibration curve relating A  49  562  to BSA protein content.  Materials and Methods  2.5.3. Antibodies 2.5.3.1 Primary Antibodies 2.5.3.1.1. Rabbit anti-B19 llkDa polyclonal antisera were prepared by Dr. Janet St. Amand (St. Amand, 1992) and raised against a 28-residue synthetic peptide conjugated to keyhole limpet hemacyanin (KLH). The peptide conjugate was used in complete Freund's adjuvant. The sequence of the synthetic peptide corresponds to amino acid residues 59-86 (PNTKDIDNVEFKYLTRYEQ HVIRMLRLC) of the B19 llkDa protein and does not contain any of the three putative SH3 ligand sequences in the llkDa protein. 2.5.3.1.2. Mouse anti-GFP monoclonal antibody (StressGen) of the IgG isotype 2  recognizes the GFP GST fusion protein and will react with GFP, EGFP, as well as GFP/EGFP fusion proteins. 2.5.3.1.3. Rabbit anti-Grb2 polyclonal antibody (StressGen) was raised against a 23residue synthetic peptide with sequence corresponding to amino acid residues 195217 (KGACHGQTGMFPRNYVTPVNRNV) of the human Grb2 protein. This peptide partially overlaps with the carboxy-terminal SH3 domain (residues 163-208) of Grb2 but does not contain the amino-terminal SH3 domain (residues 5-54) primarily responsible for the high affinity binding of Grb2 to Sos. 2.5.3.1.4. Mouse anti-GST monoclonal antibody (StressGen) of the IgG isotype a  recognizes the purified GST protein and will react with GST and GST fusion proteins. 2.5.3.1.5. Mouse anti-HA monoclonal antibody (Boehringer Mannheim) of the IgG  2bK  isotype recognizes a partial, 9-residue sequence (YPYDVPDYA) of the  hemagglutinin epitope derived from the human influenza hemagglutinin protein.  50  Materials and Methods  2.5.3.1.6. Mouse anti-Sosl monoclonal antibody (BD PharMingen) of the IgGj isotype recognizes the amino-terminal 109 amino acid residues of the mSosl protein. This peptide excludes the SH3 ligand that is known to bind Grb2. The antibody will cross-react with the human homologues hSosl and hSos2 because of a high degree of amino acid identity among the proteins.  2.5.3.2 Secondary Antibodies 2.5.3.2.1. Horse radish peroxidase (HRP)-conjugated Goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, catalog number 115-035-003) is used in conjunction with mouse anti-GFP, anti-GST, anti-HA, and anti-Sosl primary antibodies in western and far western blotting experiments. 2.5.3.2.2. Super X-absorbed horse radish peroxidase (HRP)-conjugated Donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories, catalog number 711035-152) is used in conjunction with rabbit anti-B19 llkDa and anti-Grb2 primary antibodies in western blotting experiments.  2.5.4. Western Blotting Proteins resolved  by sodium dodecyl sulfate-polyacrylamide gel  electrophoresis (SDS-PAGE) were transferred to PVDF membranes for detection of specific proteins by western blotting. The transfer was carried out by semi-dry electrophoresis in Bjerrum & Schafer-Nielsen transfer buffer (39mM glycine, 20% methanol, 1.3mM SDS, 48mM Tris-Cl pH9.2) in a Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell apparatus run at 12V for 1 hour. Post-transfer membranes were soaked for ~1 minute in methanol before being equilibrated in PBS. Non-specific proteins were then blocked by incubating the membranes in blocking buffer, which consisted of either PBS 5% skim milk powder (PBS 5% SMP) or PBS 0.05% Tween-20 supplemented with 10% goat or horse serum (referred to as PBS-T/GS or PBST/HS, respectively), for 1 hour at room temperature with shaking. After quickly  51  Materials and Methods  rinsing 3x with PBS or PBS 0.05% Tween-20 (PBS-T), the membranes were incubated with primary antibody dissolved in blocking buffer at an appropriate dilution for 1 hour at room temperature with shaking. (Dilutions vary from 1:250 for anti-Sosl antibody to 1:1000 for anti-GFP antibody to 1:5000 for anti-HA antibody to 1:10,000 for the other antibodies used in this study.) Membranes were then washed 3x 5 minutes each with PBS or PBS-T and then incubated with horse radish peroxidase (HRP)-conjugated secondary antibody dissolved in blocking buffer for 1 hour at room temperature with shaking. Membranes were washed 3x 5 minutes each with PBS or PBS-T, and proteins of interest were visualized by enhanced chemiluminescence (ECL) detection using a kit from Amersham Pharmacia Biotech according to manufacturer's instructions.  2.5.5. Far Western Blotting The far western blotting procedure permits detection of interaction between a purified protein probe in solution and another protein immobilized on a PVDF membrane. The procedure is essentially identical to that of western blotting save for an additional step prior to incubation with the primary antibody solution. After the brief rinses of the membranes following blocking, the membranes were incubated for 1 hour at room temperature with shaking in a 0.5-1.Opg/ml protein probe solution prepared by dissolving the purified protein fraction in blocking buffer. The membranes were washed 3x 5 minutes each with PBS or PBS-T, and incubation with primary antibody and subsequent steps then proceeded as per western blotting. In this case, however, the primary antibody employed would be directed against the protein probe, and far western blotting results would indicate whether the protein probe has bound. The apparent molecular weight of the protein(s) which interacted with the protein probe could then be determined.  