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Characterization of six monoclonal antibodies against the Minute Virus of Mice NS-1 protein, and the… Yeung, Douglas Edward 1990

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C H A R A C T E R I Z A T I O N OF SIX M O N O C L O N A L ANTIBODIES AGAINST THE M I N U T E VIRUS OF M I C E NS-1 PROTEIN, A N D THE USE OF ONE IN THE I M M U N O A F F I N I T Y PURIFICATION OF NS-1 EXPRESSED IN INSECT C E L L S By D O U G L A S E D W A R D Y E U N G B.Sc , The University of British Columbia, 1987 A THESIS SUBMITTED FN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Biochemistry) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A April 1990 © Douglas Edward Yeung, 1990 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 Biochemistry The University of British Columbia Vancouver, Canada Date A p r i l 19, 1990  DE-6 (2/88) ii I. Abstract Six mouse monoclonal antibodies have been isolated which react against a bacterial fusion protein containing amino acids 364 to 623 of the NS-1 protein of the prototype strain of the Minute Virus of Mice (MVMp). A l l six were found to be of the IgG class of antibodies; five being IgGi and the sixth being IgG2a. By immunoblot analyses, these antibodies all recognize an 83 kDa protein found only in MVM-infected mouse fibroblast cells, leading to the assumption that they are all NS-1 specific. Further evidence for this assumption is obtained from indirect immunofluorescence studies showing all but one of the mAbs react against a nuclear protein found in MVM-infected cells. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen. For the six monoclonal antibodies, four distinct epitopes were found (A - D). Three were clustered in a 16 amino acid region near the carboxy-terminal of the bacterial fusion protein, while the fourth was slightly more toward the amino-terminal side. Competition ELISAs against a 25 amino acid NS-1 specific peptide confirmed the mapping of the A epitope recognized by the CE10 and A C 6 monoclonal antibodies. Also in this thesis, the characterization of a N S - 1 fusion protein and a non-fused N S - 1 protein expressed in insect cells by recombinant baculoviruses is also described. The latter, a full-length N S - 1 protein designated N S - 1 A C » w a s found to be an 84 kDa cytoplasmic protein. This protein was immunoprecipitated by all six monoclonal antibodies. A CE10 monoclonal antibody immunoaffinity column was employed in the single-step purification of N S - 1 Ac from insect cells. Four elution methods (alkaline, peptide, 6 M guanidinium, and acid) were examined and the best purification was obtained using the acid elution. iii II. Table of Contents I. Abstract ii II. Table of Contents iii i n . List of Tables v IV. List of Figures v i V . List of Abbreviations viii VI . Acknowledgements xii VII. Introduction 1 A . Parvoviridae 1 B. The Autonomous Parvoviruses and the Minute Virus of Mice 2 C. The NS-1 Polypeptide 5 D . The Baculovirus Expression System 9 E . Project Purpose 12 VIII. Materials and Methods 15 A . Materials 15 B. Bacterial Strains, Cells, Viruses, and Media 16 1. Plasmids 16 2. Bacteria 18 3. Hybridomas 18 4. M V M and A9 cells 19 5. Baculovirus and Sf9 cells 20 C. Purification of lacZ/NS-1 22 1. Mini-lysates 22 2. Large scale purification 22 D . Production and Purification of Monoclonal Antibodies 23 1. Production of monoclonal antibodies 23 2. Sub-typing of monoclonal antibodies 23 3. Purification of monoclonal antibodies 23 E . ELISAs 24 F . SDS Polyacrylamide Gel Electrophoresis 25 G. Immunoblots 25 H . Immunofluorescence 26 I. Epitope Mapping 27 1. Basic Molecular Cloning Techniques 27 2. Attachment of the Termination Oligomer 28 3. Nested Deletions 30 4. Sequencing 31 J. Immunoprecipitations 32 K Immunoaffinity column construction 33 L . Immunoaffinity purification of CP/NS-1 expressed in Sf9 cells 33 M . Immunoaffinity purification of N S - 1 A C expressed in Sf9 cells 34 1. Elution by peptide and alkali 34 2. Elution by 6M guanidinium chloride 35 3. Elution by acid 35 DC. Results 36 A . Characterization of the mAbs 36 