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Investigation into the function of two small nonstructural proteins of the parvovirus V19 Zagrodney, Darren Bryce 1998

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AN INVESTIGATION INTO THE FUNCTION OF TWO SMALL NONSTRUCTURAL PROTEINS OF THE PARVOVIRUS B19. by Darren Bryce Zagrodney B.Sc, (Distinction), University of Victoria, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT 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 May 1998 © Darren Zagrodney, 1998 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. Department of [5r/>£tffe)MrVYfe.y A M McU?C\M^AiL UtOl^Ody The University of British Columbia Vancouver, Canada Date MAY M*? C M ? DE-6 (2/88) Abstract The Parvovirus B19, known to cause disease in humans, has been shown to produce two small, unique, nonstructural proteins of 7.5 and 11 kilodaltons (kDa). No functional significance, nor homology with other proteins, has yet been attributed to either the 7.5 kDa or the 11 kDa B19 proteins. However, due to the small coding capacity of B19 it is thought that these two proteins most likely play a functional role in the replication cycle of this virus. Due to its narrow host range no continuous cell line permissive for B19 infection is available for routine investigation. Therefore, studies must be performed in the absence of a permissive cell line. Previous studies have failed to demonstrate Z n + + binding or transactivation activities for either protein. Neither have been shown to be phosphorylated, nor to interact with other known viral proteins. However, the 7.5 kDa protein has been implicated in interactions with proteins within a lysate of COS-7 cells. Therefore, further investigations have focused on interactions of both the 7.5 kDa and 11 kDa B19 proteins with host cellular proteins. Far western blot experiments indicate that two COS-7 cellular proteins of 43 kDa and 55 kDa, in addition to lower molecular weight proteins, may interact with the 7.5 kDa B19 protein. Western blot experiments suggests that the 55 kDa cellular protein is most likely vimentin. Results of affinity column experiments utilizing glutathione S-transferase fusion proteins ii suggest that the 11 kDa protein may interact with two K-562 cellular proteins of approximately 27 kDa and 85 kDa. These interactions were substantiated with further batch affinity experiments. In addition, preliminary far western studies demonstrate an in vitro interaction between Growth Factor Receptor Binding protein-2 (Grb2) and the 11 kDa B19 protein. This suggests that the 11 kDa protein may enable B19 to influence its host cell environment. iii Table of Contents Abstract ii Table of Contents iv List of Tables x List of Figures xi List of Abbreviations xiii Acknowlegments xv I Introduction 1.1 The Parvoviridae Family - a group photo 1 1.2 Taxonomy 1 1.3 The Erythrovirus, B19 2 1.4 Epidemiology and clinical manifestations of B19 3 1.5 Tropism of B19 8 1.6 Life cycle of B19 8 1.7 The genome of B19 10 1.8 B19 transcription 10 1.9 The structural and nonstructural proteins encoded by B19 12 1.10 The 7.5 kDa and the 11 kDa proteins of B19 13 1.11 Previous studies on the functional role of the 7.5 kDa and 11 kDa proteins 14 1.12 Review of the functions of other nonstructural viral proteins 15 1.12.1 Alteration of protein trafficking within the host cell 16 1.12.2 Nuclear Export of mRNA Transcripts 16 1.12.3 Enhancement of viral genome replication 18 1.12.4 Processing of viral gene products 19 1.12.5 Packaging of virions 19 1.12.6 Virion emergence from the host cell 19 1.12.7 Immune response evasion 20 iv 1.13 The present study 21 II Materials and Methods Materials 2.1.1 Chemicals and supplies 22 Bacterial culture 2.2.1 Cell lines 22 2.2.2 Growth 22 2.2.3 Stocks 23 2.2.4 Electroporation 23 Yeast 2.3.1 Yeast two hybrid system 23 Tissue cell culture 2.4.1 Cell lines 24 i) Adherent 24 ii) Nonadherent 24 2.4.2 Maintenance 24 2.4.3 Storage of frozen cells 25 2.4.4 Transfections 25 2.4.5 Lysis of tissue culture cells 25 2.4.6 35S-labeled tissue culture cell lysates 25 Cloning techniques 2.5.1 Plasmid Preparation 26 2.5.2 Plasmid digestions and ligations 26 v 2.5.3 Plasmids i) pGEX2T 26 ii) pGEXll 26 iii) pGEX2T-tag 26 iv) pGEX-tag-Grb 27 v) pGEX-tag-PD 28 vi) pGEXPD 28 vii) pCMV5 29 viii) pCMV805 29 ix) pQE41 29 x) pQE7.5 29 xi) pRSETA 29 xii) pRSETll 29 xiii) pRSETllcelO 29 2.5.4 Oligonucleotides 30 2.5.5 Isolation of plasmid DNA from electrophoresis gels 30 2.5.6 DNA quantification 30 2.5.7 Polymerase chain reaction (PCR) 31 2.5.8 DNA sequencing 31 Protein preparation and characterization 2.6.1 General procedures 32 2.6.2 Protein quantification 32 2.6.3 Purification of fusion proteins 32 2.6.4 Silver staining 34 2.6.5 Western blotting 34 2.6.6 Far western blotting 34 2.6.7 Antibodies used 36 2.6.8 Affinity column procedures 36 2.6.9 Cyanogen bromide activation of sepharose 2B beads 37 v i 2.6.10 Protein solution concentration 38 2.6.11 K-562 35S-labeled lysate 2.6.12 GST vs GST-11 sepharose affinity column procedure 38 38 m Results 3.1 Characterization of fusion proteins: Dihydrofolate reductase-histidine6 (DH6) and the B19 7.5kDa-dihydrofolate reductase-histidine6 (7.5DH6) 40 3.2 Far western blots suggest that proteins within a COS-7 cell lysate interact with the 7.5 kDa B19 protein 40 3.3 Precleared far western blot of a COS-7 cell lysate with the B19 7.5 kDa protein 42 3.4 Western blotting of a COS-7 cell lysate suggests that the 55 kDa cellular protein shown to potentially interact with 7.5DH6 is vimentin 43 3.5 Far western blots fail to identify proteins that interact with the 11 kDa B19 protein 45 3.6 Purification of an 11 kDa fusion protein with a CE10 monoclonal antibody epitope tag, Hcel0H 6 47 3.7 Co-immunoprecipitation studies of the 7.5 kDa B19 protein 49 3.8 Transfection experiments 50 3.9 Immunoprecipitation of the 7.5 kDa protein expressed in COS-7 cells 50 3.10 Affinity column experiment: A COS-7 cell lysate probed for proteins that interact with the 7.5 kDa B19 protein 54 3.11 Affinity column experiment: A K-562 cell lysate probed for proteins that interact with the 7.5 kDa B19 protein 54 3.12 Affinity column experiment: A K-562 cell lysate probed for proteins that interact with the 11 kDa B19 protein 54 3.13 Affinity column experiment: An 35S-labeled COS-7 cell lysate probed for proteins that interact with the 11 kDa B19 protein 57 3.14 Batch style affinity experiments with the 11 kDa B19 protein 57 3.15 Scaled-up batch affinity experiment with the 11 kDa B19 protein 62 vi i Attempts to improve the purity of the GST-11 fusion protein preparation 3.16.1 Incubation of purified GST-11 with ATP followed by repurification on glutathione resin does not improve the purity of the GST-11 fusion protein 63 3.16.2 ATP incubation of the centrifuged bacterial cell sonicate prior to binding of glutathione resin decreased the amount of 70 kDa contamination in the GST-11 preparation 67 3.17 Western blot of GST-11 67 3.18 Batch affinity experiment: An 35S-labeled K-562 cell lysate incubated with sepharose beads covalently linked to GST or GST-11 67 3.19 Growth factor receptor-bound protein 2 (Grb2) interacts with the 11 kDa protein in far western experiments 71 3.20 The yeast two hybrid system produced no positive clones with either the 7.5 kDa or 11 kDa proteins 74 IV Discussion 4.1 Highlights of this study 75 4.2 Confirmation and extension of previous binding studies of the 7.5 kDa protein 75 4.3 Far western experiments fail to identify proteins that interact with the 11 kDa B19 protein 76 4.4 K-562 versus COS-7 cells: Approximation of B19 permissive host cells 77 4.5 Interaction of the 11 kDa B19 protein with COS-7 and K-562 cellular proteins 77 4.6 Contamination of the GST-11 protein preparation 78 4.7 Preparation of contaminant free GST-11 79 4.8 The 70 kDa contaminating protein of GST-11 preparations may be an aggregate of GST-11 79 4.9 GST-11 and GST sepharose batch affinity experiments remain inconclusive 80 4.10 The Yeast Two Hybrid System 81 4.11 GST-11 appears to interact with Grb2 in the context of far western experiments 82 viii 4.12 Future directions 82 Literature cited 84 ix List of Tables Table 1: Parvovirinae and their host species. 2 Table 2: Other pathologies associated with B19 infection. 7 Table 3: Plasmids used in this study and the proteins they encode. 28 Table 4: Oligonucleotides used in this study. 31 x List of Figures 1: Time course of experimental Erythrovirus B19 infection in a seronegative volunteer. 5 2: Transcription and coding map of B19 Parvovirus. 11 3: Map of pGEX2T-tag and its multiple cloning site sequence. 28 4(a): Far western blotting: Protein interactions and recognition by a primary antibody. 35 4(b): Far western blot illustrating a band identified by "subtractive blotting". 37 5: Characterization of D H 6 and 7.5DH6 fusion proteins. 41 6: Far western blots of a COS-7 cell lysate with the 7.5 kDa protein. 42 7: Far western blot of a precleared COS-7 cell lysate with the 7.5 kDa protein. 44 8: Western blot of a COS-7 cell lysate probed for vimentin and actin. 45 9: Far western blot of a K-562 lysate with the 11 kDa protein. 47 10: Characterization of llcel0H 6 . 48 11: A COS-7 lysate far western blot probed with llcelOH 6 or H 6 . 49 12: Co-immunoprecipitation of a COS-7 cell lysate with the 7.5 kDa protein. 51 13: Expression of the 7.5 kDa B19 protein in COS-7 cells. 52 14: Co-immunoprecipitation of cellular proteins with the 7.5 kDa protein, expressed in COS-7 cells. 53 15: Affinity column experiment: Chromatography of an 35S-labeled COS-7 cell lysate on 7.5DH6 or D H 6 bound Ni + + -NTA columns. 55 16: Affinity column experiment: Chromatography of an 35S-labeled K-562 cell lysate on 7.5DH6 or D H 6 bound Ni + + -NTA columns. 56 17: Affinity columns: Chromatography of an 35S-labeled K-562 cell lysate on GST-11 or GST glutathione-agarose columns. 58 18: Affinity columns: Chromatography of an 35S-labeled COS-7 cell lysate on GST-11 or GST glutathione-agarose columns. 60 19: Chromatography of a K-562 cell lysate incubated with GST or GST-11 bound glutathione-agarose resin using a batch affinity procedure. Silver stained gel. 60 xi 20: Chromatography of a K-562 cell lysate incubated with GST or GST-11 bound glutathione-agarose resin using a batch affinity procedure: Detection of 35S-labeled proteins using a phosphorimager. 62 21: Large scale (five fold) chromatography of a K-562 cell lysate on glutathione-agarose resin with bound GST or GST-11 fusion protein. Silver stained gel. 64 22: Large scale (five fold) chromatography of a K-562 cell lysate on glutathione-agarose resin with bound GST or GST-11 fusion protein: Detection of 35S-labeled proteins using a phosphorimager. 65 23: Purification of GST-11: Rebinding of GST-11 to glutathione-agarose resin after ATP incubation and buffer exchange. 66 24: Affinity purification of GST-11 on glutathione-agarose resin after incubation with ATP. 68 25: Western blot of GST-11. 69 26: Silver stained SDS-PAGE gel of the sepharose bound GST and GST-11 affinity experiment. 70 27: Phosphorimage of the SDS-PAGE gel of the sepharose bound GST and GST-11 affinity experiment. 71 28: Western blot of GST-11 with anti-hemagglutinin. 72 29: GST-11 far western blot probed with Grb2. 73 xii List of Abbreviations aa amino acid(s) amp ampicillin ATP Adenosine triphosphate BCIP 5-bromo-4-chloro-3-indoylphosphate bp base pair(s) BSA bovine serum albumin cDNA complementary D N A CFU-E colony forming unit-erythroid Cys cysteine dATP deoxyadenosine 5'-triphosphate D M E M Dulbecco's minimal essential medium DMSO dimethyl sulphoxide D N A deoxyribonucleic acid E. coli Escherichia coli EDTA ethylenediaminetetracetic acid EtOH ethanol FBS fetal bovine serum Grb2 growth factor receptor binding protein 2 GST glutathione S-transferase H A hemagglutinin HBS HEPES buffered saline HBsAg hepatitis B surface antigen HEPES N-2-hydroxyethyl piperazine N'-2-ethanesulfonic acid his histidine IPTG isopropyl-B-D-thiogalactopyranoside ITR inverted terminal repeats kan kanamycin kDa kilodaltons xiii m.u. map unit Met methionine MgS0 4 magnesium sulfate m R N A messenger ribonucleic acid mw molecular weight NBT nitroblue tetrazolium chloride NS nonstructural nt nucleotide ORF open reading frame PAGE polyacrylamide gel electrophoresis PBMC peripheral blood mononucleocyte PBS phosphate buffered saline PEG polyethylene glycol PKR ds RNA-activated protein kinase PMSF phenylmethylsulfonyl fluoride SDS sodium dodecyl sulfate SH3 src homology 3 SMP skim milk powder (nonfat) SV40 simian virus 40 T A C transient aplastic crisis Tris Tris(hydroxymethyl)aminomethane VP viral protein (structural) xiv Acknowledgments I would like to acknowledge the patient persistence of Dr. Caroline Astell. Thank you for the opportunity you provided for me to partake fully of this experience: one both scientific and personal. Your encouraging guidance throughout is greatly appreciated. I want to be sure that Colin Harris, Jan St. Amand and Richard Boden understand the impact they had in shaping not only my scientific endeavours and education, but also the vastly rewarding time in between: that time we call life. Through untethered conversation, discussion and debate, you helped introduce me to a variety of ideas and perceptions I may never have glimpsed without your insights. I would also like to thank John Brunstein for sharing his exhaustive knowledge. You always seemed able to answer those all to common what, where, when and how much type questions. Finally, I want to show my great appreciation for the loving support of my family and my friends. Special mention is due to my father and Shelley, as well as my mother and Dave, for support I could never adequately describe in this passage. Andrea, who bears the brunt of the burden that I present, may not realize how graciously she seems to do so, nor how greatly it is appreciated. It is our relationships that make us who we are and allow us to live the lives we do. With your support I feel free to reach toward whatever I desire. Thank you. xv Introduction B19 is a member of the family of viruses called Parvoviridae. Two small proteins of B19, one of 7.5 kDa and the other of 11 kDa, are the focus of this study. 1.1 The Parvoviridae Family - a group photo The family name, Parvoviridae, designates a distinct group of small, spherical, nonenveloped viruses with single stranded DNA genomes. Their capsids have a diameter of 18-25nm, possess T=l icosahedral symmetry and are composed of 2 or 3 proteins with overlapping sequences. Their genomes range between 4000 and 6000 base pairs and have unique palindromic hairpin termini. Parvoviridae represent many different vertebrate and insect viruses and have become a significant medical and economic concern, as their wide host range includes humans and much of our livestock. 