2.5.6. GST Protein Pull Down Assay GST protein pull down assays were carried out according to a protocol derived from the procedure described in Byrne et al, 1996. Briefly, H E K 293 cells  52  Materials and Methods  were resuspended at a concentration of roughly 3 x 10 cells per milliliter of ice-cold 6  hypotonic buffer (20mM Tris-Cl pH7.4) supplemented with protease inhibitors and lysed by dounce homogenization (see section 2.3.3). 500ul of lysate (~0.5-lmg protein) was incubated with lOul bed volume of PBS-equilibrated glutathionesepharose 4B resin, in the presence of lOug of resin-purified GST or GST fusion protein. The samples were incubated for 2 hours at 4°C with end-to-end rotation. After centrifugation at 2000rpm for 5 minutes at 4°C to pellet the resin and associated proteins, the supernatant was discarded and the pellet was washed 3x with ice-cold PBS. The resin and bound proteins were finally resuspended in 2x protein sample buffer (2x PSB; 6.7% glycerol, 1.3% SDS, 3.3% P-mercaptoethanol (pME), 0.033% bromophenol blue (BPB), 41.7mM Tris-Cl pH6.8), boiled for 5 minutes, and resolved by SDS-PAGE. The presence of the Grb2 protein in the pull down samples was investigated by western blotting using an anti-Grb2 antibody.  2.5.7. Immunoprecipitation The protocol used in immunoprecipitation studies was modified from the method described by Bonifacino et al. in Current Protocols in Molecular Biology (1999). Briefly, 15ul bed volume of equilibrated protein A-sepharose CL-4B resin was bound to 0.5-1.Oug rabbit anti-Grb2 polyclonal antibody in the presence of O.lmg BSA in lOx bed volumes (150ul) of ice-cold hypotonic buffer (20mM Tris-Cl pH7.4) supplemented with protease inhibitors by incubation for 1 hour at 4°C with end-toend rotation. The antibody-bound resin was then pelleted by centrifugation for 2-3 seconds at top speed at 4°C and washed twice with lOx bed volumes of hypotonic buffer. Parental HEK 293 cells or COS-7 cells transiently transfected with pEGFPC2 or pEGFP-11 were grown to near confluency before being harvested for cell lysate. Cells were resuspended in ice-cold hypotonic buffer supplemented with protease inhibitors at roughly 1ml per 100mm culture dish's worth of cells and lysed by dounce homogenization (see section 2.3.3). 500ul of lysate (~0.5-lmg protein) was then incubated with anti-Grb2 antibody-bound resin, in the presence or absence of 5-10ug resin-purified GST or GST-11, for 2-3 hours at 4°C with end-to-  53  Materials and Methods  end rotation, with or without a prior preclearing step involving a 1-hour incubation of the lysate with protein A-sepharose resin alone. The resin was then washed twice with hypotonic buffer.  Resins and associated proteins from both the  preclearing and actual immunoprecipitation steps were resuspended in 2x PSB, boiled for 5 minutes, and resolved by SDS-PAGE. The presence of specific proteins in the immunoprecipitation samples was investigated by western blotting.  54  Results  RESULTS  3.1. Expression of Glutathione S-Transferase (GST) and GSTTagged Fusion Proteins The proteins GST, GST-HA, GST-HA-Grb2, GST-11, GST-11 P42A, GST-11 P42,45A, GST-11 P42,45,46A, GST-11 P42,46A, GST-11 P43A, GST-11 P43,45A, GST11 P43,45,46A, GST-11 P45A, GST-11 P45,46A, and GST-11 P46A were expressed in DHlOb E. coli and purified on glutathione-agarose or glutathione-sepharose resin using a batch affinity procedure as described in the Materials and Methods section (Section 2.5.1). Selected fractions of purified proteins were resolved by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 3.1). GST appears to migrate at 27kDa, GST-HA at 32kDa, and GST-HA-Grb2 at 50kDa. Wild-type and mutant GST-11 proteins run as at least three distinct bands, at 37kDa (full-length), 32kDa, and 30kDa. The 32kDa and 30kDa species are likely products from proteolytic cleavage of the full-length GST-11 protein at the carboxyterminal.  3.2. GST-HA-Grb2 Interacts with GST-11 but not GST on a Far Western Blot Resin-purified GST, GST-11 and GST-HA-Grb2 fusion proteins described in section 3.1 were employed in far western studies to confirm preliminary observations that GST-11 interacts with GST-HA-Grb2 in vitro (Zagrodney, 1998) (Figure 3.2). Purified fractions of GST and GST-11 were resolved by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane, and probed with GSTHA-Grb2.  The membrane was then subjected to detection with an anti-  hemagglutinin (HA) antibody.  Results suggest that GST-HA-Grb2 binds to  membrane-bound GST-11 but not GST, confirming specificity of the interaction of the Grb2 fusion protein with the llkDa moiety of GST-11. The lower molecular weight species of GST-11 also fail to bind GST-HA-Grb2. This observation is consistent with our earlier hypothesis (see Figure 3.1) that the lower molecular  55  Results  CN  .Q i_  CD < X  I  <  3  < CO  <  < ^  <  CD LO LO LO LO CD C N C N C^-N C"t NICOOOCOLOLOCO 0 . 