B. Mapping of the epitopes 40 C. Recombinant baculoviruses expressing NS-1 53 1. AcSec 53 2. AcNS-1 58 D. Immunoprecipitation of [35S]-methionine labelled N S - 1 A C 63 E . Purification of NS -1 Ac using a CE10 immunoaffinity column 68 X . Discussion 75 A . Monoclonal antibodies against M V M NS-1 75 B. Recombinant baculoviruses 78 C. Immunoaffinity purifications 82 XI. Literature Cited 85 X H . Appendices 94 A . Appendix 1 94 B. Appendix H 98 III. List of Tables Table 1: Distinctions between the three genera of the family Parvoviridae Table 2: Immunoblot reaction of carboxy-terminal deleted /acZ/NS-1 proteins versus the monoclonal antibodies vi IV. List of Figures Page Figure 1: Map of the M V M p genome. 4 Figure 2: The construction of pUC19/D. 12 Figure 3: Coomassie blue-stained gel of /acZ/NS-1 and 37 immunoblots using the six monoclonal antibodies. Figure 4: Coomassie blue-stained gel of mock- and MVM-infected 39 cell lysates, and immunoblots using the six monoclonal antibodies. Figure 5: Immunofluorescence time-course study of MVM-infected, 41 synchronous L A 9 cells using the CE10 monoclonal antibody. Figure 6: Immunofluorescence in MVM-infected L A 9 cells using the 42 CE10, AC6, BE2, EA2, and CH10 monoclonal antibodies. Figure 7: The construction of pUC19/D.t and its unidirectional 44 deleted clones. Figure 8: Immunoblots of the six monoclonal antibodies against 45 thirteen deletion clones of pUC19/D.t. Figure 9: Predicted positions of the epitopes of the six monoclonal 47 antibodies on lacZ/NS-l and on M V M NS-1. Figure 10: Immunoblots of the CH10 and BC4 monoclonal antibodies 49 against a NS-l /NS-2 fusion protein. Figure 11: Titration curves of the six monoclonal antibody solutions 50 used in the competition ELISAs. Figure 12: Graphs of the peptide 1 competition ELISAs showing 52 absorbance at 405 nm versus the competitor concentration in each well. Figure 13: Coomassie blue-stained gel of mock-, A c N P V - and 54 AcSec-infected total cell lysates. Figure 14: Immunofluorescence time course study of AcSec-infected 56 Sf9 cells using the CE10 monoclonal antibody. vii Figure 15: Coomassie blue-stained gel and immunoblot using the 57 CE10 monoclonal antibody, of fractions taken during the immunoaffinity purification of CP/NS-1 expressed in insect cells. Figure 16 Immunoblot of mock-, AcNPV-, and AcSec-infected Sf9 cell fractionation study Figure 17: Coomassie blue-stained gel of mock-, AcNPV- , and AcNS-1-infected Sf9 cell lysates, and immunoblots using the CE10 monoclonal antibody. Figure 18: AcNPV- and AcNS-1 infected Sf9 cell fractionation study. 59 61 Figure 19: Immunofluorescence time course study of AcNS-1- 62 infected Sf9 cells using the CE10 monoclonal antibody. Figure 20: Immunoprecipitation of AcNS-1- infected cell lysates 64 using the six monoclonal antibodies. Figure 21: Immunoprecipitation of AcNS-1-and AcNPV-infected cell 65 lysates using the CE10 mAb and an anti-TP sera, and double immunoprecipitation studies on CE10 mAb immunoprecipitated AcNS-1 infected Sf9 cell lysates using the CE10 mAb and an anti-TP sera. Figure 22: Coomassie blue-stained gel and immunoblot using the 67 CE10 monoclonal antibody, of fractions taken during the immunoaffinity purification of N S - 1 A C expressed in insect cells and eluted by peptide and alkali. Figure 23: Coomassie blue-stained gel and immunoblot using the 70 CE10 monoclonal antibody, of fractions taken during the immunoaffinity purification of N S - 1 A C expressed in insect cells and eluted by 6M Guanidinium chloride. Figure 24: Coomassie blue-stained gel and immunoblot using the 72 CE10 monoclonal antibody, of fractions taken during the immunoaffinity purification of N S - 1 A C expressed in insect cells and eluted by acid (pH 2.5). List of Abbreviations A28O absorbance at 280 nm A405 absorbance at 405 nm a a amino acid (s) A c N P V Autographa californica nuclear polyhedrosis virus Amp ampicillin bp base pair (s) B S A bovine serum albumin cm centimeter CNBr cyanogen bromide CO2 carbon dioxide dATP deoxyadenosine triphosphate D E A E diethylaminoethyl dGTP deoxyguanosine triphosphate (IH2O