1.2 Taxonomy The family Parvoviridae is divided into two subfamilies based on viral host range. The Parvovirinae infect vertebrates and the Densovirinae require insect hosts. These subfamilies are further divided into genera based on genome organization,19 specific host range, and whether the infection is dependent or independent of a co-infecting helper virus. The subfamily Parvovirinae include three genera. The Parvovirus, which independently infect their host, the Dependovirus, which requires its host cell to be co-infected by a helper virus, and the Erythrovirus, which include autonomous viruses that infect a narrow range of erythroid progenitor cells. The B19 virus is a member of the genus Erythrovirus (Table 1). Introduction Table 1: Parvovirinae and their host species. Parvoviridae Parvovirinae Parvovirus Aleutian Mink Disease Virus Bovine Parvovirus Canine Parvovirus Feline Panleukopenia Virus Goose Parvovirus HI Virus Kilham Rat Virus LuIII Minute Virus of Canines Minute Virus of Mice Mouse Parvovirus Mink Enteritis Virus Porcine Parvovirus Dependovirus Adeno-Associated Virus Avian Adeno-Associated Virus Bovine Adeno-Associated Virus Erythrovirus B19 Pig-tailed Macaque Parvovirus Simian Parvovirus Hosts Known mink cattle dog cat goose rodent rat unknown dog mouse mouse mink Pig most animals (including humans) bird cattle human monkey monkey 1.3 The Erythrovirus. B19 During routine screening of asymptomatic blood donors for evidence of hepatitis B, a particle smaller than a Dane particle was found in sample number 19 of panel B,21 hence the name B19. The B19 virus was later characterized as a Parvovirus53 and remains the only Parvovirus known to infect humans. This virus has since been renamed Human Parvovirus 2 (HPV-2), however the name B19 appears to be used more frequently. 2 Introduction 1.4 Epidemiology and clinical manifestations of B19 Although B19 infection is very prevalent, its manifestations vary considerably with the influence of different host immunological and hematological states. Most infections are either asymptomatic or mistakenly attributed to influenza infection due to symptom similarities. In fact, a study of the elderly revealed that although 80% of that population is seropositive for IgG against B19, signifying a previous exposure,24 few were aware of having had a B19 infection. To understand the course of B19 infection volunteers were inoculated intranasally with 1x10s viral particles.49 The first sign of viremia was detected at 6 days and peaked after 8-9 days (Figure 1). The serum titre can reach 1011 particles/ml. Patients experienced flu-like symptoms of headache, myalgia and chills at 6 - 8 days and by day 10, the bone marrow was almost devoid of erythroid precursors. At the apex of viremia the reticulocyte count had dropped significantly, indicating a cessation of red cell production. The viremia cleared by day 15-17. The second phase of infection was marked by pruritus of the limbs and trunk, evolving into fine maculopapular cutaneous eruptions lasting 2-4 days. Some volunteers, mostly female, experienced arthralgia or mild arthritis, lasting 4-6 days.49 From the above description of a B19 infection in normal hosts, it can be seen that the pathology caused by B19 may be classified into three groups based on their probable mechanisms. First, the flu-like symptoms are thought to arise from inflammatory cytokines, although a-interferon is not detected. Second, the cessation of red cell production is assumed due to direct killing of erythroid progenitor cells, possibly caused by cytotoxic effects of the large nonstructural protein NS-1, expressed during B19 infection. Third, cells infected with B19 appear to undergo apoptosis,81 a cytotoxic effect that correlates directly to the expression of NS-1 in tissue culture.71 Transfection of Hela cells with the left portion of the B19 genome encoding NS-1, is toxic to 3 Introduction the cells. However, this toxicity is not observed when cells are transfected with a similiar genome, mutated to produce a single amino acid change within the nucleotide binding fold of NS-1. 7 2 Finally, the appearance of the second stage of pathogenesis, denoted by a skin rash and sometimes arthropathy, actually occurs once the viremia is cleared, but coincides with the peak of neutralizing IgM and the appearance of IgG (Figure 1). Therefore it is thought that this pathology is an indirect result of infection mediated by immune complexes.83 There is still much work to be done to elucidate the mechanisms of B19 pathology, however it is evident that much of the disease caused by B19 infection is mediated by the hosts immune system. 4 Introduction If 3 ; A • i r- I I I I I I I I 100-1 0) .2 19 antibo< (units) 50-m 10-0 J CD 1.0; eticulo la/ (To 0.2 : 15n 111 11 I 1 1 1 1 1 1 1 igM 10 i I I 1 1 1 1 1 1 1 1 1 1 1 ^ _, e?& 13H E — © i2 cT 1 1 5-1 I I I I I I I I i I 11 o-> 1 1 1 1 1 1 1 u 1111 a l 340H • i £ jo o 100 J I I I I I I i I I I I M w E o a E >> w 1 (ever, chills rash, headache arthralgia myalgia A2 6 10- 20 i* days inoculation Figure 1: Time course of experimental Erythrovirus B19 infection in a seronegative volunteer.49 5 Introduction In children, symptomatic B19 infections will most often manifest as Erythema Infectiosum (EI), or Fifth Disease, the fifth of six classical childhood exanthema, EI begins as a self-limiting rash on the cheeks that may extend to the trunk and extremities and is often accompanied with flu-like symptoms of nausea, headache, fever and diarrhea. These symptoms may be transient or recur for 1 to 3 weeks.23 Adults rarely suffer from EI but are more likely to acquire arthropathies than are children. Women, more than men, are affected by a symmetrical arthropathy causing pain, swelling and stiffness, usually in the small joints of the hands and feet.23 Symptoms have lasted as long as two years, however, usually the arthropathy is resolved within 1-3 weeks.28 Patients with underlying hemolytic conditions can experience more severe effects of B19 infection. The first major illness associated with B19 was Transient Aplastic Crisis (TAC), 5 0 in which severe anemia with absent reticulocytes abruptly appears in patients with sickle-cell disease, thalassemia and hereditary spherocytosis.25'26 Anemia is rarely found in patients with normal erythroid cell turnover but with increased cell turnover, even a short interruption in erythropoiesis may result in life threatening anemia. The TAC is concurrent with viremia and is readily treated by blood transfusion to replenish the red cells. However, if the patient is unable to clear the viremia, the aplastic crisis will become chronic. Immunocompromised patients infected with B19 who are unable to effectively clear the virus, usually suffer chronic red cell aplasia. Blood transfusion is the usual treatment for these patients, although B19 infection usually recurs.27 Immune globulin therapy has also proven effective.98 B19 infection during pregnancy may lead to hydrops fetalis or miscarriage if the virus crosses the placenta. Infection during the first trimester results in an equal probability of losing the fetus as without 6 Introduction infection. However, infection during the second trimester increases that probability to 20 times that of noninfected pregnant women.30 This is thought due to the increased need of red blood cell production in the fetus during the second trimester and the immaturity of the fetal immune system.84 There also exist an exhaustive number of less prevalent disorders that have been linked to B19 infection (Table 2). These range from cardiovascular manifestations to respiratory and even renal disorders. Cardiovascular manifestations • Acute congestive heart failure • Myocarditis • Pericarditis Cutaneous manifestations • Peripheral edema • Vascular purpura • Vesicular lesions Hematologic disorders • Aplastic anemia • Autoimmune hemolytic anemia • Chronic neutropenia • Idiopathic thrombocytopenic purpura Hepatobiliary tract disorders • Acute hepatic sequestration • Fulminant liver failure Neurologic disease • Coma • Seizures • Sensorineural abnormalities Renal disease • Acute renal failure • Nephrotic syndrome Rheumatic disease • Juvenile rheumatoid arthritis • Rheumatoid arthritis • Vasculitis Table 2: Other Pathologies associated with B19 infection. 7 Introduction The above summary demonstrates that although B19 infection is very prevalent, its pathology is dependent on the state of the host and is often asymptomatic. In fact, the most common clinical manifestations of B19 are either self-limiting as with EI, or fairly easily treated, as is usually the case with TAC. However, B19 is responsible for more debilitating and devastating illnesses that affect significant populations of our society, making B19 an important pathogen. 1.5 Tropism of B19 B19 appears to replicate only in human progenitor cells of the erythroid lineage.31 More specifically, pluripotent progenitor cells must differentiate into mature erythroid progenitor cells (CFU-E) and erythroblasts, to become targets of B19 infection.32 Unfortunately, this narrow host range makes culturing of B19 difficult and expensive, and to date there is no continuous cell line found capable of supporting B19 propagation. Researchers must utilize cell sources enriched with erythroid progenitor cells, such as human bone marrow,31 fetal liver,33 and umbilical cord blood.34 Alternatively, megakaryocytoblastoid cell lines, UT-735 and MB-02,36 as well as the erythroleukemic cell line, JK-1,37 have been shown to support low level persistent infection and replication. However, most likely due to the requirement of B19 for mitotically active host cells, these cell lines require an initial adaptation in erythropoietin to support more than nominal levels of viral replication.37 Unfortunately, these cell lines still produce low levels of B19 replication compared to cultured bone marrow and the high cost of the erythropoietin adaptation makes their routine, long-term use impractical. 1.6 Life cycle of B19 The basic replication cycle of any virus requires that it enter the host species, bind a susceptible cell and cross at least one membrane barrier. The virus has to transcribe its proteins as well as replicate and package its genome 8 Introduction before it manages an escape from the cell. To make matters more difficult, this must all be accomplished while evading the hosts immune system. Only then may the virus be poised for transmission to another unwitting host organism. In the case of B19, evidence suggests that the natural mode of transmission is through respiratory secretions in the form of fomites, more than aerosol. DNA is consistently found in the respiratory secretions of viremic patients38,39 and the nonenveloped capsids of parvoviruses are very stable. In fact, the transmission of Porcine Parvovirus (PPV) from an area contaminated four months prior to infection has been documented.40 B19 has also been shown to be transferred with blood, blood products,41 and procedures that puncture the skin, like tattooing.42 Once in proximity to a susceptible host cell, B19 has been shown to adsorb to the blood group P antigen, a glycolipid known as globoside.43 The requirement of P antigen for B19 infectability was most dramatically demonstrated by the complete resistance to B19 of seronegative Amish individuals who genetically lacked P antigen.44 Also, in hematopoietic progenitor assays excess globoside, or monoclonal antibody to globoside, conferred protection to B19 infection.43 It is thought that once bound to P antigen B19 is endocytosed via clathrin coated pits in a cell cycle independent manner. However, it is unknown how the virus enters the nucleus, or where it is uncoated.45 Replication of the genome requires cellular factors present during S phase,46 so that B19, like all autonomous parvoviruses, require mitotically active host cells. Replication and packaging of the genome into the capsid occurs in the nucleus,47 from which the progeny virions escape after membrane rupture48 via an undefined mechanism. 9 Introduction 1.7 The genome of B19 B19 has the characteristic single stranded, DNA genome of parvoviruses. These genomes, capable of forming terminal hairpin structures, have inverted terminal repeat sequences, containing internal palindromic sequences. By varying extraction conditions, it was demonstrated that B19 packages its genome as either a minus or a positive sense DNA strand.53 This genome has been cloned51 and sequenced, and found to be about 5000 nucleotides (nt) in length,69 with identical ITRs, considerably larger than those of other parvoviruses. The B19 ITRs are 383nt long, of which 365nt have the potential to form a hairpin of 176bp.54 In comparison with B19 ITRs, the left end of the Minute Virus of Mice (MVM) genome has a 50bp hairpin structure and the right end, a hairpin structure of approximately lOObp.55 1.8 B19 transcription As with other parvoviruses, B19 has two large open reading frames (ORFs). One ORF spans the left half of the genome and encodes the major nonstructural proteins and another ORF is located on the right side of the genome and encodes two major structural proteins.56 Unlike other parvoviruses, B19 was shown to have only one promotor. Its single promotor distinguishes B19 from MVM which has two promotors and AAV-2 which has three. Initiation of all B19 transcripts has been localized to map unit 656 and further refined to nucleotides (nt) 350 -351.57 Therefore, to produce at least nine different transcripts, B19 utilizes extensive alternate splicing and end processing.58 All transcripts begin with a 56 nt leader sequence and are 3' end processed either at the middle of the genome, or at the far right.58 By sequencing cDNA libraries created from B19 infected CML cells, or COS-7 cells transfected with SV40/B19 hybrid plasmids, the splice sites for all nine 10 Introduction transcripts were identified and a transcription map (Figure 2) was established.59'60 open 1 reading 2 frames 3 map units 0 H nucleotides 0 20 40 1 0 0 0 T 2000 60 80 3000 4000 1 0 0 5000 350 2659 nucleotide protein 2309 NS 350 406 350 406 350 406. 350 406 350 406 350 406 1910 2659 2030 2659 1910 2030 1910 2183 3045/3051 2030 2183 3045/3051 1910 2183 2030 2183 5010 5010 5010 5010 4704 5010 807 7.5 KOa 687 ? 3156 VP-1 7.5 KDa 2980 VP-1 2282/2288 VP-2 7.5 KDa 2162/2168 VP-2 638 11 KDa 7.5 KDa 518 11 KDa 4704 5010 Figure 2: Transcription and coding map of B19 parvovirus.60 ORFs encoded by three different reading frames from the plus strand are shown above the genome map. The same labeled boxes representing corresponding ORFs are shown below, on each spliced transcript. 