0 . 0 . 0 . 0 . 0 . 0 . 0 . 0 . 0 .  < X h- — I — I — I — I — I — I — I — I h— 1— — I — I — I W W W M W W W W W O O W W W W  kDa  oooooooooooooo  66.2 45  37kDa GST-11 (full-length) 32kDa GST-11 30kDa GST-11  31 21.5 14.4 6.5  Figure 3.1. Coomassie-stained polyacrylamide gel of purified GST/GST-fusion proteins. Bacterially expressed and resin-purified fractions of GST and various GST-fusion proteins were resolved by 12.5% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. The apparent migration of GST-11 wild-type or mutant proteins is indicated.  56  Results  CO  kDa  CD  h-  co O  CO  CO  CD  45-  <r- GST-11 binds GST-HA-Grb2  31  <- G S T does not bind GST-HA-Grb2  21.5-  WB  FW  anti-GST  anti-HA  Figure 3.2. Western blot and far western blot demonstrating interaction of GST-11 but not GST w i t h G S T - H A - G r b 2 . Resin-purified protein preparations of GST and GST-11 were resolved by 12.5% S D S - P A G E , transferred to P V D F membrane, and subjected to either western blotting w i t h an anti-GST antibody at 1:10,000 dilution (left) or far western blotting (right). For far western blotting, the membrane was first incubated w i t h a protein probe consisting of resin-purified G S T - H A - G r b 2 diluted i n blocking buffer to a final concentration of l . O u g / m l . A n y bound GSTH A - G r b 2 was subsequently detected using an a n t i - H A antibody at 1:5000 dilution as per western blotting.  57  Results  weight species represent degradation products of GST-11 lacking part or all of the llkDa portion.  3.3. GST-HA-Grb2, but not G S T - H A , Interacts with GST-11 on a Far Western Blot Resin-purified GST, GST-11, GST-HA and GST-HA-Grb2 fusion proteins described in section 3.1 were employed in far western studies to confirm specificity of the interaction with Grb2 (Figure 3.3). Purified fractions of GST and GST-11 were immobilized on PVDF membrane and probed with either GST-HA or GST-HAGrb2.  Results demonstrate that GST-HA-Grb2 but not GST-HA binds to  membrane-bound GST-11, suggesting that specificity of the interaction with GST-11 resides in the Grb2 portion of the GST-HA-Grb2 fusion protein.  3.4.  Anti-Hemagglutinin ( H A ) Antibody Recognizes Both  G S T - H A and GST-HA-Grb2 In order to rule out the possibility that the lack of signal observed in the far western using GST-HA was due to the inability of the anti-HA antibody to detect GST-HA as compared with GST-HA-Grb2, both proteins were resolved by SDSPAGE and subjected to western blotting using the anti-HA antibody (Figure 3.4). From figure 3.4, it can been seen that GST-HA and GST-HA-Grb2 are both detected with similar sensitivity by either the anti-HA antibody or the anti-GST antibody. The bands at ~31kDa in the GST-HA-Grb2 lanes likely represent degradation products of the protein. The higher molecular weight species detected by the antiHA antibody in the GST-HA and GST-HA-Grb2 lanes may be dimerization products due to incomplete denaturation of proteins in the samples.  58  Results  kDa  H r—  r— r—  H H  CO C/)  CO CO  CO CO  CD CD ( D O CD CD  66.2 45  31  Grb2 but not G S T - H A  21.5 14.4 6.5 H  < <  <N <  9 1  h i  CD • + =  CQ  ^  ^ >  § S  G_  <S H  CO  CD  Figure 3.3. Western blot and far western blots demonstrating interaction of GSTHA-Grb2 but not GST-HA with GST-11. Resin-purified protein preparations of GST and GST-11 were resolved by 12.5% SDS-PAGE, transferred to PVDF membrane, and subjected to either western blotting with an anti-GST antibody at 1:10,000 dilution (left) or far western blotting (center and right). For far western blotting, the membranes were first incubated with a protein probe consisting of either resin-purified GST-HA (center) or resin-purified GST-HA-Grb2 (right) diluted in blocking buffer to a final concentration of l.Oug/ml. Any bound GST-HA or GST-HA-Grb2 was subsequently detected using an anti-HA antibody at 1:5000 dilution using the western blotting protocol (Sections 2.5.4 and 2.5.5).  59  Results  CN  CN  CD  CD <  _Q  .Q  < < H  kDa  H  r—  r—  h-  CO CO CO CO CO  CD  CD  CD  CD  H  CO  CD CD  GST-HA-Grb2  <- G S T - H A <-GST  WB  WB  anti-GST  anti-HA  Figure 3.4. Western blots demonstrating ability of anti-HA antibody to recognize H A epitope i n both G S T - H A and GST-HA-Grb2. Resin-purified protein preparations of GST, GST-HA and GST-HA-Grb2 were resolved by 12.5% SDSP A G E , transferred to PVDF membrane, and subjected to western blotting with either an anti-GST antibody at 1:10,000 dilution (left) or an anti-HA antibody at 1:5000 dilution (right).  