autoclaved, distilled water D M E M Dulbecco's Modified Eagle's Medium D N A deoxyribonucleic acid (s) DTT dithiothreitol E. coli Escherichia coli E D T A ethylenediaminetetra-acetic acid E L I S A enzyme-linked immunosorbent assay E V excluded virus F C S fetal calf serum G gauge g gram (s) H G P R T - hypoxanthine-guanine phosphribosyl transferase deficient ix h.p.i. hours post-infection h.p.r. hours post-release Ig immunoglobulin lie" isoleucine deficient I M D M Iscove's Modified Dulbecco's Media kb kilobase K Q potassium chloride kDa kiloDalton KH2PO4 potassium phosphate, monobasic KOAc potassium acetate 1 litre (s) liq N2 liquid nitrogen M molar mA milliampere (s) mAb monoclonal antibody M C S multiple cloning site MgCl2 magnesium chloride MgSC>4 magnesium sulfate min minute ml millilitre (s) m M millimolar mm millimetre (s) MO I multiplicity of infection M V M Minute Virus of Mice M V M p Minute Virus of Mice, prototype strain NaCl sodium chloride NaDOC sodium deoxycholate X Na2HP04 sodium phosphate, dibasic NaN3 sodium azide N a O H sodium hydroxide (NH4)2S04 ammonium sulfate nm nanometer (s) NP-40 Nonidet P-40 O R F open reading frame O V occluded virus P4 M V M p promoter at 4 map units P38 M V M p promoter at 38 map units pfu plaque forming unit (s) pmol picomole p - N P P para -nitrophenyl phosphate rccmlg rabbit anti-mouse immunoglobulin RNase ribonuclease rpm revolution (s) per minute RT room temperature SDS sodium dodecyl sulfate sec second (s) t time Tris tris (hydroxymethyl) aminomethane U unit (s) V volt (s) v / v volume to volume ratio W watt (s) w / v weight to volume ratio x g times gravity xi Z11SO4 zinc sulfate M-g microgram (s) [il microlitre (s) °C degree (s) Celsius VI. Acknowledgements First and most of all, I would like to thank my supervisor, Dr. Caroline Astell, for giving me the opportunity and the means to work in her lab. With her correct mix of guidance, advice, encouragement, and independence I was able to take this project (and my golf game) as far as I did. Secondly, I would like to thank the many colleagues who have passed through this lab: Roland, Matt, Caroline, Jan, and Rosemary thanks for all the help (especially with the analytical balance)! To those still in the lab, thanks for all the ideas, assistance, and your ability to put up with me. To Grant, thanks for a project. Thirdly, I owe a large debt of gratitude to the various students, faculty, and staff of the Biochemistry department at U.B.C. whom I have prodded and bothered for help, equipment, supplies, or participation in intramural teams. Thanks for all the friendship, memories, and support. Finally, I'd like to thank the two other members of my graduate committee, Dr. R. Molday and Dr. P. Bragg for their suggestions and reading of this thesis; Dr. I. Clark-Lewis for synthesizing the peptides; Dr. P. Tattersall and S. F. Cotmore for their gifts of various sera against M V M proteins; Dr. J. W. Bodnar for the anti-TP polyclonal sera; Dr. R. Molday for the 2B2 monoclonal antibodies; Dr. F. Tufaro and his lab, and Dr. S. Gillam and her lab for their advice and harassment; and Michael "Doc" Weis for the painfully long hours he spent protecting the E . M . lab teaching me how not to blow up the Zeiss. "Without the heart, there can be no understanding between the mind and the hand." Anonymous 1 VII. Introduction A . Parvoviridae The family Parvoviridae consists of a group of physically similar viruses that are among the smallest known D N A viruses. These viruses infect a wide range of animals from insects to the higher vertebrates, including humans. They have been identified in many important veterinary diseases generally causing fetal and neonatal abnormalities by destroying rapidly dividing cell populations. In contrast, the human parvovirus, B19, causes a rather mild rash known as erythema infectiosum (Anderson et al. 1984) and a type of aplastic crisis in people with hemolytic anemias (Tijssen et al. 1990). Apart from the clinical and pathological importance of the parvoviruses, it was thought that the small size of its D N A genome and its limited coding capacity would allow