11 Introduction In addition to the transcripts containing the large open reading frames common to other parvoviruses, B19 produces two relatively abundant classes of small mRNA transcripts unique to B19 (Figure 2). These classes of mRNAs were found in both erythroid progenitor cells infected with B1958 and COS-7 cells transfected with an SV40/B19 hybrid vector.61 The mRNAs of the 700 -800 nt transcript family terminate in the middle of the genome and those of the 500 - 600 nt family terminate at the far right of the genome.60 Clones were made of the 687nt and 807nt transcripts from the 700 - 800 nt transcript family, as well as of the 638nt and 518nt transcripts from the 500 -600 nt transcript family. All were found to be polyadenylated and to have small ORFs.59 Since these small ORFs had in frame ATG start codons, the transcripts were used in in vitro and in vivo (transfected cells) experiments to determine if they are translated. The putative proteins were then detected, with antibodies raised to peptides, in western blot, or immunoprecipitation experiments. These studies revealed that the 807nt mRNA is translated into a 7.5 kDa protein, the 518nt mRNA is translated from each of three start codons into a family of three 11 kDa proteins, and the 638nt mRNA is translated into both 7.5 kDa and 11 kDa proteins. The 687nt mRNA does not appear to be translated.59'60'62 1.9 The structural and nonstructural proteins encoded by B19 The B19 capsid contains a total of 60 copies of two capsid proteins, VP1 and VP2. These proteins are both translated from alternate start codons of the same ORF, resulting in an additional 226 N-terminal amino acid (aa) residues in VP1, not found in VP2. These residues are highly antigenic65, indicating a probable localization on the exterior of the virion.64 VP1 is an 84 kDa protein of 781 amino acids and comprises only 5% of the capsid. The remaining 95% of the capsid is made up of VP2, a 58 kDa protein of 554 amino acids, that is capable of spontaneously assembling into capsid structures, even in the absence of VP1.66 12 Introduction The ORF of NS-1, the major nonstructural (NS) protein of B19, encodes a protein of 671 amino acids, with a calculated mass of 74097 daltons. Observations suggest that B19 produces three NS proteins of about 71 kDa, 55 kDa and 34 kDa. However, the transcription map (Figure 2) of B19 indicates no splice sites for smaller mRNA transcripts, as found in M V M and A A V , 6 7 that may direct expression of smaller NS proteins. Therefore, these additional lower molecular weight proteins may result from post-translational processing or possibly degradation of the full length NS-1 protein. B19 NS-1 has a 135 amino acid region which displays 41% homology to a similiar region of M V M , 51% homology with AAV-2 Rep 6 9 and 67% homology to the NS protein of the Erythrovirus, SPV 6 8 . A comparison of these replicative proteins indicate that this 135 amino acid region is a nucleotide binding fold. 7 2 1 1 6 Studies to investigate the function of B19 NS-1 have been impeded by its cytotoxicity in mammalian cells, common to the NS-1 proteins of all parvoviruses.71 It has however been demonstrated, that B19 NS-1 transactivates the P6 promotor of the B19 genome. This would be expected by a comparison with the transactivation activity of NS-1 from M V M and the Rep protein from A A V - 2 . 7 3 Also, the 135 amino acid domain, or nucleotide binding region mentioned above, appears to play a role in the function of B19 NS-1, as deletion of this region abrogates its cytotoxic effect in mammalian cell culture.72 1.10 The 7.5 kDa and the 11 kDa proteins of B19 As mentioned above, B19 also produces two families of small abundant transcripts that are translated into a 7.5 kDa protein of 72 amino acids and a family of 11 kDa proteins of approximately 94 amino acids. Both proteins have been shown to be expressed in B19 infected human cell lines, 6 2 1 1 5 as well as in COS-7 cells61 transfected with an SV40/B19 hybrid vector. Unfortunately 13 Introduction to date, neither protein has significant homology to any sequences in the Genbank or Swissprot databases. Although unknown, the function of the 11 kDa protein may be conserved within the genera Erythrovirus. A putative ORF of comparable size and location to the B19 11 kDa ORF was found in SPV and is predicted to express a 104 amino acid protein with three regions of homology to the 11 kDa protein.78 The primary sequences of the 7.5 kDa and 11 kDa proteins yield few clues as to their function. Neither have obvious hydrophobic or basic regions, nor significant homology with known protein motifs, with one exception. Both proteins have a high content of proline residues, known to be important in some protein - protein interactions. In fact, the 11 kDa protein has three consensus sequences with the Src Homology 3 (SH3) ligand motif, xxxPxxPx. One of these SH3 ligand motifs is very homologous to a motif of the SH3 ligand Sos-1, shown to interact with Growth Factor binding protein 2(Grb2).76 Grb2 is involved in recruiting the Ras protein to the cell membrane through an interaction with Sos-1, linking Sos-1 to important cell cycle control mechanisms.79 It is possible that B19 infection interferes with this process to facilitate the viral replication cycle. It has also been suggested that the HkDa protein may play a scaffolding role in capsid formation. However, a mutation that oblates the expression of the 11 kDa protein in COS cells transfected with a plasmid that expresses the remaining non-structural and structural proteins, does not affect capsid formation.59 To date, no function in B19 infection or pathogenesis has yet been ascribed to either the 7.5 kDa or the 11 kDa proteins.63 1.11 Previous studies on the possible functional roles of the 7.5 kDa and 11 kDa B19 proteins Expression of the 7.5 kDa or of the 11 kDa proteins does not affect expression of other viral proteins, nor do they appear to interact with other viral proteins. When missense mutations were engineered into either of 14 Introduction these proteins, their expression in transfected COS-7 cells was abolished with no apparent effect on expression of the other viral proteins.62 In immunoprecipitation studies neither the 11 kDa nor 7.5 kDa proteins were found to co-immunoprecipitate the other viral proteins VP1 or VP2. 2 2 Localization studies using fluorescent staining with B19 infected peripheral blood mononuclear cells (PBMC) showed that the 7.5 kDa protein is cytoplasmic and the 11 kDa protein is predominantly nuclear. In transfected COS-7 cells expressing the 11 kDa protein there was some fluorescence of the reticular network in the cytoplasm.63 Further studies into possible transactivation, Z n + + binding and D N A binding activities of both the 7.5 kDa and 11 kDa proteins failed to detect these properties. Also, neither protein appears to be phosphorylated.22 Preliminary investigations into the interaction of the 7.5 kDa protein with host cellular proteins began with far western studies. Two COS-7 cellular proteins of 43 kDa and 55 kDa, were tentatively identified as interacting with the 7.5 kDa protein.22 1.12 Review of the functions of other nonstructural viral proteins There are many examples of nonstructural, viral proteins that enhance infection through interactions with host cell or viral components. The net effects of these interactions, be they increased pathology and replication, or inhibition of the host immune response, are driven by myriad mechanisms with often surprising results. In order to develop an understanding of the possible functions of the B19 11 kDa and 7.5 kDa proteins, it may be helpful to review some of the wide variety of functions attributed to a number of the many nonstructural, viral proteins characterized to date. 15 Introduction 1.12.1 Alteration of protein trafficking within the host cell There exist small, nonstructural, viral proteins that act via vastly diverse mechanisms to modify trafficking of viral and cellular proteins, within the host cell. Vpu, a 16 kDa HIV-1 late gene product, has been shown to alter the host cell trafficking of viral envelope (Env) precursors. These Env proteins are retained in the endoplasmic reticulum (ER) by cellular CD4 molecules that inhibit their transport to the cell surface.90 In order to ensure proper transport of the Env protein, a C-terminally phosphorylated Vpu in the ER causes degradation of CD4 molecules.91 The Poliovirus 2BC polyprotein is most likely responsible for vesicle proliferation observed in Poliovirus infected cells. It is suggested that protein trafficking through the golgi apparatus is altered by this vesicle formation, such that cellular protein trafficking is inhibited.92 1.12.2 Nuclear Export of mRNA Transcripts In most cases the export of mRNAs from the nucleus of cells is limited to fully spliced transcripts. According to the 'spliceosome retention' hypothesis, partially or nonspliced pre-mRNA transcripts are retained in the nucleus by a mechanism involving the splicing machinery. However, splicing is not always necessary. A variety of nonspliced pre-mRNAs of several virus groups have been shown to be efficiently exported from the nucleus of infected cells. In HIV-1 infections it has been found that unspliced pre-mRNAs are exported from the nucleus. This is thought to be mediated by the 19 kDa, 116aa Rev protein encoded by HIV-1.81 It has been shown that without Rev only spliced viral mRNAs are transported from the nucleus, while the unspliced and partially spliced viral mRNAs are retained.82 Rev appears to act as a multimer70 with its N-terminal arginine rich region binding a specific sequence of the mRNA called the Rev Responsive Element (RRE). Rev also possesses a C-terminal region, conserved among Rev-like proteins of the 16 Introduction lentiviruses, that most likely interacts with cellular factors involved in nuclear export. This interaction, combined with binding of specific RRE containing pre-mRNAs, could selectively facilitate the export of viral mRNAs. One such cellular factor may be the U l small nuclear R N A (snRNA), which is part of the cellular splicing machinery and has been shown to be necessary for the REV-dependent expression of Env mRNA. 7 4 Some viral proteins capitalize on the nuclear retention of nonspliced mRNAs to preferentially retain mRNAs of a cellular origin while exporting those encoded by the virus. The 63 kDaHerpes Simplex Virus Type 1 (HSV-1) immediate early (alpha) protein, ICP27, has been shown to impair the splicing of host cell mRNAs, resulting in decreased host protein synthesis.120 Similiarly, the Influenza Virus NS1 protein inhibits splicing of cellular pre -mRNAs by binding a key spliceosomal RNA, the U6 small nuclear RNA(snRNA). 9 9 In a variation on the mRNA retention scheme, Adenovirus proteins E1B (55 kDa) and E4 (34 kDa) form a complex that is thought responsible for the observed increased export of late viral mRNAs at the expense of cellular mRNAs. 8 0 In this case, it is the spliced cellular mRNAs that are retained in the nucleus. One hypothesis used to explain this phenomenon is that the E1B/E4 complex vigourously recruits a nuclear factor or factors to sites of viral replication and transcription. This could deprive cellular mRNAs of such export factors, while promoting efficient export of viral mRNAs. In order for mRNAs to be exported from the nucleus they usually require 3' end processing. This is another point at which the HSV-1 protein ICP27 appears to act. ICP27 not only inhibits host cell protein synthesis as discussed above, but is also able to stimulate 3' end RNA processing at selected poly(A) sites, thereby specifically stimulating the production of late viral gene products and not cellular gene products.94 17 Introduction 1.12.3 Enhancement of viral genome replication Stimulation of viral replication is another important function of nonstructural viral proteins. The HSV-1, ICP27 has also been implicated in a replicative role, as ICP27 defective mutants display inhibited genome replication,94 although the mechanism remains unclear. Another activator of viral replication is the small, highly basic, C protein of Vesicular Stomatitis Virus (VSV). This protein is thought to stabilize the viral transcriptase/genome complex, resulting in the production of more full length, genomic RNA transcripts. 9 6 Interestingly, the vesicle proliferation caused by the Poliovirus 2BC protein (discussed above in relation to host protein synthesis inhibition) implicates 2BC in a replicative function. It is thought that these vesicles, formed in floret shaped structures, serve as sites of Poliovirus R N A replication.97 In support of this, it was shown that blocking the synthesis of these vesicles with cerulenin, an inhibitor of phospholipid synthesis, immediately blocks Poliovirus RNA synthesis.93 Another replicative enhancer, the viral protein Nef of HIV-1, appears to stimulate replication in primary PBMCs and macrophages.111 It has been suggested that Nef stimulates proviral DNA synthesis.101 The mechanism of this stimulation appears to involve cellular serine kinases and a subset of the Src family of tyrosine kinases. These kinases have been shown to physically interact with Nef1 1 3 and have been implicated in Nef-mediated enhancement of HIV-1 infectivity.112 Finally, the viral protein R(Vpr) of HIV-1 has been shown to connect the pre-integration complex of HIV-1 with the nuclear import pathway of the host cell. 1 0 4 This enables the virus to cross the intact nuclear membrane of quiescent cells and integrate its genome with that of the host. Without Vpr the host cells must be actively dividing to allow entry of the viral RNA into the nucleus during the normal degradation of the nuclear envelope. It 18 Introduction should be noted that the matrix protein (MA) also serves a nuclear import function, however Vpr and M A recognize distinct cellular receptors and V p r / M A double mutants cannot establish stable infections in quiescent T lymphocytes.105 1.12.4 Processing of viral gene products The nonstructural protein NS3 of Hepatitis C Virus (HCV) aids in the processing of the single viral gene product. HCV translates a single polyprotein that is cleaved by cellular and viral proteases into its integral protein components. NS3, a 70 kDa protein of two domains: an N-terminal protease and a C-terminal ATP-dependent RNA helicase, first cleaves itself from the polyprotein, then intermolecularly cleaves the remaining polyprotein into distinct viral proteins.117 1.12.4 Packaging of virions Packaging or virion assembly, is another point at which small viral proteins have been found to enhance the viral replication cycle. For instance, the virion infectivity protein factor (Vif) of most lentiviruses has been shown to be important in viral packaging. Between 60 - 100 molecules of the 23 kDa protein, Vif, are packaged per HIV-1 particle and co-localize with sedimented viral core structures. Although vif-mutated HIV-1 efficiently produces viral particles, these virions were shown to have improperly packaged nucleoprotein cores102 and could not synthesize proviral DNA in target cells.103 1.12.5 Virion emergence from the host cell Some small nonstructural viral proteins have also been shown to help virions escape from the host cell, as seen with the HIV-1 viral protein U (Vpu). Vpu is a 16 kDa, 81 amino acid (aa), integral membrane phosphoprotein, that associates with internal host cell membranes and is known to be involved in proper maturation, targeting and release of virions from the host cell.85 In the absence of Vpu, virions bud into vacuolar 19 Introduction compartments instead of from the plasma membrane and often contain multiple cores.89 From studies of deletion mutations and amino acid substitutions, the ion channel formed by the transmembrane domain of Vpu was shown to be essential for the release of mature virions. 