60  Results  3.5. GST-11 but not GST Interacts with Cellular Grb2 i n a GST Protein Pull D o w n Assay Resin-purified GST and GST-11 fusion proteins described in section 3.1 were employed in GST fusion protein pull down assays to investigate the ability of GST11 to co-precipitate endogenous Grb2 from mammalian cells. This experiment assesses the interaction of soluble GST and GST-11 with cellular Grb2. lOug of GST or GST-11 were incubated with an aliquot of the dounce-homogenized lysate from about 1.5 x 10 human embryonal kidney (HEK) 293 cells (~0.5-lmg protein) in the 6  presence of glutathione (GSH)-sepharose resin. GST, GST-11 and any associated proteins were pelleted by centrifugation and analyzed by SDS-PAGE (Figure 3.5). Figure 3.5 shows that cellular Grb2 is effectively co-precipitated by GST-11 but not by GST, further providing support for an interaction between Grb2 and the llkDa protein.  3.6. Exogenously A d d e d GST and GST-11 D o N o t Appear to Interact w i t h Cellular Grb2 i n Co-Immunoprecipitation Studies 10pg of resin-purified GST or GST-11 fusion proteins described in section 3.1 were added exogenously to a dounce-homogenized HEK 293 cell lysate (~0.5-lmg protein) in an immunoprecipitation experiment using an antibody directed against Grb2. Grb2 and any associated proteins were pelleted by centrifugation and analyzed by SDS-PAGE (Figure 3.6). In the absence of preclearing (lanes labeled "IP w/o PrCl"), GST-11 was co-immunoprecipitated with Grb2 (Figure 3.6B). However, this interaction was non-specific as GST was also co-immunoprecipitated in a parallel control experiment using GST in place of GST-11 (Figure 3.6C). Moreover, once the samples have been precleared, neither GST nor GST-11 were observed to co-immunoprecipitate with Grb2 under otherwise identical conditions (Figures 3.6B and C) even though Grb2 was clearly present in precleared samples (Figure 3.6A). Bands in the 50-55kDa range in the IP lanes (Figure 3.6B) likely  61  Results  kDa  — I  — I — I  — I  CO  CO  CO  CO  opullodowns oo tffc<-  26 -  GST  G r b 2  WB anti-Grb2  GST-11  WB anti-GST  Figure 3.5. Western blots demonstrating interaction of exogenously added GST-11 but not GST w i t h cellular Grb2 i n the GST protein p u l l d o w n assays. H E K 293 cell lysates were incubated w i t h resin-purified G S T or GST-11 i n the presence of glutathione-sepharose resin. A n y interacting proteins were co-precipitated upon centrifugation. Proteins from p u l l d o w n samples were resolved b y 12.5% SDSP A G E , transferred to P V D F membrane, and subjected to western blotting w i t h either an anti-Grb2 antibody (left) or an anti-GST antibody (right) at 1:10,000 dilution.  62  Results  o o CL  m -*—» TO  CL  o 8. * w  co  uj  kDa  O  O  o Q.  CL  CL CL  O CL  +GST-11  +GST  66.2 45  mmm  31 21.5 14.4  m*m+-  Grb2  | |  WB anti-Grb2  66.2 45 31 21.5 14.4  <- 37kDa GST-11  •  66.2 45  — —  31  —"  WB anti-11kDa  — k  21.5  -  14.4  —  <- 37kDa GST-11 <- 30kDa GST-11  k G  S  T  WB anti-GST  Figure 3.6. [Please see next page for figure legend.]  63  Results  Figure 3.6. Western blots of anti-Grb2-immunoprecipitated samples in the presence of exogenously added GST or GST-11. HEK 293 cell lysates with added resinpurified GST or GST-11 were immunoprecipitated with an anti-Grb2 antibody in the presence of protein A-sepharose resin, with (post PrCl) or without (w/o PrCl) a prior preclearing step (using protein A-sepharose beads alone). Any interacting proteins were co-immunoprecipitated upon centrifugation. Proteins from immunoprecipitation samples were resolved by 12.5% SDS-PAGE, transferred to PVDF membrane, and subjected to western blotting with either an anti-Grb2 antibody (A), an anti-llkDa antibody (B), or an anti-GST antibody (C) at 1:10,000 dilution. Bands in the PrCl lanes correspond to proteins that bound nonspecifically to protein A-sepharose in the absence of antibody.  64  Results  represent IgG heavy chains of the anti-Grb2 antibody. However, it is unclear what the other bands of a similar molecular weight in the PrCl lanes are (Figure 3.6A).  3.7. Addition of Exogenous GST and GST-11 Proteins Do Not Affect Grb2/Sos Interaction If the llkDa proteins affect the interaction of Grb2 with Sos as proposed in our hypothesis (Section 1.8), then one might expect the amount of Sos bound to Grb2 would be reduced in the presence of the llkDa proteins.  Hence  immunoprecipitation samples from the experiment described in section 3.6 were subjected to western blot analysis for the Grb2-binding protein Sos.  Briefly,  proteins precipitated with an anti-Grb2 antibody from dounce-homogenized HEK 293 cell lysates in the absence or presence of 10pg of resin-purified GST or GST-11 fusion proteins were resolved by SDS-PAGE and probed with an antibody directed against Sos (Figure 3.7). The levels of Sos protein co-precipitated with Grb2 appear unchanged in the presence of exogenously added GST or GST-11.  