these viruses to serve as a simple model for D N A replication and transcription. Rather than succumbing to expectation, these viruses have proven to be much more complex than first thought. Parvoviruses have characteristic non-enveloped, isometric particles, 20 - 25 nm in diameter enclosing a linear, single-stranded D N A genome that is 4.7 to 6.0 kb long (Siegl 1984, Tijssen et al. 1990). The particles exhibit icosahedral symmetry and do not appear to contain carbohydrates or lipids. In terms of total mass, the D N A comprises 19 - 32% and the capsid proteins the rest. Recent reports indicate that there is, in fact, a very small proportion of viral encoded enzymes packaged as well (Cotmore and Tattersall 1989, Faust et al. 1989). The small size of the viruses makes them relatively resistant to environmental extremes, showing stability from pH 3 to pH 9, at 56 °C for at least 60 minutes (Siegl et al. 1984). The family Parvoviridae is divided into three genera, Parvovirus, Dependovirus, and Densovirus. Classification into these three genera is dependent on three factors: 1) autonomous or helper virus-dependent replication, 2) encapsidation of mainly the negative sense strand or both positive and negative sense strand in equal proportion, and 3) host being vertebrate or invertebrate (see Table 1). Now that more is known about the 2 parvoviruses, several of the distinctions outlined in Table 1 no longer absolutely define the three genera (reviewed by Tijssen 1990). Throughout the rest of this thesis, the genera will be referred to by their common names, autonomous for Parvovirus, helper-dependent for Dependovirus, and densonucleosis for Densovirus. Table 1: Distinctions between the three genera of the family Parvoviridae Parvovirus -autonomously replicating -mainly negative sense strand packaged -vertebrate hosts Dependovirus -helper virus-dependent -both positive and negative sense strands packaged -vertebrate hosts Densovirus -autonomously replicating -both positive and negative sense strands packaged -invertebrate hosts B . The Autonomous Parvoviruses and the Minute Virus of Mice The Minute Virus of Mice (MVM) is a representative member of the autonomously replicating parvoviruses. Typical of all parvoviruses, it has a linear, single-stranded D N A genome with palindromic sequences at the 3' and 5' termini capable of forming hairpin duplex structures, but unlike the helper-dependent subgroup of viruses, the 3' palindrome and the 5' palindrome are non-identical (Bourguignon et al. 1976). Like most of the members of the autonomous parvoviruses, M V M does not require co-infection with helper virus for productive infection, packages predominantly negative sense D N A (i.e. D N A which is the complement of that encoded into RNA) greater than 99% of the time (Cotmore and Tattersall 1987), and infects a vertebrate host. The relatedness of the members of the autonomous sub-group becomes even more apparent in the genomic organization and polypeptides encoded. To date the autonomous parvovirus genomes sequenced, or partially sequenced include the prototype strain of M V M (MVMp), the immunosuppressive strain of M V M (MVMi) , the rat virus H - l , bovine parvovirus (BPV), the human parvovirus B-19, canine parvovirus 3 (CPV-2), and feline panleukopenia virus (FPV). Comparisons of the sequences indicate that these parvoviruses show a high degree of similarity in their genomic organization with respect to hairpin sequences, promoter locations, protein gene position, intron positions, and polyadenylation signals (reviewed by Rhode III and Iversen 1990). For M V M , the highest degree of relatedness is shown with CPV, FPV, and the rodent parvoviruses (Cotmore and Tattersall 1987). Comparison of all seven genomes reveals a similar positioning of two large blocks of open reading frame (ORF) spanning approximately 80% of the genome, and a number of smaller ORFs (Shade et al. 1986). The major, right-hand ORF encodes the capsid polypeptides and the major left-hand ORF encodes the nonstructural polypeptides. SDS-polyacrylamide gel analysis of M V M virions revealed the existence