8 6 , 8 7 1.12.6 Immune response evasion Yet another role for viral nonstructural proteins is avoidance of the hosts immune system. Host cells are able to respond to the presence of double stranded RNA (dsRNA), a sign of viral infection, through the activation of dsRNA-activated protein kinase (PKR). PKR is responsible for phosphorylation of the translation initiation factor elF-2, resulting in protein synthesis inhibition. To evade this host response viruses may express proteins such as the Influenza NS1 protein, that bind ds-RNA molecules, thereby inhibiting the activation of PKR. 1 0 0 The Tat protein of HIV-1 appears able to suppress the immune system of its host organism. It has been shown that the small nuclear protein Tat increases the expression of interleukin-4 receptor in a human B cell line, 1 0 6 as well as the secretion of interleukin-2 from activated T cells.107 It is thought that Tat acts as a transcription factor to transactivate cellular genes, helping to mediate the progressive deterioration of the immune system in HIV-infected patients.108 Nonstructural viral proteins have also been implicated in suppression of our innate immune response. HSV-1 does so by producing glycoprotein C (gC) which binds the C3b fragment of the complement cascade.110 Usually a heterodimer of C3b and Bb form the enzyme convertase, a component within the complement cascade. However, convertase is unstable and decays quickly without the addition of a third cellular protein, properdin.1 0 9 It has been shown that when gC binds the C3b protein, it blocks the subsequent binding of properdin. This destabilization of the C3bBb convertase complex interrupts the complement pathway, inhibiting complement-mediated lysis of infected host cells.98 20 Introduction As can be seen by this brief review many viral proteins serve multiple functions through diverse mechanisms, all to achieve the same ultimate goal of viral replication. In order to do so the active protein must interact, either directly or indirectly, with machinery of the host organism. 1.13 The present study In the case of B19, we hypothesize that the small nonstructural 7.5 kDa and 11 kDa proteins most likely interact directly with proteins of the host cell to aid in the replication cycle and subsequent pathogenesis of B19. This study charts the progression toward identification of these putative cellular proteins. In order to investigate these possible interactions, far western blot, co-immunoprecipitation and affinity purification experiments were conducted. In addition, the yeast two hybrid system was used to attempt to identify interacting cellular proteins. 21 Materials and Methods II Materials and Methods Materials 2.1.1 Chemicals and supplies Chemicals were purchased from Fisher Scientific, BDH, Sigma Chemical, or Rose Scientific unless otherwise specified. Polyacrylamide and agarose gel supplies, were purchased from GIBCO/BRL or Bio-Rad Laboratories. Bacterial and yeast culturing supplies were from Difco Laboratories. Tissue culture supplies were purchased from Stem Cell Technologies, GIBCO/BRL, or Sigma Tissue Culture. Ampicillin (Penbritin) was supplied by Ayerst Laboratories. All restriction and deoxyribonucleic acid(DNA) modifying enzymes as well as antibodies were purchased from either GIBCO/BRL, Boehringer Mannheim, or New England Biolabs. Sequencing kits and the Sequenase enzyme were both purchased from United States Biochemical Company. Glutathione Agarose Resin was purchased from Sigma and Probond™ Resin from Invitrogen. Bacterial culture 2.2.1 Cell lines Recombinant deficient strains of E.coli, DH50C1 and Sure® (Stratagene) were employed for all routine cloning and plasmid propagation. Protein expression was done using protease deficient E.coli BL21(DE3) 1, UT560015 or M15(pREP4) (that expresses the T7 RNA polymerase)13 or with DH5a. 2.2.2 Growth Bacteria were routinely grown on YT agar plates (8g tryptone, 5g yeast extract, 5g NaCl and 15g agar per liter) or 2xYT media (16g tryptone, lOg yeast extract, 5g NaCl per litre) for protein expression purposes. For plasmid 22 Materials and Methods isolation and propagation L-broth (lOg tryptone, 5g yeast extract, 5g NaCl, per litre with 0.1% D-glucose) or L-broth agar plates (L-broth with 1.5% Agar) were utilized. When required for the selection of plasmids, media was supplemented with 25 Lig/ml kanamycin, or 100 Lig/ml ampicillin. 2.2.3 Stocks Bacterial cultures were routinely frozen with 25% glycerol on dry ice, then stored at -65°C. 2.2.4 Electroporation For transformation of bacteria with plasmid DNA, cells were prepared and used in accordance with the manufacturers instructions received with the Bio-Rad Gene Pulser. Yeast 2.3.1 Yeast two hybrid system Clontech's Matchmaker® Two-Hybrid System and the Clontech Human Bone Marrow Matchmaker® cDNA library were utilized following the manufacturer's recommended protocols, unless otherwise specified9,10. 23 Materials and Methods Tissue cell culture 2.4.1 Cell lines i) Adherent COS-7 cells3 were grown with low glucose Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum and lOmM HEPES (pH 7.4). ii) Nonadherent K-562 cells4 were grown with RPMI-1640 medium supplemented with 10% fetal bovine serum and lOmM HEPES (pH 7.4). 2.4.2 Maintenance COS-7 cells were propagated in 100mm culture dishes in a water jacketed incubator held at 37°C under 5% CO z . Frozen stocks of approximately 107 cells in a 1 ml volume were quickly thawed at 37°C, then incubated overnight in 10 mis media. The media was then changed to remove the dimethyl sulfoxide (DMSO). When cells reached approximately 90% confluency, usually after three days, they were passaged by trypsinization and diluted 1:10 in fresh media.29 Frozen stocks of K-562 cells were quickly thawed at 37°C, briefly centrifuged, then resuspended in a 1:10 dilution in fresh media. Cultures of less than 10 ml were incubated as described above for COS-7 cells. For large numbers of cells, the cells were propagated in suspension in sealed spinner flasks purged with 5% C02/balance air. Cells were passaged by simple dilution of the culture with fresh media. Neither COS-7 nor K-562 cells were passaged more than six times. 24 Materials and Methods 2.4.3 Storage of frozen cells One millilitre frozen stocks of approximately 107 cells in a 1 ml volume were made in the growth media supplemented with 10% DMSO. DMSO was added after resuspension of the cells. These stocks were packed in styrofoam, frozen over night at -65°C, then transferred directly to liquid nitrogen for long term storage. 2.4.4 Transfections A modified DEAE-Dextran transfection procedure was used to transfect plasmids into COS-7 cells. Cells were seeded at a 1:5 dilution in 60mm dishes and grown overnight to approximately 40% confluency. The cells were washed twice with D M E M with no FBS and incubated in 1 ml D M E M with no FBS, supplemented with 1 ug plasmid DNA complexed to 200 ug DEAE-Dextran. After incubation at 37°C for 8 hours, the media was removed and the cells were shocked for three minutes in 1 ml of 10% DMSO-90% HBS (21mM HEPES/135mM NaCl/5mM KCl/0.8mM Na 2 HP0 4 /5mM dextrose). The cells were washed three times in phosphate buffered saline, pH7.4 (PBS) then grown at 37°C in 5% C 0 2 for 48 to 72 hours before harvesting. 2.4.5 Lysis of tissue culture cells All tissue culture cell lysates were prepared as previously described using low salt lysis buffer (1.0% Nonidet P-40, 50 mM Tris, pH 8.0).5 Lysates were made no more than two hours before use unless otherwise specified. 2.4.6 35S-labeled tissue culture cell lysates Tissue culture cells were labeled using Tran 3 5 S 3 5 S-label™ from New England Nuclear(NEN) as per the manufacturer's specifications following a previously described protocol.6 25 Materials and Methods Cloning techniques 2.5.1 Plasmid Preparation Large-scale plasmid DNA preparations described previously1 were followed by polyethylene glycol(PEG) precipitation. Plasmid minipreps were collected using a rapid version of the modified alkaline lysis protocol2 with an additional phenol/chloroform extraction step1 for miniprep DNAs that were used for cloning procedures. Protocols for plasmid isolation from yeast were described previously.9 2.5.2 Plasmid digestions and ligations All plasmid restriction digestions and ligations were done using the buffers and protocols provided by the manufacturer. T4 DNA ligase was used for all ligations. Sticky end ligation reactions were incubated 2 - 4 hours at room temperature or 8 hours at 16°C. Blunt end ligations were incubated for 10 - 20 hours at 16°C. 2.5.3 Plasmids See Table 3 for quick reference to plasmids and the proteins they encode. i) pGEX2T utilizes the tac promotor to direct the expression in E.coli of glutathione S-transferase (GST), a 26 kDa protein from Schistosoma japonicum. GST binds strongly to glutathione. This vector allows the expression of GST or GST-fusion proteins, which are easily purified by affinity chromatography on glutathione-agarose resin.12 ii) pGEXll, constructed by Jan St. Amand, directs the expression of GST-11. It was constructed by cloning the ORF for the 11 kDa protein of the parvovirus B19 into the BflmHI/EcoRI sites of pGEX2T. iii) pGEX2T-tag (Figure 3) was a generous gift from Ken Harder. This plasmid contains a Hemagglutinin(HA) epitope tag and polylinker in 26 Materials and Methods the BamHI/EcoRI sites of pGEX2T, such that the HA tag may be removed by Sma I digestion and religation. This vector results in expression of a fusion protein of glutathione S-transferase(GST) and HA. Fusion protein derivatives utilizing this vector are recognized by anti-HA monoclonal antibodies raised against the peptide sequence YPYDVPDYA. Plasmid pGEX2T pGEX2T pGEX2T-tag pGEX-tag-Grb2 pGEX-tag-PD pGEXPD pCMV5 pCMV805 PQE41 pQE7.5 pRSETA pRSETll Protein Expressed N-GST-C N-GST-11 kDa protein-C N-GST-Hemagglutinin epitope-C N-GST- Hemagglutinin epitope-Grb2-C N-GST-Hemagglutinin epitope-PD-C N-GST-PD-C this is a mammalian expression vector 7.5 kDa B19 protein N-(histidine)6-dihydrofolate reductase-C (DH6) N-(histidine)6-dihydrofolate reductase-7.5-C (7.5DH6) vector for expression of (histidine)6 fusion proteins N-(histidine)6-ll kDa-C (11H6) N-(histidine)6-cel0 monoclonal epitope-11 kDa-C (HcelOH6) pRSETllcelO Table 3: Plasmids and the proteins they encode The plasmids used in this study are listed adjacent to the corresponding fusion protein expressed by each. N and C refer to the amino and carboxy terminals of the fusion proteins, respectively. iv) pGEX-tag-Grb2 was a generous gift from Dr. Schrader of the Biomedical Research Centre, UBC. This plasmid was constructed by insertion of a cDNA encoding human Grb2 into the Nhel/EcoKL sites of pGEX2T-tag as 27 Materials and Methods previously described.18 This plasmid directs the expression of a GST-Hemagglutinin epitope-Grb2 fusion protein referred to as GST-HA-Grb2. v) pGEX-tag-PD, a gift from Ken Harder, is a vector into which the sequence encoding the phage display Src homology 3 (SH3) ligand was ligated. This plasmid therefore directs the expression of GST-HA-PD, which has been shown to bind to most known SH3 domains through its SH3 ligand consensus sequence "RPLPPLP".1 6 vi) pGEXPD is a derivative of pGEX-tag-PD in which the H A epitope tag has been removed by Sma I digestion and religation. The resulting fusion protein is referred to as GST-PD and was used as a positive control in this study for SH3 ligand/SH3 domain binding studies. 71 BspM I Dsa I 4904 Bsu36l 4795 463 Mscl 654 BslBI 930 BamH I 940 Asp718 940 Kpnl 946 Spll 956 BspM I 978 NheI 984 AccI 984 Sail 1171 Tth111 I 1179 BsaAl AlwN I 2673 Thrombin Hemagglutinin epitope tag I Leu Val Pro Arg Gly Ser ' Pro Gly 'Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Sei^  Pro Gly C T G G T T C C G C G T G G A T C C C C C G G G T A C C C G T A C G A C G T T C C G G A C T A C G C A T C C C C C G G G Bam HI Sma I Kpn I Sma I Ala Ser Val Asp Arg Ser Glu Phe He Val Thr Asp ?i2E G C T A G C G T C G A C A G A T C T G A A T T C A T C G T G A C T G A C T G A Nhel Sail Bgl II Eco RI Figure 3: Map of pGEX2T-tag and its multiple cloning site sequence. 28 Materials and Methods vii) pCMV5 is a mammalian expression vector that directs the transcription and subsequent protein expression with the Cytomegalovirus (CMV) immediate early promotor and enhancer sequences.118 viii) pCMV805 directs the synthesis of the 7.5 kDa B19 protein, due to the insertion of an 805 bp cDNA into the EcoRI/Sall site of pCMV5. The 805bp cDNA encodes the ORF of the 7.5 kDa B19 protein. ix) pQE41 from Qiagen directs the expression of a histidine tagged, dihydrofolate reductase(DHFR) fusion protein (DH6). Expression of this fusion protein is controlled by a regulable promotor/operator element, consisting of the E.coli phage T5 promotor and two lac operator sequences.13 This fusion protein is expressed in the E.coli strain M15(pREP4). x) pQE7.5 directs the expression of 7.5DH6, a fusion protein including the 7.5 kDa B19 protein, DHFR for protein stability and a histidine tag. This plasmid was constructed in this laboratory by Carl Yong utilizing the BamHI/EcoRI site of pQE41. The 7.5DH6 fusion protein is expressed in E.coli, M15(pREP4). xi) pRSETA, from Invitrogen, utilizes the T7 promotor to express histidine tagged fusion proteins.17 xii) pRSETl l was constructed by C. Yong. This construction utilized the BamHI/EcoRI sites of pRSETA to clone the ORF of the 11 kDa B19 protein. This plasmid directs the expression of a N-terminally histidine tagged 11 kDa fusion protein. xiii) pRSETllcelO is a modified pRSET 11 vector, in which an antibody epitope of the NS1 protein of M V M was inserted between the histidine tag and the 11 kDa viral protein. The amino acid sequence of this epitope is LALEPWST. This plasmid therefore directs the expression of the HkDa-cel0-H 6 (llcelOHJ fusion protein to allow visualization of the B19 11 kDa fusion protein with the monoclonal antibody CE10. 29 Materials and Methods 2.5.4 Oligonucleotides All synthetic oligonucleotides used in this study (Table 4) were prepared using ABI 371 or 374 DNA synthesizers (Applied Biosystems). zl 5' - GAT CCC TGG CTT TAG AGC CTT GGA GCA CAG - 3' z2 5' - GAT CCT GTG CTC CAA GGC TCT AAA GCC AGG - 3' z3 5' - pGAT CCC TGG CTT TAG AGC CTT GGA GCA CAG - 3' z4 5' - pGAT CCT GTG CT C CAA GGC TCT AAA GCC AGG - 3' z5 5' - GGA ATT CAT GCC CTC CAC CCA GAC C - 3' z6 5' - CGG GAT CCT ACA ACT TCG GAG GAA A - 3' Table 4: Oligonucleotides used in this study. Sequences are listed 5' to 3' with p signifying a 5'phosphate. 2.5.5 Isolation of plasmid DNA from electrophoresis gels Bands of plasmid DNA were routinely excised from agarose gels and recovered by spinning through silanized glass wool in a microcentifuge at lOOOOg for 3 minutes. The eluant was used directly for ligation reactions and transformations, or was ethanol (EtOH) precipitated to achieve a higher concentration of DNA. If the fragment of DNA was smaller than 500bp, transfer RNA (tRNA, lOug/ml) was added to the precipitation solution. 2.5.6 DNA quantification The concentration of medium to large scale DNA and oligonucleotide preparations were determined spectrophotometrically. Determination of the DNA concentration of routine plasmid miniprep DNA was done by comparison with bands of known concentration from a standard DNA ladder, run on an agarose gel. 30 Materials and Methods 2.5.7 Polymerase chain reaction (PCR) PCR was performed using VENT DNA polymerase (New England Biolabs) with the supplied buffer. DNA was PEG precipitated and milli-Q filtered, sterile, double distilled (sdd) water was used wherever possible. Reaction volumes were of 50ul including O.lng plasmid DNA, 500nM each dNTP, BSA at 10Llg/ml and 25 nmol each primer. The PCR reaction was carried out in 500 ill eppendorf tubes in an MJ Research Minicycler. The reactions were warmed for 1 minute at 95°C before initiation of 25 cycles of 1 minute incubations at 95°C, 52°C and then 72°C These incubations allowed denaturation, annealing and extension reactions to proceed, respectively. The cycling was followed by 3 minutes at 72°C to allow complete blunt end formation, followed by 1 minute at 4°C to cool the reaction before termination. 