3.8. Neither the Enhanced Green Fluorescent Protein (EGFP) Nor the E G F P - l l k D a Fusion Protein (EGFP-11) Interact with Cellular Grb2 i n Co-Immunoprecipitation Studies In Vivo African green monkey kidney COS-7 cells transiently expressing either EGFP or EGFP-11 were dounce-homogenized and subjected to immunoprecipitation experiments using the anti-Grb2 antibody. Grb2 and any associated proteins were pelleted by centrifugation and analyzed by SDS-PAGE (Figure 3.8). In the absence of preclearing, EGFP-11 was co-immunoprecipitated with Grb2, but this interaction was non-specific as EGFP was also co-immunoprecipitated in a parallel control experiment. Different efficiencies of transfection were observed for each of the pEGFP-C2 and pEGFP-11-transfected cells, which necessitated the dilution of the EGFP-expressing cell lysate twenty-five fold with non-transfected COS-7 cell lysate in order to obtain roughly equivalent concentrations of EGFP and EGFP-11 in these  65  Results  Figure 3.7. Western blots of anti-Grb2-immunoprecipitated samples in the absence or presence of exogenously added GST or GST-11. H E K 293 cell lysates with or without added resin-purified GST or GST-11 were immunoprecipitated with an anti-Grb2 antibody in the presence of protein A-sepharose resin. Any interacting proteins were co-immunoprecipitated upon centrifugation. Proteins from immunoprecipitation samples were resolved by 12.5% (A) or 7.5% (B) SDS-PAGE, transferred to PVDF membrane, and subjected to western blotting with either an anti-Grb2 antibody at 1:10,000 dilution (A) or an anti-Sosl antibody at 1:250 dilution (B).  66  Results  -I—<  CO  0  co  O v  ^0=0:9= kDa  Q_ Q_  diluted  COS-7 + pEGFP-C2  o  "5;  C O S - 7 + pEGFP-11  — <- Grb2  WB anti-Grb2  <- EGFP-11  WB anti-11kDa  C.  ^EGFP-11 <-EGFP  WB anti-GFP  Figure 3.8. [Please see next page for figure legend.]  67  Results  Figure 3.8. Western blots of anti-Grb2-immunoprecipitated samples in COS-7 cells transfected with pEGFP-C2 or pEGFP-11. Undiluted (EGFP and EGFP-11expressing) or 25-fold diluted (EGFP-expressing) cell lysates were immunoprecipitated with an anti-Grb2 antibody in the presence of protein Asepharose, with (post PrCl) or without (w/o PrCl) a prior preclearing step (using protein A-sepharose beads alone). Any interacting proteins were coimmunoprecipitated upon centrifugation. Proteins from immunoprecipitation samples were resolved by 12.5% SDS-PAGE, transferred to PVDF membrane, and subjected to western blotting with either an anti-Grb2 antibody at 1:10,000 dilution (A), an anti-llkDa antibody at 1:10,000 dilution (B), or an anti-GFP antibody at 1:1000 dilution (C). Bands in the PrCl lanes correspond to proteins that bound nonspecifically to protein A-sepharose in the absence of antibody.  68  Results  studies.  Once the samples have been precleared and transgene expression  normalized against each other by dilution of the pEGFP-C2 lysate, neither EGFP nor EGFP-11 were observed to co-immunoprecipitate with Grb2 under otherwise identical conditions.  3.9.  Levels of Sos Protein Co-Immunoprecipitated by A n t i -  Grb2 Antibody Are Comparable i n COS-7 Cells Transiently Expressing EGFP or EGFP-11 Immunoprecipitation samples from the experiment described in section 3.8 were subjected to western blot analysis for the Grb2-binding protein Sos. Briefly, proteins precipitated with an anti-Grb2 antibody from dounce-homogenized COS-7 cells previously transfected with constructs expressing either EGFP or EGFP-11 were resolved by SDS-PAGE and probed with an antibody directed against Sos (Figure 3.9). The levels of Sos protein co-precipitated with Grb2 are comparable in COS-7 cells transfected with either construct.  3.10. The G S T - H A - G r b 2 / G S T - l l Interaction on a Far Western Blot Requires l l k D a Proline-Rich Region Up to this point, it has been assumed that the interaction between Grb2 and the llkDa proteins observed in far western blots and GST pull down experiments involved interactions of an SH3 domain (in Grb2) with the putative SH3 ligand sequences in the llkDa proteins. To further investigate the nature of these interactions, resin-purified GST, GST-11 wild-type (wt) and mutant proteins described in section 3.1 were employed in far western studies (Figure 3.10). Purified fractions of GST and GST-11 wt/mutant proteins were resolved by SDSPAGE, transferred to PVDF membrane, and probed with GST-HA-Grb2. The membrane was then subjected to detection with an anti-hemagglutinin (HA) antibody. Results indicate that GST-HA-Grb2 binds with reduced affinity to membrane-bound GST-11 proteins mutated in certain proline residues, as  69  Results  WB anti-Grb2  WB anti-Sosl  Figure 3.9. Western blots of anti-Grb2-immunoprecipitated samples from COS-7 cells transfected with pEGFP-C2 or pEGFP-11. Undiluted cell lysates were immunoprecipitated with an anti-Grb2 antibody in the presence of protein A sepharose. A n y interacting proteins were co-immunoprecipitated upon centrifugation. Proteins from immunoprecipitation samples were resolved by 12.5% (A) or 7.5% (B) SDS-PAGE, transferred to PVDF membrane, and subjected to western blotting with either an anti-Grb2 antibody at 1:10,000 dilution (A) or an anti-Sosl antibody at 1:250 dilution (B).  70  Results  < CM  < CN It  CL h— IcnsB  < CD in •*  CM"  CM"  < co  CL  0_  CL  I— I— I— I—  W  C  O  W  < CD  W  I— W  < LO  <*  < CD m"  co" co" in  in"  < CD  CL  Q_  Q_  0_  I—  I— W  Q.  