of three capsid proteins designated VP-1, VP-2, and VP-3 (for yjral proteins 1 through 3 respectively). The apparent molecular weights of these proteins were 83 kDa, 64 kDa, and 62 kDa respectively (Tattersall et al. 1976). VP-1 and VP-2 have been shown to be primary translation products, while VP-3 was the result of proteolytic cleavage of VP-2 (Tattersall, Shatkin, and Ward 1977). The largest capsid protein, VP-1, makes up approximately 18% of total capsid protein; the rest being accounted for by the two smaller capsid proteins, although in variable proportions (Tattersall 1978). The exact structure of the capsomeres and the exact number of capsomeres forming the M V M virion is still unknown. Identification of two nonstructural polypeptides distinct from the capsid proteins was obtained by in vitro translation of viral mRNAs (Cotmore et al. 1983). Three classes of M V M transcripts have been found, RI , R2, and R3 (Pintel et al. 1983) (see Figure 1). The R3 transcripts code for the capsid proteins and are initiated from a promoter at 38 map units (P38). Note that in M V M , one map unit corresponds to -51 nucleotides (Astell et al. 1983, Astell et al. 1986). The RI and R2 transcripts are initiated by a second promoter found at 4 map units (P4), with the R2 transcripts containing a large splice of 1476 nucleotides not found in the RI class (Pintel et al. 1983). A l l three classes of transcripts contain a small splice of approximately 150 nucleotides at 43 map units and have polyadenylation sites clustered near 4 map units 0 25 50 75 100 I 1 ¥ • 1 1 nucleotides Figure 1: Map of the M V M p genome The cytoplasmic transcripts of M V M , denoted R l , R2, R3, and R3', are aligned beneath a line diagram of the viral D N A strand which illustrates the extent of the 3' and 5' terminal hairpin palindromes and the positions of the two promoters at map units 4 and 38. The sequences encoded in the viral proteins NS-1, NS-2, VP-1, and VP-2 are illustrated with the reading frames expressed in each part of the molecule shade-coded ( • = 1 , • =2 , • =3). Figure modified from S.F. Cotmore and P. Tattersall, 1987. 5 93 map units (Clemens and Pintel 1987, Pintel et al. 1983). Translation of the viral rriRNAs in rabbit reticulocyte lysates and immunoprecipitation of the products with sera from animals infected with different autonomous parvovirus serotypes indicated the presence of two nonstructural polypeptides, NS-1 and NS-2. The NS-1 protein co-migrated with VP-1 (MW =83 kDa) and NS-2 at an apparent molecular weight of 25 kDa (Cotmore et al. 1983). C. The NS-1 Polypeptide The large nonstructural protein was shown to be the primary translation product of the R l transcripts (Cotmore et al. 1983). The first A U G codon in the R l transcripts occurs at nucleotide 261 and the first in-frame stop codon at nucleotide 2277, just prior to the small splice site (Astell et al. 1983, Ben-Asher and Aloni 1984). Bacterial fusion proteins have been made including regions within the R l transcript, in all three reading frames, and subsequently used to generate polyclonal antibodies. Antibodies directed against regions in the predicted large open reading frame immunoprecipitate NS-1 from in vitro translations making it highly unlikely that NS-1 contains sequences downstream of the 2277 termination codon, initiates at an A U G codon downstream of the 261 codon, or contains an undetected splice resulting in a reading frame shift (Cotmore and Tattersall 1986b, Cotmore and Tattersall 1987). Although the NS-1 protein has an apparent molecular weight of 83 kDa, the predicted protein encoded by the R l transcripts would be 672 amino acids long and have a molecular weight of only 77 kDa. Sub-cellular fractionation and immunoprecipitation of MVM-infected L A 9 cells identified the predominant NS-1 species as a nuclear-localized phospoprotein with an apparent molecular weight of 83 kDa and a highly phosphorylated form running at 85 - 88 kDa (Cotmore and Tattersall 1986a). Figure 1 illustrates the positions of the transcripts and the proteins encoded by the M V M p genome. To maximize the use of a