2.5.8 DNA sequencing DNA sequencing was done using the Sanger dideoxy method as previously described.1 Two micrograms of template DNA was extracted with an equal volume of phenohchloroform and EtOH precipitated. The D N A was denatured by 30min incubations at 37°C in 0.2 M NaOH/0.2mM ethylene-diaminetetra-acetic acid (EDTA) and neutralized. The primer (lpmol) was annealed to the template in the sequencing buffer provided in the Sequenase 2.0 kit (United States Biochemicals/Amersham). Protocols supplied with the kit were followed using a-32P dATP as the labeling nucleotide. 31 Materials and Methods Protein preparation and characterization 2.6.1 General procedures All general molecular biological techniques, such as SDS poly acrylamide gel electrophoresis (SDS-PAGE), western blotting via the semi dry method, silver staining, immunoprecipitation and co-immunoprecipitation have been described previously5 and were modified only as specified. 2.6.2 Protein quantification The concentration of purified protein solutions were determined using the Pierce BCA kit following either the supplied protocol, or a version scaled down to one tenth the prescribed volume. This allows reactions to proceed in the wells of a 96 well plate with the absorbance easily measured in an ELISA reader. 2.6.3 Purification of fusion proteins Glutathione S-transferase (GST) fusion proteins were purified using the Pharmacia protocols12 supplied with the pGEX2T plasmid, with minor modifications. Briefly, prewarmed 2xYT media was inoculated with a saturated overnight culture of transformed bacteria at a 1:50 dilution and grown at 37°C to an O D 6 0 0 of 0.5, in the absence of antibiotics. The culture was induced with isopropyl (3-D-thiogalactopyranoside(IPTG) at 0.1 mM to 1 m M (most often 0.2mM) and incubated for 2.5 hours at 37°C before harvesting by centrifugation. To prepare for sonication the bacterial pellet was resuspended in PBS with 2u,g/ml antipain, 0.5 mM PMSF, lug/ml leupeptin and 4|ig/ml aprotinin, at a volume of 70 |il per milliliter of the initial bacterial culture. Sonication was done in 15ml falcon tubes on ice for no more than two 12 second bursts with an incubation on ice of at least 1 minute between bursts. After the solubilization and centrifugation steps, the protein was bound to the resin either at 4°C for 1 hour, or at room temperature for 30 minutes in a 32 Materials and Methods batch style using 15ml falcon tubes. The protein bound resin was washed three times in 10 bed volumes of PBS with protease inhibitors in an eppendorf tube and was either loaded onto a column, or left in tubes and used in a batch style affinity procedure. The elution of the protein from the resin either proceeded in a batch style, or by washing the elution buffer through an affinity column. Protein was eluted from the column with 3-4 incubations of 1 bed volume of glutathione elution buffer (GEB: lOmM reduced form glutathione/5OmM Tris-HCl pH 8.0, freeze thawed no more than twice). During batch style elution the eppendorf tube containing the protein bound resin was gently mixed in a rotating wheel (end over end). Fusion proteins expressed by this procedure are often contaminated with the DnaK protein with an apparent mass of 70 kDa. In order to attempt to purify DnaK-free GST-11 fusion proteins, a 10 minute, 37°C incubation with 2 mMATP/10mMMgSO4/50mM Tris pH8.0 was added to the GST-fusion purification scheme, just prior to incubation with glutathione resin. Similiarly, subsequent wash and elution steps were performed in 5mM ATP to avoid co-purification of GroEL (57kDa).12 Purification of histidine tagged fusion proteins followed the protocols previously described.13 The native purification system was used to purify the 7.5DH6 and D H 6 fusion proteins. The HcelOH 6, 11H6 and H 6 proteins were purified under denaturing conditions using buffers B and C. Renaturation was performed while the protein was bound to the column and employed a linear gradient maker to achieve gradients of 8M - 4M urea, 4M - IM urea and finally IM - 0M urea. All proteins were eluted from the columns with either 250mM imidazole, or a gradient of imidazole from lOOmM to 400mM. 33 Materials and Methods 2.6.4 Silver staining Silver staining of SDS-polyacrylamide gels was done according to the modified procedure previously described.14 This procedure includes a sensitizing step that increases the sensitivity of the staining procedure. 2.6.5 Western blotting The transfer of proteins from SDS-PAGE minigels to Immobi lon-P™ utilized the Transblot SD®, semidry Transfer Cell (Bio-Rad) at 15V for 20 - 30 minutes, following the manufacturers recommendations. The Immobilon-P™ was presoaked for 15 seconds in methanol, 2 minutes in sterile double distilled (sdd) water, and 10 minutes in Tris/Glycine transfer buffer (25mM Tris/192 mM Glycine/20%v/v Methanol). After transfer in the same buffer, the blot was washed briefly in KBS (137 mM NaCl, 1.5mM KH 2 P0 4 , 7.2mM Na 2 HP0 4 , 2.7mMKCl, 0.02% (w/v) NaN 3 , 0.05% (v/v) Tween-20) with 5% (w/v) no fat, skim milk powder (SMP), then incubated in the same buffer overnight at 4°C, or 1 hour at room temperature. KBS was used for subsequent washes and 3% SMP-KBS was used to dilute the primary and secondary antibodies. A system using alkaline phosphatase conjugated secondary antibodies (GIBCO/BRL) with BCIP and NBT, allowed visualization of the blot in accordance with the manufacturers suggestions. Concentrations of the primary and secondary antibodies were optimized for each application. Monoclonal antibodies raised against vimentin and actin (a generous gift from Dr. Michel Roberge) were used in western blot analysis of the COS-7 cell lysate. 2.6.6 Far western blotting Far western blotting procedures were used to investigate if a purified protein (X) in solution would bind an protein (Y) immobilized on a western blot. Briefly, protein Y was immobilized onto an Immobilon-P™ membrane (see Section, 2.6.5) and the membrane was incubated in a solution of purified protein X diluted in 3% SMP-KBS. Binding of protein X to immobilized 34 Materials and Methods protein Y was visualized as in a western blot with a primary antibody directed against protein X and a suitable secondary antibody (Figure 4(a)). Therefore a band should appear on the far western blot corresponding to the molecular weight of protein Y, if it is bound to protein X. If a cell lysate is immobilized in place of protein Y, then this protocol may be used to identify the molecular weight of protein(s) that interact with a purified protein X. Figure 4(a): Far western blotting: Protein interactions and recognition by a primary antibody. Far western blots used to identify cell lysate proteins that interact with the 7.5 kDa or 11 kDa B19 proteins employed "subtractive blotting". For example, the 7.5DH6 fusion protein was used with the test lanes and the D H 6 protein with control lanes. Briefly, cell lysate was run in all lanes by SDS-PAGE and blotted onto a membrane. The membrane was cut in sections to allow incubation of identical lanes with either 7.5DH6 or DH 6 . This allowed the lysate proteins interacting with D H 6 to be "subtracted" from those interacting with 7.5DH6, to reveal the molecular weight of those proteins that 35 Materials and Methods appear to interact due to the 7.5 kDa moiety of the 7.5DH 6 fusion protein (Figure 4(b)). 2.6.7 Antibodies used i) polyclonal rabbit anti-7.5DH 6 ii) polyclonal rabbit anti-GST. A generous gift of Dr. Ivan Sadowski. i i i ) monoclonal mouse anti-vimentin. A generous gift of Dr. M i c h e l Roberge. iv) monoclonal mouse anti-actin. A generous gift of Dr. Michel Roberge. v) monoclonal mouse anti-HA. Recognizes the partial hemagglutinin amino acid sequence, Y P Y D V P D Y A . v i ) polyclonal anti-llkDa. Raised against the synthetic peptide corresponding to the partial amino acid sequence of the 11 kDa B19 protein, P N T K D I D N V E F K Y L T R Y E Q H V I R M L R L C . 2.6.8 Affinity column procedures Affinity columns were prepared either from N i + + - N T A resin, or glutathione agarose resin, for histidine tagged or GST fusion proteins, respectively. Freshly prepared fusion proteins were immobi l ized on the appropriate resin in a batch style, washed to remove nonspecifically b inding bacterial proteins, then either loaded into a column or into a falcon tube for incubation wi th a fresh 3 5S-labeled cell lysate. Lysate was added to the washed protein bound resin either by r u n n i n g the lysate through the column, or by adding the lysate directly to a falcon tube wi th protein bound resin, along wi th an equal volume of PBS wi th protease inhibitors. Nonspecifically interacting proteins were washed from the column and the fusion proteins, along wi th any interacting cellular proteins from the lysate, were eluted using the appropriate buffers. 36 Materials and Methods Protein interacting with the 7.5 kDa protein ^ ^ - ^ ^ 7.5DH 6 D H 6 Immomilon P™ membrane—• Figure 4(b): Far western blot illustrating a band identified by "subtractive blotting". Bands that appear in the 7.5DH6 lanes but not in the DH 6 lanes represent cellular proteins immobilized on the membrane to which the 7.5 kDa protein presumably binds. 2.6.9 Cyanogen bromide activation of sepharose 2B beads Cyanogen bromide (CNBr) and sepharose 2B beads were a generous gift of Dr. B. Molday. (Note that cross linked beads do not appear to work well for this application.) Briefly, 8ml of beads were washed 4 times with 15 ml dH zO. The beads were then gently stirred with a stir bar in 8 ml dH z O IN A FUME HOOD. A pH of between 10 and 11 was obtained using 0.2 N NaOH before adding 0.15g CNBr (weighed out in a fume hood). The pH of the slowly mixing solution was held between 10 and 11 with 0.2 N NaOH until it stabilized, or for 30 minutes. About 30 ml cold borate buffer (0.02 M Sodium Borate, pH8.4) was then added and this wash step was repeated 5 times. A known volume of packed beads was then mixed with the purified protein solution of interest at 2 mg/ml protein per ml of beads. The protein solution 37 Materials and Methods was diluted in borate buffer as needed. The coupling reaction continued at least 8 hours at 4°C with gentle mixing on a rotation device. After removal of the protein solution supernatant, remaining free active sites were blocked with an identical incubation in TBS-Glycine-NaN3 buffer (0.02 M Tris/0.05 M glycine/0.15 M NaCl, 0.02% NaN 3 , pH 8.0). 2.6.10 Protein solution concentration Proteins were concentrated in the appropriate Micron microconcentrators, for volumes of 500ul or less, and in the appropriate Ultrafree - 15 centrifugal filter device for volumes between 15ml and 2ml, as instructed in the literature provided. These filtration devices were supplied by Millipore. 2.6.11 K-56 2 35S-labeled lysate Approximately 1.5 x 108 K-562 cells were washed once in RPMI-1640 media without cysteine or methionine, then resuspended in 10 ml of the same media and incubated 30 minutes at 37°C under 5% C0 2 . 300ul (~3.3mCi) fresh Tran35S35Slabel (35S-labeled Cys and Met) was added and the cells were incubated 3.5 hours as above with gentle agitation, or gentle shaking every 20 minutes. Cells were then harvested, washed once in PBS and lysed in low salt lysis buffer (1% Nonidet P40, 50mM Tris, pH 8.0) supplemented with protease inhibitors as previously described5. 2.6.12 GST vs GST-11 sepharose affinity column procedure Either GST, or GST-11 bound sepharose beads (= 3.25 mis) were incubated 60 minutes at 4°C, with 8 ml fresh 35S-labeled K-562 lysate and 10ml PBS with protease inhibitors. This incubation was done in a rotation device to ensure constant gentle mixing of the beads with the lysate. The beads were washed 4 times in cold PBS with protease inhibitors and eluted by boiling 5 38 Materials and Methods minutes with 2ml of protein sample buffer (2% SDS, 10% Glycerol, 60mM Tris pH6.8, 0.001% bromophenol blue, 5% B-mercaptoethanol). 3 9 Results III Results 3.1 Characterization of fusion proteins: Dihydrofolate reductase-histidine4 (DH )^ and the B19 7.5kDa-dihydrofolate reductase-histidine^ (7.5DHL) The His 6 tagged dihydrofolate reductase (DH6) and His 6 tagged 7.5 kDa dihydrofolate reductase (7.5DH6) fusion proteins were expressed in bacteria and purified by affinity chromatography on N i + + - N T A columns as described in the Materials and Methods section (Section, 2.6.3). Fractions of purified D H 6 and 7.5DH6 were each run on two duplicate 12.5% SDS polyacrylamide gels. As indicated on western blots, D H 6 runs as three bands between 25 kDa and 30 kDa (Figure 5A) and 7.5DH6 runs as two bands; one at 29 kDa and another of lesser intensity at 32.5 kDa (Figure 5B). 3.2 Far western blots suggest that proteins within a COS-7 cell lysate interact with the 7.5 kDa B19 protein The affinity purified D H 6 and 7.5DH6 fusion proteins described above (Section, 3.1) were used in far western blotting experiments to determine if proteins within a COS-7 cell lysate interact with the 7.5 kDa B19 protein. By comparing the difference between interactions found using the 7.5DH6 fusion protein with those found using the D H 6 fusion protein, one may presumably elucidate interactions due only to the 7.5 kDa moiety of the 7.5DH6 fusion protein. Hence, this approach may identify cellular proteins that interact with the 7.5 kDa B19 protein. Using this procedure, it appears that proteins of 55 kDa, 43 kDa (Figure 6A) and of lower molecular weight (Figure 6A) interact with the 7.5DH6 and not with D H 6 fusion proteins. 40 Results A: DH6Western ^ Eluant Samples, 32.5 B: 7.5DH6 Western ^ Eluant Samples 32.5i 25 H C: DH6 Coomassie Stained Gel Eluant Samples, D: 7.5DH6 Coomassie Stained Gel Eluant Samples, 62T~ 32.5 16.5 Figure 5: Characterization of DHfe and 7.5DHC fusion proteins. The upper panels are 12.5% SDS-polyacrylamide gels of affinity-purified DH6 (A) or 7.5DH6 (B), western blotted and probed with rabbit anti-7.5DH6 antisera as primary antibody, followed by an alkaline phosphatase conjugated Goat anti-rabbit IgG secondary antibody. The blots were developed using BCIP and NBT as described in the Materials and Methods (Section, 2.6.5). The lower panels are Coomassie stained 12.5% SDS-polyacrylamide gels of DH6 (C) or 7.5DH, (D). 41 Results A: B: Putatively Interacting Cellular Proteins Figure 6: Far western blots of a COS-7 cell lysate with the 7.5 kDa protein. Proteins from a COS-7 cell lysate were separated by 12.5% SDS-PAGE and blotted onto Immobilon-P™. This membrane was cut in half for incubation with either the 7.5DH6 or DH6 fusion protein, followed by incubation with rabbit anti-7.5DH6 antisera and secondary antibody as described in Figure 5. Bands corresponding to 43 kDa and 55 kDa proteins (Figure 6, A and B), as well as a 27 kDa protein (Figure 6A only) are shown. These far western blots suggest that COS-7 cell lysate proteins of these molecular weights interact with the 7.5 kDa B19 protein. 3.3 Precleared far western blot of a COS-7 cell lysate with the B19 7.5 kDa protein In an attempt to decrease the background in the far western procedure (Figure 6) a preclearing method was adopted. Fresh COS-7 cell lysate was incubated with the fusion protein D H 6 followed by rabbit anti-7.5DH6 antisera. 42 Results The resulting antibody/protein complexes were precipitated with Immunoprecipit in™ or protein A-agarose. This cleared the lysate of many proteins that interact with either D H 6 or the primary antibody used in the far western procedure, the anti-7.5DH6 polyclonal antibody. The above procedure resulted in an overall decrease in background (compare Figure 7 with Figure 6). The appearance of 50 kDa and 25 kDa bands, corresponding to the large and small chains of the preclearing antibody, may obscure bands that represent interacting cellular proteins. However, (Figure 7) a 55 kDa, a 43 kDa, and possibly some smaller molecular weight cellular proteins, appear to interact with the 7.5 kDa protein, using this procedure. 