I— I— I— W W W W  <  < CD  W  i—  c j ) 0 0 ( D O O O O O O O C )  W  4531 -  21.5FW anti-HA B. 45 mm mm «•  «*» m»  * **•*  31 -  37kDa GST-11 (full-length) 32kDa GST-11 30kDa GST-11  GST-5  21.5  -  mr «*»•  WB anti-GST  45 37kDa GST-11 (full-length)  31  21.5WB anti-11kDa  Figure 3.10. [Please see next page for figure legend.]  71  Results  Figure 3.10. Far western blot and western blots demonstrating differential strength of interaction of GST-11 wt/mutant proteins with GST-HA-Grb2. Resin-purified protein preparations of GST, GST-11, and GST-11 mutants were resolved by 12.5% SDS-PAGE, transferred to PVDF membrane, and subjected to either far western blotting (A) or western blotting (B and C). For far western blotting, the membrane was first incubated with a protein probe consisting of resin-purified GST-HA-Grb2 diluted in blocking buffer to a final concentration of 0.5ug/ml. Any bound GSTHA-Grb2 was subsequently detected using an anti-HA antibody at 1:5000 dilution. For western blotting, the membranes were detected with either an anti-GST antibody (B) or an anti-llkDa antibody (C) at 1:10,000 dilution.  72  Results  compared with wt GST-11. While the binding of Grb2 to GST-11 is not greatly affected by single mutations at proline residues 43 or 46, multiple mutations in the GST-11 protein appear to significantly reduce the interaction. In particular, a marked decrease in interaction with GST-HA-Grb2 is observed in GST-11 proteins mutated at proline positions 42 and 46; 43, 45 and 46; and 45 and 46. These results strongly suggest this proline-rich region is involved in mediating interactions of the llkDa protein with Grb2 in far western experiments.  3.11.  The G r b 2 / G S T - l l Interaction i n a GST Protein P u l l  D o w n Assay Requires l l k D a Proline-Rich Region The effect of mutating specific proline residues on Grb2/llkDa interactions as observed in GST protein pull down experiments was also examined. Resinpurified GST, GST-11 wild-type (wt) and GST-11 mutant proteins described in section 3.1 were compared for their ability to co-precipitate endogenous Grb2 from mammalian cells. 10ug of GST, GST-11 or GST-11 mutant were incubated with a dounce-homogenized lysate from about 1.5 x 10 human embryonal kidney (HEK) 6  293 cells in the presence of glutathione (GSH) sepharose resin. GST, GST-11 (wt/mutant) and any associated proteins were pelleted by centrifugation and analyzed by SDS-PAGE (Figure 3.11). Results again suggest that Grb2 binds with reduced affinity to GST-11 proteins mutated in certain proline residues, as compared with wt GST-11. Single mutations at proline positions 43 or 46 do not appear to interfere with the ability of GST-11 to co-precipitate Grb2. These results parallel those from the far western blotting experiments (Section 3.10) and provide further support that the proline-rich region is involved in mediating interactions of the llkDa protein with Grb2.  73  Results  < CM T t  Q_  — I  CO  I  — I  CO  I  — I  CO  <  < CO T t  <  CD  if)  If)  T t  T t  CM"  CM"  T t  T t  CM"  CL  CL  CL  I  — I  CO  I  — I  CO  T t  T t  I  — I  CO  < co  T t  CL I  — I  CO  < LO  < CD T t  ID"  T t  T t  co"  co"  T t  CL I  — I  CO  T t  I  — I  C/3  < LO T t  CL I  — I  CO  <  CD LO"  < CD  CL  CL  T t  T t  I  — I  CO  T t  I  — I  CO  WB anti-GST  Figure 3.11. Western blots demonstrating differential strength of interaction of exogenously added GST-11 wt/mutant proteins with cellular Grb2 in a GST protein pull down assay. H E K 293 cell lysates were incubated with resin-purified GST, GST-11, or GST-11 mutants in the presence of glutathione-sepharose resin. Any interacting proteins were co-precipitated upon centrifugation. Proteins from pull down samples were resolved by 12.5% SDS-PAGE, transferred to PVDF membrane, and subjected to western blotting with either an anti-Grb2 antibody (A) or an antiGST antibody (B) at 1:10,000 dilution.  74  Discussion  DISCUSSION In this study, further evidence is provided in support of the hypothesis that the small, unique l l k D a proteins produced by parvovirus B19 may play a role in the viral life cycle by interfering with the host environment through its interactions with cellular signaling molecules such as the growth factor receptor-binding protein 2 (Grb2).  4.1. Characterization of the B19 l l k D a / G r b 2 Interaction 4.1.1. Far Western Studies The initial observation that the l l k D a protein in the form of a G S T - l l k D a fusion protein (GST-11) interacted with GST-HA-Grb2 in a far western experiment provides the basis for the investigation carried out in this study. This interaction has since been confirmed. Membrane-immobilized GST-11 interacts with GST-HAGrb2 in solution, and this interaction is specific to the l l k D a portion of the protein as GST alone fails to bind GST-HA-Grb2 in the far western. Similarly, while an antibody directed against the hemagglutinin (HA) epitope readily detects GST-HAGrb2 which has bound to immobilized GST-11, no signal is observed if GST-HAGrb2 is replaced with GST-HA in the far western protocol. This demonstrates that specificity of the interaction resides in the Grb2 portion of the fusion protein as predicted.  4.1.2. Pull D o w n Assays A GST fusion protein pull down assay was carried out to investigate the ability of GST-11 to interact with endogenous, cellular Grb2 in mammalian cells. A human embryonal kidney (HEK) cell line, 293, was used in these studies. Results from the assay indicate that Grb2 is readily co-precipitated with GST-11 but not GST, once again confirming the specific binding of l l k D a to Grb2.  75  Discussion  4.1.3. Immunoprecipitation Studies Numerous attempts were made to characterize this interaction further in an in vivo cell model, but all immunoprecipitation experiments to date have failed to yield conclusive results. While pilot studies suggested an interaction in a COS-7 cell model between the transfected EGFP-llkDa fusion protein (EGFP-11) and cellular Grb2, later experiments have shown this binding to be nonspecific and removable by an earlier preclearing step. In these studies, efficiency of transfection was consistently low despite various attempts at optimization by varying transfection conditions. To compound the difficulties, transfection of pEGFP-11 (expressing the EGFP-11 protein, -2% fluorescing cells) typically yielded 10- to 20-fold fewer fluorescing cells than pEGFP-C2 (expressing the EGFP protein, -20-50% fluorescing cells), suggesting a significant decrease in transfection efficiency for pEGFP-11. The reduced number of fluorescing cells may also be due to misfolding of a subpopulation of expressed EGFP-11, which may affect the excitation/emission properties of the fusion protein. However, this second possibility is not supported by data from western blotting experiments which indicate a 10- to 20-fold increase in EGFP expression as compared with EGFP-11 expression from a comparable number of cells, consistent with observed differences in the apparent levels of fluorescence between pEGFP-C2 and pEGFP-ll-transfected cells. These findings may indicate a reduced half life of the fusion protein and leave one to question if over-expression of the llkDa fusion protein is toxic in these cells. A different model system was investigated in another attempt to demonstrate the Grb2/llkDa interactions in vivo. Untransfected HEK 293 cell lysates were subjected to immunoprecipitation in the presence of bacterially expressed and affinity purified GST or GST-11 protein. Similar observations of nonspecific binding were noted. While we were able to show the Grb2/llkDa interaction in both far western and pull down experiments, we have not been successful in using co-immunoprecipitation to demonstrate an interaction between endogenous Grb2 and the llkDa protein expressed as either an EGFP or GST fusion  76  Discussion  construct. This suggests that while the binding between the two proteins is strong enough to be detected by far western and pull down approaches, it may not be stable enough to allow co-immunoprecipitation of the two proteins under the conditions tested.  4.2. Src Homology 3 (SH3)-Mediated Interactions and B19 l l k D a Proteins The binding of Grb2 to llkDa is thought to perturb the normal function of the Grb2 protein in binding Sos. Interactions of Grb2 with the guanine nucleotide exchange factor Sos involves the binding of proline-rich motifs (known as Src homology 3 or SH3 ligands) in Sos with the cognate domains (SH3 domains) in Grb2. Sos contains multiple such SH3 recognition sites, consisting of proline-rich PxxP motifs which adopt a polyproline type II (PP-II) helix conformation (Ren et al, 1993; Yu et al, 1994). The B19 llkDa proteins are also rich in similar proline-rich sequences, and in fact, one of these motifs (RPPVPPR) strongly resembles a known SH3 binding site on Sos with the sequence PPPVPPR. It therefore seems likely that the binding of Grb2 to llkDa may be mediated by the same type of SH3 domain/ligand interactions as those responsible for Grb2/Sos interactions during normal host signaling. To test the requirement for specific proline residues within the aforementioned SH3 ligand-like sequence of the llkDa in mediating interactions with Grb2, PCR-based site-directed mutagenesis was carried out to substitute selected proline residues with alanine residues.  The resulting mutant proteins were  expressed as GST-fusion proteins and subjected to far western studies to test their ability to bind the GST-HA-Grb2 fusion protein in vitro.  77  Discussion  4.3.  Effects  of Proline-to-Alanine Mutations on B19  l l k D a / G r b 2 Interactions 4.3.1. Far Western Studies Ten mutants were derived from GST-11 where one or more proline residues were selectively replaced with alanine residues. These were tested alongside GST and GST-11 in far western experiments to determine the requirement of the HkDa/Grb2 interaction for the proline-rich region in question. Singly mutated GST-11 proteins appeared to retain the ability to interact with GST-HA-Grb2, while a reduced interaction was observed for several of the double and triple mutants. The interaction was markedly decreased for GST-11 proteins mutated at proline positions 42 and 46; 43, 45 and 46; and 45 and 46. These results suggest that while no single proline residue is critical for SH3 binding, multiple prolines together contribute to the interaction.  