3.4 Western blotting of a COS-7 cell lysate suggests that the 55 kDa cellular protein shown to potentially interact with 7.5DHfeis vimentin A western blot of a COS-7 cell lysate was run to determine if the proteins that appear to interact with the 7.5 kDa B19 protein (Figure 6) are vimentin (55 kDa) and actin (43 kDa). It appears that the 55 kDa band may represent vimentin (Figure 8, Lane #2), however actin appears (Figure 8, Lane #1) to run higher than the potentially interacting 43 kDa band. 43 Results A B C 7.5DH6 D H 6 None *m m Figure 7: Far western blot of a precleared COS-7 cell lysate with the 7.5 kDa protein. Precleared COS-7 cell lysate was run in all lanes of a 12% SDS-PAGE gel and subsequently blotted onto Immobilon-P™. Sections of the blot were incubated with either : A) 7.5DH6, B) DH6 or C) no protein solution, before visualization with rabbit anti-7.5DH6 antisera and an appropriate secondary antibody, as previously described (Figure 6). Two potentially interacting cellular proteins are indicated. 44 Results #1 MW # 2 Actin -Vimentin Figure 8: Western blot of a COS-7 cell lysate probed for vimentin and actin. Proteins within a COS-7 cell lysate were separated by 12.5% SDS-PAGE and blotted onto Immobilon-P™. Sections of the membrane were incubated with mouse monoclonal antibodies that recognize either actin (Lane #1) or vimentin (Lane #2) and further developed with alkaline phosphatase conjugated Goat anti-mouse IgG and BCIP/NBT. 3.5 Far western blots fail to identify proteins that interact with the 11 kDa B19 protein Far western experiments were carried out to identify either COS-7 or K-562 cellular proteins that potentially interact with the 11 kDa B19 protein. These experiments utilized the 11H6 fusion protein expressed in bacteria transformed with pRSETll. To serve as the negative control in these far western experiments a preparation (H6) from bacteria transformed with pRSETA was made following the protocol used to purify the 11H6 fusion protein. 45 Results Unfortunately, visualization of these far western experiments failed to reveal any bands on the membrane (data not shown). Considering the high background observed in similiar 7.5DH6 experiments (Figure 6), this was surprising. Either the 11 kDa protein does not bind any cellular proteins immobilized on the membrane, or the primary antibody does not recognize this 11 kDa fusion protein in far western experiments. However, this antibody does recognize the same fusion protein in western blots. To further investigate these possible explanations, far western experiments with two other fusion protein systems were carried out. First, the fusion proteins GST-11 and GST were utilized in far western experiments visualized with rabbit anti-GST antisera (a generous gift of Dr. Ivan Sadowski) as the primary antibody (Figure 9). This resulted in visualization of faint bands, due either to primary antibody recognition of GST-11, or recognition of cellular proteins. Although a banding pattern is shown, it is too faint to confidently distinguish bands that appear on the GST-11 and not the GST incubated sections of the blot. However, these results seem to suggest that the 11 kDa B19 protein binds cellular proteins in far western experiments. 46 Results A B GST GST-11 Postitive Control Figure 9: Far western blot of a K-562 lysate with the 11 kDa protein. Proteins within a K-562 cell lysate were separated by 12.5% SDS-PAGE, blotted onto Immobilon-P™ and sections of the blot were incubated with either A) GST-11 or B) GST fusion proteins. GST-11 was included in the gel as a positive control(+). To visualize this far western experiment rabbit anti-GST polyclonal antisera was used as the primary antibody followed by further development as described in Figure 5. 3.6 Purification of an 11 kDa fusion protein with a CE10 monoclonal antibody epitope tag, llcelOH 6 DNA sequence encoding an antibody epitope recognized by the monoclonal antibody CE10 was inserted into the plasmid pRSETll. This plasmid expresses HcelOH 6 , a fusion protein in which the 11 kDa B19 protein is expressed with a CE10 epitope and His6 tag. When separated by SDS-PAGE this fusion protein was readily visualized on western blots probed with monoclonal antibody CE10 (Figure 10A). Unfortunately, only faint bands develop in far western experiments 47 Results using the llcelOH 6 fusion protein visualized with the same monoclonal antibody (Figure 11). However, the fact that bands appear only on the section of membrane incubated with HcelOH 6 (Figure 11, A and not B) suggests that the HcelOH 6 fusion protein does bind cellular proteins, although either with low affinity, or in a way that obscures its subsequent antibody recognition. No conclusion about the interaction of the 11 kDa protein with cellular proteins can be drawn from this experiment, as the negative control protein preparation H 6 , incubated with section B of the membrane, (Figure 11, B) contains no CE10 epitope tagged proteins. The positive control lane (Figure 11 "+") demonstrates that the antibody recognized the HcelOH 6 fusion protein. A . llcelOH6Western £>. l lcelOH 6 Western Figure 10: Characterization of llcelOH .^ Proteins in fractions of llcelOH^ purified on a Ni++-NTA column, were separated on two 12.5% SDS-PAGE gels. The gels were either blotted onto Immobilon-P™ (A) and probed with CE10 monoclonal antibody, (further developed with alkaline phosphatase conjugated goat anti-mouse IgG and BCIP/NBT) or stained with Coomassie blue (B). The banding pattern indicates that even after boiling in SDS sample buffer, aggregates of the 16 kDa protein, llcelOH6, remain. 48 Results A B l l c e l O H e 47.5H! 32.5H 16.5H Figure 11: A COS-7 cell lysate far western blot probed with llcelOH0 or Hc. Proteins within a COS-7 cell lysate were separated by 12.5% SDS-PAGE. A positive control lane of llcelOH6 (+) was included. The gel was blotted onto Immobilon-P™ and sections were incubated in purified protein preparations of either A) HcelOH6 or B) H6. The blot was then visualized with the primary antibody, monoclonal CE10 and further developed as in Figure 10. The black appearance of the posititive control lane indicates the overexposure of the section of the blot including H6 and +. 3.7 Co-immunoprecipitation studies of the 7.5 kDa B 1 9 protein The conditions of immunoprecipitation allow protein interactions to occur with a more native form of protein than may be found after its 49 Results immobilization on a membrane following SDS-PAGE. Therefore, co-immunoprecipitation experiments were carried out to immunoprecipitate cellular proteins that may be interacting with the 7.5 B19 protein. Again the subtractive control method was utilized in which a 7.5DH6 experiment was run side by side with a D H 6 control experiment, thus identifying interactions presumably due to the 7.5 kDa protein moiety of the 7.5DH6 fusion protein. In this experiment, an 35S-labeled COS-7 cell lysate was precleared by incubation with D H 6 followed by incubation with the rabbit polyclonal antibody anti-7.5DH6. Resulting immune complexes were then precipitated with Immunoprecipitin™. The "cleared" lysate was used in a co-immunoprecipitation experiment, the results of which suggests that proteins within a COS-7 cell lysate may interact with the 7.5 kDa B19 protein (Figure 12). However, no assignment of molecular mass of these interacting proteins may be drawn from these results. 3.8 Transfection experiments In order to more closely represent the in vivo situation in which the 7.5 kDa protein may function, attempts were made to express this protein in COS-7 cells. Thus, after gentle lysis of the cells, the 7.5 kDa protein may be precipitated, bound to interacting cellular proteins. The vector pCMV807, which directs the expression of the 7.5 kDa protein and the control vector pCMV5 were transfected into COS-7 cells. Western blots (Figure 13) suggest that expression of the 7.5 kDa protein was low, most likely due to low transfection efficiency (data not shown). 3.9 Immunoprecipitation of the 7.5 kDa protein expressed in COS-7 cells Although expression of the 7.5 kDa protein was low in transfected COS-7 cells (Figure 13), one attempt was made to co-immunoprecipitate 35S-labeled 50 Results cellular proteins within a lysate of COS-7 cells expressing the 7.5 kDa protein. As expected, no bands corresponding to cellular proteins that may interact with the 7.5 kDa protein are shown (Figure 14). A B 7.5DH6 + -#1 #2 + #3 DHL Figure 12: Co-immunoprecipitation of a COS-7 cell lysate with the 7.5 kDa protein. Fresh 35S-labeled COS-7 cell lysate was precleared by incubation with DH6 followed by incubation with the polyclonal antibody anti-7.5DH6. The resulting immune complexes were precipitated with Immunoprecipitin™. Samples of this precleared lysate were then incubated with one of three protein solutions, 7.5DH6 (Panel A), DH6 (Panel B) or no protein (Panel C). After a second incubation with either anti-7.5DH6 sera ("+") , or prebleed sera ("-"), immune complexes were precipitated with protein A-agarose and washed. Samples were analyzed by separation on a 12.5% SDS-PAGE gel and subsequent exposure to a phosphorimager screen for 48hrs. The arrows indicate the general location of cellular proteins that potentially interact with the 7.5 kDa B19 protein, however no more specific information may be drawn from these results. 51 Results pCMV5 pCMV807 #1 #2 Figure 13: Expression of the 7.5 kDa B19 protein in COS-7 cells. Proteins within a lysate of COS-7 cells transfected with either the control vector pCMV5 (Lane #1) or the 7.5 kDa expression vector pCMV807, (Lane #2) were separated by SDS-PAGE and blotted onto Immobilon-P™. The blot was probed with antisera raised against the 7.5DH6 fusion protein and further developed as described in Figure 5. The band corresponding to the 7.5 kDa protein (Lane #2) is indicated. 52 Results #1 A pCMV5 CONTOL + # 2 47.5H 32.5H 2fH B pCMV807 # 3 mm •*v.u«*s*/!,-:,tt Figure 14: Co-immunoprecipitation of cellular proteins with the 7.5 kDa protein, expressed in COS-7 cells. COS-7 cells transfected with either a (A) pCMV5 control vector, or (B) pCMV807 which expresses the 7.5 kDa B19 protein were labeled with 35S-methionine and lysed. The lysates were incubated with either (-) prebleed sera or (+) anti-7.5DH6 sera and protein/antibody complexes were precipitated with protein A-agarose. Protein samples were separated by 12.5% SDS-PAGE and exposed to a phosphorimager screen for 28 hours. Bands corresponding to labeled interacting cellular proteins were not observed. 53 Results proteins were utilized as described below (Figure 17). These experiments suggest that at least two K-562 cellular proteins of approximately 85 kDa and 26 kDa may interact with the 11 kDa B19 protein. Figure 15: Affinity column experiment: Chromatography of an 35S-labeled COS-7 cell lysate on 7.5DH,. or DH£ bound Ni^-NTA columns. In one column, 7.5DH6 (A, Lane #1) was bound to Ni++-NTA resin and unbound protein was removed by washing. A second column using DH6 (B, Lane #1) was similiarly prepared. An 3SS-labeled COS-7 cell lysate was passed through each column to allow any interacting cellular proteins to bind 7.5DH6 or DH6. The columns were washed (Lanes #2 - #4) and proteins were eluted with imidazole. Fractions of both the 7.5DH6 and DH6 eluants (Lanes #5) were collected and samples were separated by 12.5% SDS-PAGE. After a 24 hour exposure on a phosphorimager screen no potential interacting cellular proteins were detected. 55 Results A: 7.5DPL Bl DHL Eluant Eluant #1 #2 #3 #4 #5 #1 #2 #3 #4 #5 Cellular Proteins Figure 16: Affinity column experiment: Chromatography of an 35S-labeled K-562 cell on 7.5DH, or DH,. bound Ni^-NTA columns. In one column, 7.5DH6 was bound to Ni++-NTA resin and washed. (A, Lane #1) In second column DH6 was bound and washed. (B, Lane #1) An 35S-labeled K-562 cell lysate was passed through the columns to allow any interacting cellular proteins to bind the 7.5DH6 or DH 6 proteins. The columns were washed (Lanes #2 - #4) and then eluted with imidazole. Fractions of the 7.5DH6 or the DH6 eluants (Lanes #5) were collected and samples were separated by 12.5% SDS-PAGE. After a 24 hour exposure to a phosphorimager screen comparison of the results showed two potential interacting cellular proteins, as indicated by arrows. 56 Results 3.13 Affinity column experiment: An 35S-labeled COS-7 cell lysate probed for proteins that interact with the 11 kDa B19 protein An affinity experiment, similiar to that described above (section 3.13) was carried out in which COS-7 cells were substituted for the K-562 cells used previously. This was done to determine if the potential interacting proteins expressed in K-562 cells (Figure 17) may also be expressed in COS-7 cells and to identify other cellular proteins that may interact with the 11 kDa protein. It is shown (Figure 18) that a COS-7 cell protein around 26 kDa, as well as those of higher molecular weight, potentially interact with the 11 kDa B19 protein. 3.14 Batch style affinity experiments with the 11 kDa B19 protein After optimizing the expression of GST-11 (data not shown), batch style affinity experiments were run in order to further investigate K-562 cellular proteins interacting with the 11 kDa B19 protein. Batch style affinity experiments are carried out with free affinity resin within a tube instead of settled at the bottom of a column. This allows more stringent washing and elution of proteins bound to the affinity resin. The silver stained gel (Figure 19) suggests that K-562 cellular proteins may interact with the 11 kDa B19 protein. This interaction is substantiated by the phosphorimage of this gel, (Figure 20) in which only the 35S-labeled K-562 lysate proteins are visualized. The data within these two figures suggest that at least two proteins of approximately 85 kDa and 27 kDa interact with the 11 kDa B19 protein (indicated by arrows). 57 Results A l G S T - 1 1 Eluant G S T Eluant unconc cone unconc cone #1 #2 #3 #4 #5 #1 #2 #3 #4 #5 Potential Interacting Cellular Proteins Figure 17: Affinity columns: Chromatography of an 35S-labeled K-562 cell lysate on GST-11 or GST glutathione-agarose columns. Two glutathione-agarose bead affinity columns with either GST-11 (A) or GST (B) were prepared and washed. A K-562 35S-labeled cell lysate was passed through the column and unbound proteins were washed off the column (Lanes #l-#3). A glutathione solution (GEB) was used to elute GST or GST-11 along with interacting cellular proteins. The eluted protein samples were concentrated, separated by 12.5% SDS-PAGE and exposed to a phosphorimager screen for 30 hours. Samples separated on the gel include all wash samples (Lanes #1 - #3), the nonconcentrated eluant (Lane #4) and the concentrated eluant (Lane #5). By comparing the concentrated eluant sample of GST-11 with that of GST, K-562 cellular proteins that potentially interact with the 11 kDa B19 protein can be discerned as indicated. 58 Results A : GST-11 B : GST Eluant Eluant #1 #2 #3 #4 #1 #2 #3 #4 Potential Interacting Cellular Protein Figure 18: Affinity columns: Chromatography of an 35S-labeled COS-7 cell lysate on GST-11 or GST glutathione-agarose columns. Two glutathione-agarose bead affinity columns with either GST-11 (A) or GST (B) were prepared and washed. A COS-7 3SS-labeled cell lysate was passed through the columns which were then washed (Lanes #l-#3). GEB was used to elute GST-11 or GST along with interacting cellular proteins. The eluted protein samples were concentrated, separated by 12.5% SDS-PAGE and exposed to a phosphorimager screen for 27 hours. Samples separated on the gel included all wash samples (Lanes #1 - #3) and the concentrated eluant (Lane #4). By comparing the concentrated eluant sample of GST-11 with that of GST, COS-7 cellular proteins that potentially interact with the 11 kDa B19 protein can be discerned as indicated. Also, several higher molecular weight bands are detected. 59 Results Post Resin Eluant Eluant Contaminant # 1 # 2 • # 3 # 4 ' # 5 # 6 m # 7 #8 , #9 #10 ,#11 #12 ,#13 #14 Potential Interacting Cellular Proteins Figure 19: Chromatography of a K-562 cell lysate incubated with GST or GST-11 bound glutathione-agarose resin using a batch affinity procedure. Silver stained gel. Please see the folloing page for the text. 60 Results Figure 19: Chromatography of a K-562 cell lysate incubated with GST or GST-11 bound glutathione-agarose resin using a batch affinity procedure. Silver stained gel. An 35S-labeled K-562 cell lysate was incubated with GST (even numbered lanes) or GST-11 (odd numbered lanes) bound glutathione-agarose resin using a batch affinity procedure (see Materials and Methods, section 2.6.7). Protein samples were analyzed by 12.5% SDS-PAGE and bands were detected by silver staining ( for detection of 35S see Figure 20). In the first two lanes, the purified affinity proteins GST (Lane #1) and GST-11 (Lane #2) were analyzed. These proteins are free of contaminating protein with the exception of a high molecular weight band in the GST-11 preparation (Lane #2). Samples of the final wash (Lanes #3 and #4) demonstrate the lack of protein in these samples. In the eluant sample (Lanes #5 and #6) the major proteins are GST and GST-11. In addition, potential interacting cellular proteins are indicated (arrows). Unfortunately, when these samples were concentrated, these proteins were lost (Lanes #7 and #8, #11 and #12). Also, further analysis of the resin following elution demonstrated that significant amounts of both GST and GST-11 are not eluted by washing with GEB (Lane #9 and #10). 61 Results #1 #21 #3 #4 175-8 3 -6 2 -47.5" 32.5H 25 16.5-6.5" Post Resin Eluant Eluant #5 #6 MW #7 #8. #9 #10 .#11 #12 . #13 #14 GST-11 GST Potential Interacting Cellular Proteins Figure 20: Chromatography of a K-562 cell lysate incubated with GST or GST-11 bound glutathione-agarose resin using a batch affinity procedure: Detection of 35S-labeled proteins using a phosphorimager. The silver stained gel in Figure 19 was exposed to a phosphorimage screen for 35 hours. Bands that appear in the GST-11 sample (Lanes #6 and #10) and not in the GST sample (Lanes #5 and #9) lanes, correspond to cellular proteins that may interact with the 11 kDa B19 protein. The position of migration of unlabeled GST and GST-11 is indicated. 3 .15 Scaled-up batch affinity experiment with the 11 kDa B 1 9 protein The batch affinity procedure that tentatively identified an 8 5 kDa and 2 7 kDa K - 5 6 2 cellular proteins as interacting with the 1 1 kDa protein, (Section, 6 2 Results 3.14) was scaled-up five-fold. This was done to repeat these results and to attempt to isolate enough cellular protein for micro-sequencing. Scaling-up this experiment enhanced bands corresponding to both the 85 kDa and 27 kDa cellular proteins, supporting the suggestion that they interact with the 11 kDa B19 protein. However, in both the silver stained gel (Figure 21) and its phosphorimage, (Figure 22) the band at 27 kDa was obscured by distortion of the gel. This was due to the large amount of GST protein eluted with the cellular proteins and subsequently loaded onto the gel. 3.16 Attempts to improve the purity of the GST-11 fusion protein preparation 3.16.1 Incubation of purified GST-11 with ATP followed by repurification on glutathione resin does not improve the purity of the GST-11 fusion protein Shown in the purified GST-11 fusion protein sample (Figure 19, Lane #2) is a protein of 70 kDa, presumed to be a contaminant. This protein, not present in the GST preparation, may be responsible for the cellular proteins found binding the GST-11 affinity matrix, but not the GST affinity matrix, (see Sections, 3.14 and 3.15) This contaminant was thought to be the bacterial chaperonin, DnaK, an ATP-dependent protein that binds improperly folded or foreign protein molecules in E.coli. In an attempt to rid the GST-11 preparation of this 70 kDa protein, an ATP incubation step was added to the purification protocol of GST-11. It can be seen (Figure 23) that purification of GST-11 followed by exchange of buffers, incubation with ATP and rebinding to glutathione-agarose, failed to remove the bands migrating at approximately 70 kDa from the GST-11 preparation. 63 Results Post Lysate Wash Glutathione Eluant Resin Unconc. 45x Cone. Eluant #1 #2 . mw . #3 #4 #5 #6 #7 #8 175-83-62^ 4 Potential Interacting Celluli r Proteins GST-11 GST Figure 21: Large scale (five fold) chromatography of a K-562 cell lysate on glutathione-agarose resin with bound GST or GST-11 fusion protein. Silver stained gel. Nonconcentrated eluant samples from the GST (Lane #3) or GST-11 (Lane #4) experiment, and concentrated (45 fold) eluant samples from the same experiments (GST, Lane #5 and GST-11, Lane #6) were separated by 12.5% SDS-PAGE and visualized by silver staining. The absence of proteins in the final wash, after incubation with K-562 cell lysate (Lanes #1 and #2), indicates that cellular proteins bound nonspecifically, were adequately washed from the column. Bands corresponding to potential interacting cellular proteins are shown in the GST-11 experiment (Lane #6) but not the GST experiment (Lane #5). GST protein, GST-11 fusion protein and potential interacting cellular proteins are indicated. 64 Results Post Lysate Wash #1 #2 . mw Glutathione Eluant 175-83-62-47.5-32.5-25 -16.5~ Unconc. #3 #4 45x Cone. #5 #6 Resin Eluant #7 #8 Potentia Cellular Interacting Proteins -GST-11 -GST Figure 22: Large scale (five fold) chromatography of a K-562 cell lysate on glutathione-agarose resin with bound GST or GST-11 fusion protein: Detection of 35S-labeled proteins using a phosphorimager. See Figure 21 legend for details. Note the appearance of prominent bands corresponding to 85 kDa and 27 kDa 35S-labeled K-562 cellular proteins binding to GST-11 (Lane #6), but not GST (Lane #5). Unfortunately, the large band of GST protein (Lane #5, see silver stained gel, Figure 22) obscures the 27 kDa band. 65 Results Eluant Post Elution Resin Figure 23: Purification of GST-11: Rebinding of GST-11 to glutathione-agarose resin after ATP incubation and buffer exchange. Both GST (Lane #1) and GST-11 (Lane #2) were purified on glutathione-agarose. The glutathione buffer was exchanged and both proteins were incubated with ATP/MgS04/Tris (see Materials and Methods Section, 2.6.3) before repurification. Recovery of GST (Lane #3) and GST-11 (Lane #4) from the second affinity purification column was poor, presumably due to the lack of elution from this second resin binding step. A large amount of both GST (Lane #7) and GST-11 (Lane #8) remained on the resin after the final elution. In addition, the repurified GST-11 preparation contains the same high molecular weight contaminants (Lane #8) found in the initial purification (Lane #2). 6 6 Results 3.16.2 ATP incubation of the centrifuged bacterial cell sonicate prior to binding of glutathione resin decreased the amount of 70 kDa contamination in the GST-11 preparation One method to rid a protein preparation of DnaK (70 kDa) contamination is to incubate the bacterial cell sonicate in an ATP/MgS0 4 solution, prior to binding GST fusion proteins to glutathione resin.1 2 However, when this method was used, increased degradation of GST-11, as well as a concomitant decrease in the amount of 70 kDa protein contamination, was observed (Figure 24). It is not yet established that these are independent events. 3.17 Western blot of GST-11 A western blot experiment was carried out to determine if the 70 kDa contaminating protein may be an aggregate of the GST-11 fusion protein. This blot (Figure 25) supports such a theory, as a band corresponding to a 70 kDa protein is recognized by the rabbit polyclonal antibody raised against a synthetic peptide corresponding to partial sequence of the HkDa peptide. Since the immunizing antigen was a synthetic peptide, there should not be antibodies in the serum that recognize bacterial chaperonins. 3.18 Batch affinity experiment: An 35S-labeled K-562 cell lysate incubated with sepharose beads covalently linked to GST or GST-11 In order to obtain a significant amount of cellular protein interacting with GST-11 without large amounts of GST or GST-11 contaminating the cellular protein and distorting the gel pattern (compare Figure 20 and 21), both GST and GST-11 were covalently linked to cyanogen bromide (CNBr) activated sepharose beads. This experiment is as yet inconclusive ( Figure 26 and 27). 67 Results Figure 24: Affinity purification of GST-11 on glutathione-agarose resin after incubation with ATP. The GST-11 fusion protein was treated as described in the text, (Section, 3.18) affinity purified, separated by 12.5% SDS-PAGE and silver stained. Incubation of the sonicate prior to purification on glutathione-agarose resin appeared to dramatically decrease the amount of the 70 kDa contaminating protein (Lane #2), when compared to purification without an ATP incubation step (Lane #1). A GST control was included (Lane #5). 68 Results M W GST-11 175-83-62-47.5-32.5H 25^  GST-11 16.5H Figure 25: Western blot of GST-11. A GST-11 preparation was separated by 12.5% SDS-PAGE and blotted onto Immobilon-P™. The primary antisera used to probe this blot was rabbit anti-HkDa antisera, raised against a synthetic peptide corresponding to amino acids 58-86 (PNTKDIDNVEFKYLTRYEQHVIRMLRLC) of the 11 kDA B19 protein.75 This blot was further developed as described in Figure 5. It can be seen that a band around 70 kDa is recognized by this antibody. 69 Results Prelysate Characterization Eluant 47 . 5 H 32.54. 25^ 16.54 Contaminants GST-11 G S T Figure 26: Silver stained SDS-PAGE gel of a sepharose affinity column covalently bound to GST and GST-11. Samples of sepharose coupled GST and GST-11 were characterized before incubation with 35S-labeled K-562 cell lysate (Lane #1 and Lane #2, respectively). After incubation of the resins with K-562 lysate and washing, proteins that remained bound to the resin were eluted with SDS reducing sample buffer and separated on a 12.5% SDS-PAGE gel. Silver staining of this gel suggests that the GST and GST-11 fusion proteins were eluted from the sepharose. Also, no proteins other than contaminating 70 kDa and 80 kDa proteins elute from the GST-11 (Lane #4) but not the GST (Lane #3) resin. Samples analyzed were not concentrated. 70 Results G S T GST-11 #1 #2 32.5- ' * 25-16.5- . 6.5- , , w , , Figure 27: Phosphorimage of the SDS-PAGE gel of the sepharose bound GST and GST-11 affinity experiment. There is no indication of 35S-labeled cellular proteins eluted with GST-11 (Lane #2) that did not elute with GST (Lane #1). 3.19 Growth factor receptor-bound protein 2 (Grb2) interacts with the 11 kDa protein in far western experiments To determine if Grb2 is able to bind the 11 kDa protein in a far western experiment, GST-11, GST and the positive control protein GST-PD (containing an SH3 ligand) were separated by 12.5% SDS-PAGE and transferred to Immobilon-P™. The membrane was incubated with GST-HA-Grb2 and the presence of bound GST-HA-Grb2 was subsequently detected using the anti-HA monoclonal antibody (Figure 29). The Grb2 containing protein interacts with the GST-11 and GST-PD proteins and not GST alone. 71 Results A western blot was performed to demonstrate that GST-11 is not recognized by the monoclonal antibody used to detect bound GST-HA-Grb2 in the far western experiment. GST-11 is detected by a polyclonal antibody that recognizes GST but not by the anti-HA monoclonal antibody. (Figure 28) B: anti-GST anti-HA GST-11 GST-HA-PD mw GST-11 GST-HA-PD #4 No GST-11 "detected ST-HA-PD 16.54 Figure 28: Western blots of GST-11 with anti-GST or anti-hemagglutinin. The fusion proteins GST-11 (Lanes #1 and #3) and a positive control GST-HA-PD (Lanes #2 and #4) were separated by 12.5% SDS-PAGE and transferred to Immobilon-P™. Sections of the blot were incubated with either a primary polyclonal antibody that detects GST (A) or a primary monoclonal antibody that detects the HA tag (B) .This blot suggests that GST-11 is not recognized by the monoclonal antibody used to visualize the Grb2/GST-ll far western. (The section of the blot probed with anti-GST was developed as described in Figure 5 and the section probed with anti-HA was developed as described in Figure 10.) 72 Results A: Anti-GST #1 #2 #3 GST-11 B: Anti-Hemagglutinin #4 #5 h47.5 32.5 K6.5 Figure 29: GST-11 interacts with Grb2 on a far western blot. Protein preparations of GST-11 (Lane #1 and #4), GST (Lane #2 and #5) and the positive SH3 ligand control, GST-PD (Lane #3 and #6), were separated by 12.5% SDS-PAGE, blotted onto Immobilon-P™ and incubated with a solution of purified GST-HA-Grb2. Sections of the blot were incubated with either primary polyclonal antibody anti-GST (A) or monoclonal anti-HA (B). All three proteins were visualized with the anti-GST antisera as expected (A), but only GST-HA-Grb2 (Lane #6) and GST-11 (Lane #4) were detected with the anti-HA antibody. The negative control GST (Lane #5) was relatively undetected by the anti-HA antibody. (The section of the blot probed with anti-GST was developed as described in Figure 5 and the section probed with anti-HA was developed as described in Figure 10.) 73 Results 3.20 The yeast two hybrid system produced no positive clones with either the 7.5 kDa of 11 kDa proteins Over 8 million or 9 million primary transformants were screened for interactions between proteins encoded by a human bone marrow cDNA library using the 11 kDa, or the 7.5 kDa protein respectively. Of more than 900 11 kDa and 450 7.5 kDa transformants that passed the first screening stage, no authentic positive clones were identified. 74 Discussion IV Discussion 4.1 Highlights of this study This study describes experiments conducted to identify functions of the small 7.5 kDa and 11 kDa proteins expressed by the virus B19. Because previous studies indicated these functions may be mediated through interactions with host cellular proteins, the ultimate goal of this study became the identification, isolation and characterization of such proteins. While many experiments failed to identify these cellular proteins, some results suggest further experiments are warranted. In affinity experiments, molecular weights of cellular proteins that appear to interact with the 7.5 kDa, or the 11 kDa protein were determined and in one case tentatively identified. The most promising of these proteins include one of 85 kDa and others ranging from 26 kDa to 30 kDa (Figure 20). In addition, a protein named Son of Sevenless (Sosl) was identified in the literature to have an SH3 ligand almost identical to that of the 11 kDa protein. Because Grb2 had been previously shown to interact with Sosl, 7 6 this finding prompted far western experiments to be conducted to study the possibility of an interaction between Grb2 and the 11 kDa B19 protein. These studies suggest that the 11 kDa protein may be capable of interacting with the cellular protein, Grb2. If such an interaction occurs in vivo, this could be a mechanism through which B19 may modulate its host cell environment. 