4.3.2. Pull D o w n Assays The GST-11 mutants were also subjected to pull down assays to further investigate the ability of various GST-11 mutant constructs to pull down endogenous Grb2 from mammalian cell lysates. Results obtained here generally mirror those from the far western studies, namely that most of the GST-11 mutant proteins displayed reduced interaction with Grb2, as compared with wild-type GST-11. Single mutations at proline positions 43 or 46 did not appear to interfere with the ability of GST-11 to co-precipitate Grb2, and indeed, no single-proline mutation studied here was capable of completely abrogating the interaction. Nevertheless, these results, which mimic the results of the far western studies (Section 4.3.1), provide strong support for the involvement of the proline-rich region of the B19 llkDa proteins as a conformationally defined unit in mediating interactions with Grb2.  78  Discussion  4.4. Implications of Present Findings on Possible Functions of B19 l l k D a Proteins The process of elucidating the function of the B19 llkDa proteins is ongoing. These proteins do not seem to be involved in structural aspects of virion production as their expression does not appear to affect viral capsid formation (Cohen et ah, 1995). The ability of the llkDa proteins to interact with cellular Grb2, however, suggests a possible mechanism of the B19 parvovirus to manipulate the host cell environment by modulating signaling pathways. The llkDa proteins contain multiple proline-rich regions analogous to SH3 recognition sites on the signaling molecule Sos, which may facilitate binding to Grb2 in a similar manner. As such, it is possible that interaction of llkDa with Grb2 may affect downstream signaling events including those stemming from mitogenic stimuli (Figure 1.8). Examples of virally encoded nonstructural proteins which interact with SH3 domain proteins and/or disrupt mitogenic signaling include the hepatitis C virus NS5A protein (Tan et al., 1999) and the human immunodeficiency virus type 1 Nef protein (Saksela et ah, 1995; Manninen et al., 1998), both of which are implicated in modulation of host signaling cascades involved in virulence and pathogenesis. The B19 llkDa proteins may function in a similar way to enhance viral replication and propagation.  4.5. Future Directions Far western and GST pull down experiments indicate an interaction between the B19 llkDa proteins and Grb2, and studies using proline-to-alanine mutants of the llkDa proteins implicate a role for the proline-rich region in binding the SH3 domain of Grb2.  The inability to observe  this interaction in co-  immunoprecipitation studies suggests that these interactions may not be sufficiently stable to be detected by this approach. If the interaction is relatively weak and not stable to the multiple washing steps, it may be useful to include a cross-linking step prior to washing. Having established the involvement of at least one of the three proline-rich sequences of the llkDa proteins in binding to Grb2 in far western and pull down  79  Discussion  assays, it will be instructive to mutate the remaining two regions individually or in combination to examine the requirement for, and possible synergistic or compensatory effects from, these proline-rich regions in the Grb2/llkDa interaction. Some additional problems to be addressed include the need to obtain higher levels of transfection efficiency and possibly the selection of stable cell lines with inducible expression, as we suspect the llkDa proteins may be toxic in cells. It is also important to scale up the purification of cellular proteins shown to interact with the llkDa proteins in affinity chromatography experiments (Zagrodney, 1998) and to use mass spectrometry analysis to determine the identities of the ~83kDa and ~26kDa putatively interacting proteins.  4.6. Conclusions This study was undertaken in an attempt to further elucidate the functional role of the small llkDa proteins produced by B19 parvovirus. Preliminary findings pointing to an interaction between the llkDa proteins and cellular Grb2 have been confirmed, and the nature of this interaction has been determined to require a proline-rich region within the llkDa proteins. Progress has been made to further our understanding of the function of these proteins, though much remains to be learned from this class of small proteins so unique to the parvovirus B19.  80  References  REFERENCES Agbandje, M.; Kajigaya, S.; McKenna, R.; Young, N.S. and Rossmann, M.G. (1994). "The Structure of Human Parvovirus B19 at 8A Resolution". Virology. 203(1): 106115. Ahn, K.; Angulo, A.; Ghazal, P.; Peterson, P.A.; Yang, Y. and Fruh, K. (1996). "Human Cytomegalovirus Inhibits Antigen Presentation by a Sequential Multistep Process". 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