4.2 Confirmation and extension of previous binding studies of the 7.5 kDa protein Previous far western experiments indicate that 55 kDa and 43 kDa COS-7 cellular proteins potentially interact with the 7.5 kDa B19 protein.22 In this study, similiar experiments were conducted that confirmed these previous results (Figure 6 and Figure 7). 75 Discussion Because the cytoskeletal proteins vimentin and actin, are known to migrate by SDS-PAGE at approximately 55 kDa and 43 kDa, respectively, western blot experiments were performed that tentatively identify vimentin as the 55 kDa band (Figure 8). These same studies also suggest that the actin present in the COS-7 lysate probed, migrates at approximately 47 kDa. This is a higher molecular weight than the band corresponding to the 43 kDa protein that appears to interact with the 7.5 kDa B19 protein in far western experiments (Figure 6). The apparent binding of vimentin by the 7.5 kDa protein is most likely not meaningful. Vimentin is an abundant cell protein that forms filaments which span the cell, increasing its tensile strength. During mitosis, vimentin undergoes structural differentiation and its polymerization into filaments dissolves as it forms aggregates within the cytoplasm.52 Without further evidence that vimentin interacts with the 7.5 kDa protein, the interaction found by far western blots may be assumed due to the abundance of vimentin and its denatured state when immobilized on a blot. Further studies did not substantiate the interaction of the 7.5 kDa protein with a 55 kDa cellular protein corresponding to vimentin. Both i n vitro (Figure 12) and in vivo (Figure 14) co-immunoprecipitation studies failed to confidently indicate an interaction with a 55 kDa protein, or proteins of other sizes. However, affinity column experiments utilizing the 7.5DH6 fusion protein with Ni + + -NTA resin (Figure 16), suggest interactions of the 7.5 kDa B19 protein with proteins within a K-562 cell lysate. Based on studies with the 11 kDa protein, further investigations of the 7.5 kDa protein should probably utilize GST fusion proteins rather than His 6 fusion proteins. 4.3 Far western experiments fail to identify proteins that interact with the 11 kDa B19 protein Unlike the 7.5 kDa protein, far western experiments utilizing GST-11 (Figure 9) and 11H6 (data not shown) failed to suggest that the HkDa protein 76 Discussion interacts with any immobilized K-562 or COS-7 cellular proteins. It is probably not unexpected that far western experiments failed to demonstrate such interactions if it is assumed that all 11 kDa protein interactions are mediated through its SH3 ligand motif. SH3 ligand/domain interactions appear not to be demonstrable in far western experiments in which the SH3 domain containing protein is immobilized/ 6 possibly due to a failure of denatured SH3 domains to interact with SH3 ligands. In this study it was the SH3 domain proteins that were immobilized. However, far western blots (Figure 11) suggest interactions do occur between the HcelOH 6 fusion protein and cellular proteins as discussed earlier, (see Section, 3.6) Therefore, it may be instructive to construct a celOH6 negative control and repeat the HcelOH 6 far western experiment to see if these interactions are specific to the 11 kDa moiety of the fusion protein (Figure 10). However, other experiments with the 11 kDa B19 protein appear much more promising (see below). 4.4 K-562 versus COS-7 cells: Approximation of B19 permissive host cells In order to more closely approximate the B19 permissive host cell, lysates of K-562 cells were studied in addition to COS-7 cells. K-562 cells are of a lymphoblast morphology and spontaneously differentiate into precursors of the erythroid, granulocytic and monocytic series.4 Although they do not differentiate into the mature erythroid progenitor cells known to be permissive for B19 infection,32 K-562 cells presumably approximate these cells better than COS-7 cells do. COS-7 cells are of fibroblast morphology and were derived from African green monkey kidney cells, transformed with SV40.3 4.5 Interaction of the 11 kDa B19 protein with COS-7 and K-562 cellular proteins Affinity column experiments appear to indicate that the 11 kDa protein interacts with at least two cellular proteins within a K-562 lysate (Figure 17) as well as one within a COS-7 lysate (Figure 18). The cellular protein around 26 77 Discussion kDa tentatively shown to interact with the 11 kDa protein, may be found in both COS-7 and K-562 lysates, as a band around 26 kDa was observed in far western experiments using both K-562 and COS-7 cell lysates (Figure 17 and Figure 18). Although this observation may suggest a nonspecific interaction, this band could also correspond to Grb2, a 25 kDa protein shown to interact with the 11 kDa protein by far western blotting (Figure 29). The higher molecular weight 85 kDa K-562 cellular protein (Figure 17) not only appears to bind in affinity column experiments, but also in batch style affinity experiments (Figure 19 and Figure 20). Hence, further investigation of this interaction is warranted. In order to better evaluate the results of batch affinity experiments, (Figure 19 and Figure 20) these protocols were scaled-up five fold and the eluant was concentrated. The scale-up version (Figure 21 and Figure 22) resulted in a more definitive assignment of an interacting protein of 85 kDa. However, a large amount of GST eluted from the column and distorted the SDS-PAGE gel banding pattern, preventing observation of the lower molecular weight 30 kDa protein. 4.6 Contamination of the GST-11 protein preparation While evaluating the possibility of recovering and sequencing interacting cellular protein from an isolated band of an affinity experiment, it was noted that the GST-11 protein preparation (Figure 19, Lane #2) was contaminated. GST-11 was shown to contain proteins that are not present in the GST preparation. In order to be sure that the differential binding of cellular proteins shown between GST and GST-11 is due to the 11 kDa moiety of GST-11, this preparation must be free of contaminants not found in the GST preparation. 78 Discussion 4.7 Preparation of contaminant free GST-11 To achieve a contaminant free GST-11 preparation, different variations on the GST-11 purification scheme were attempted. It was proposed that DnaK could be the contaminating 70 kDa band. DnaK is a 70 kDa chaperonin protein of E.coli that binds improperly folded or foreign proteins to aid with their refolding or proteolysis. It has been shown that an ATP/MgS0 4 incubation step may result in purification of a DnaK free, GST fusion protein preparation.12 This ATP incubation step was added at three steps of the GST-11 purification protocol with varying success. First, GST-11 was purified on glutathione resin and eluted with GEB. This purified protein was incubated with an ATP/MgS0 4 solution and repurified with fresh resin. This scheme did not reduce the amount of the 70 kDa contaminating protein (Figure 23, Lane #8). In fact, the second purification step did not result in recovery of GST-11 (Figure 23, Lane #2). In a different approach, in which GST-ll-bound resin was incubated with ATP/MgS0 4 prior to GEB elution, no decrease in 70 kDa contamination was found (data not shown). Only when the ATP incubation step directly preceded the resin binding step was a significant decrease in the 70 kDa band found (Figure 24, Lane #2 versus Lane #1). Note however, this procedure also resulted in a larger increase in proteins around 30 kDa and 32.5 kDa, assumed to be degradation products of GST-11. 4.8 The 70 kDa contaminating protein of GST-11 preparations may be an aggregate of GST-11 When an ATP incubation step was added to the GST-11 purification protocol, the intensity of bands representing putative degradation products of GST-11 increased. However, this increase appeared to be substantially greater than the corresponding decrease in the GST-11 band (Figure 24). The possibility that the contaminating 70 kDa band may in fact be an aggregate of GST-11 was therefore investigated. , 79 Discussion Rabbit polyclonal antisera "J" was raised against a synthetic peptide corresponding to a sequence within the 11 kDa B19 protein (see text, Figure 25). In a western blot probed with antisera "]" (Figure 25), the GST-11 protein is recognized at 70 kDa, in addition to 40 kDa and 53 kDa. This indicates that the 70 kDa protein may not be DnaK, but instead an aggregate of GST-11. The 11 kDa protein was previously shown to form aggregates when Hcel0H 6 was separated by SDS-PAGE under the same denaturing and reducing conditions (Figure 10). To further investigate this, western blots probed with antibody directed against DnaK could definitively refute, or at least support this hypothesis. 4.9 GST-11 and GST sepharose batch affinity experiments remain inconclusive In order to avoid the distortion (Figure 22 and Figure 23) found in the SDS-PAGE gels of large volume GST-11 affinity experiments, both GST and GST-11 were covalently linked to CNBr-activated sepharose beads. This should allow elution of interacting cellular proteins without elution of GST or GST-11 fusion protein. However, samples of GST- and GST-ll-bound sepharose used in this experiment indicate significant amounts of contaminating protein in the GST-11 fusion protein sample bound to the sepharose (Figure 26, Lane #2). This may indicate that more stringent purification of GST-11 is necessary before binding the fusion protein to activated separose. Unfortunately, samples of the SDS sample buffer eluant do not suggest any 11 kDa interactions (Figure 27). In addition, these samples contain a large amount of GST or GST-11. This protein was eluted due to the reducing condition of the elution buffer. Nonreducing elution buffer should be used to avoid cleavage of GST or GST-11 from the sepharose beads. The fact that cellular proteins were shown to be bound to the column (Figure 27), although nonspecifically, indicates that the post lysate wash step was not too stringent. When this experiment was repeated using a larger amount of protein 80 Discussion and cell lysate and 4% SDS nonreducing elution buffer no interaction cellular proteins were indicated.(data not shown) Unfortunately concentration of the eluant was unsuccessful. This experiment should be modified to allow concentration of a large volume of eluant. Utilization of a low pH elution buffer, neutralized before concentration, may facillitate such a concentration step. 4.10 The Yeast Two Hybrid System The Yeast Two Hybrid System used in this study utilizes GAL4 binding domain (BD) and activation domain (AD) plasmids that were designed with truncated ADH1 promotors driving expression of the fusion proteins. When C L O N T E C H constructed this promotor they included an adjacent fragment of pBR322, which unknowingly acts as a transcriptional enhancer in yeast. When CLONTECH incorporated this promotor into the pGADIO based vectors used in this experiment, the enhancing fragment of pBR322 was lost. This resulted in very low expression of fusion proteins, undetectable in western blots of yeast extacts.10 Although the two hybrid system is designed to detect interactions between proteins of low abundance, if more two hybrid screening is to be done with the 11 kDa and 7.5 kDa proteins, use of plasmids with a stronger promotor is suggested. This could increase the activation of the genes used for selection of yeast with interacting proteins, making the selection of positive clones easier. These positive colonies would grow faster and be more easily distinguished from the background of false positive clones. 81 Discussion 4.11 GST-11 appears to interact with Grb2 in the context of far western experiments The 11 kDa protein of B19 contains an SH3 ligand motif similar to one found in the guanine nucleotide exchange factor, Son of Sevenless (Sosl).133 Figure 30: Comparison of the SH3 ligand sequences of the 11 kDa protein of B19 and Human Sosl. mSosl N-terminal - P P P V P P R - C-terminal B19- l lkDa N-terminal - R P P V P P R - C-terminal Note that all but the N-terminal proline of the mSosl motif, is identical to that of the 11 kDa protein (Figure 30). This similiarity provided incentive to conduct far western experiments that demonstrate the 11 kDa B19 protein binds Grb2, in vitro (Figure 29). Sosl has been shown to act as a positive regulator of the Ras protein, an upstream regulator of MAP kinases. MAP kinases are believed to play a central role in cell cycle regulation.18 Although the result suggesting that the 11 kDa protein interacts with Grb2 is preliminary, it opens the door to speculation about how the 11 kDa protein may affect host cell cycle events to enhance the B19 replication cycle. This result also justifies further study into the HkDa / Grb2 interaction, in vitro and in vivo. Additional support for the hypothesis that the 11 kDa B19 protein may interrupt Ras signaling, is found in a study in which Ras signaling was monitored in cells transiently expressing the Grb2 binding, C-terminal domain of Sosl. It was shown that downstream events of Ras signaling, including cell proliferation, cell differentiation and the ERK MAP kinase signaling pathways were interrupted.119 Since the C-terminal portion of Sosl expressed in these experiments contains the SH3 ligand mimicked by the 11 82 Discussion kDa B19 protein, these results may indicate that the 11 kDa B19 protein may influence the cell signaling pathways in a similiar manner. Also of interest are subcellular fractionation studies that demonstrate the presence of Grb2 in the nucleas.121 These results locate Grb2 in the same cellular space as the 11 kDa B19 protein. Also shown in this study is an association between Grb2 and heterogeneous nuclear ribonucleoprotein C,' presenting another important interaction that the B19 11 kDa protein may interrupt. 4.12 Future directions The investigations into the 11 kDa protein show promise of revealing a function of this protein. The affinity columns using pure GST-11 will likely result in confident isolation of interacting cellular proteins, if in fact they exist. Further investigation into the Grb2 / 11 kDa protein interaction should include affinity columns, utilizing either glutathione-agarose or activated sepharose systems, as discussed previously. By utilizing monoclonal antibodies that recognize Grb2, the 27 kDa protein shown to interact with the 11 kDa protein may be probed to investigate if it may be Grb2. In addition, bidirectional mammalian expression vectors could be used in K-562 or COS-7 cells to attempt co-immunoprecipitation of over- and equally- expressed Grb2 and 11 kDa proteins. The yeast two hybrid system could also be employed to quickly substantiate this interaction. However, a yeast two hybrid system with the promotor modifications mentioned above should be used instead of the system used in this study. 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