limited size genome, the transcription strategy of M V M utilizes overlapping ORFs. NS-1 and NS-2 both have the same initiation codon and share a common amino-terminus, but due to the large splice present in the R2 transcripts and 6 the resulting reading frame shift, NS-2 has a carboxy-terminus different than NS-1. Similarly, the capsid proteins VP-1 and VP-2 are both translated from P38-initiated mRNAs, but due to alternative splicing patterns possible at the small splice site, the proteins initiate at different A U G codons starting at nucleotides 2287 and 2795 respectively (Jongeneel et al. 1986, Morgan and Ward 1986). The NS-1 polypeptides of parvoviruses show a high degree of structural conservation and amino acid homology. Sera from animals infected with canine parvovirus, porcine parvovirus, or the rodent parvoviruses, H - l , H-3, or K R V all recognize the M V M NS-1 molecule. On the contrary, capsid polypeptides from different viruses rarely cross-react, indicating a higher degree of structural conservation of the NS-1 polypeptides than the capsid proteins. (Cotmore et al. 1983). The leftward open reading frame has been shown by heteroduplex mapping (Banjeree et al. 1983) and by amino acid homology (Shade et al. 1986) to be highly conserved among the parvoviruses. The region of highest homology corresponds to a 405 nucleotide sequence (nt 1428-1833 in M V M ) that, at the amino acid level, shares greater than 99% homology with H - l , 95.5% with FPV, 41% with the human parvovirus B19, and 51% with the helper-dependent parvovirus A A V (Astell et al. 1987, Cotmore and Tattersall 1987). A sequence corresponding to the purine triphosphate binding site consensus sequence, G(X)4GKT/s(X)5_6I/L/v> has been identified in this region (Anton and Lane 1986, Astell et al. 1987). Downstream of the 405 nucleotide conserved region, the homology between parvoviral NS-1 molecules drops to -16% among B19, M V M , and A A V , but remains relatively high at -60% among the rodent parvoviruses. The highly conserved nature of NS-1, both in primary amino acid sequence and in antigenic structure points to the importance of NS-1 structure and function. The role of NS-1 is not well understood although several experiments indirectly suggest possible functions for NS-1. First, co-transfection experiments with plasmids containing the NS-1 gene and a reporter gene under the control of either of the two viral promoters have shown that NS-1 up regulates the P38 capsid promoter and down regulates its own P4 7 promoter and several cellular promoters (Doerig et al. 1990, Doerig et al. 1988, Rhode III 1985, Rhode III and Richard 1987). As would be expected with this type of regulation, MVM-infected cells show the P38-derived R3 transcripts appearing slightly after the P4-derived R l and R2 transcripts (Clemens and Pintel 1988, Tunis et al. 1988). The R3 transcripts are also approximately four times more abundant than the R l and R2 transcripts late in the infection cycle (Pintel et al. 1983). Contrary to the expected pattern, expression of NS-1 and the capsid proteins is not found to be temporally regulated. They are detected almost simultaneously early in infection (Cotmore and Tattersall 1987) although later in infection, levels of NS-1 expression fall and capsid expression rises. In light of all of these studies, it has been proposed that NS-1 is a transcriptional regulator. The second postulated function of NS-1 is in viral D N A replication. First, frameshift mutations which affect the NS-1 gene, but not the NS-2 gene, were made in an infectious plasmid containing the entire genome of M V M and found to prevent viral duplex D N A replication (Merchlinsky et al. 1983). In the related rodent virus, H - l , similar mutations also prevented D N A replication, but this function could be restored by co-transfection with a second plasmid containing an unmutated NS-1 gene (Rhode III 1982). Second, when some transformed cell lines are infected with M V M , there is increased viral D N A synthesis correlating with an increase in NS-1 production even though the full productive infection appears to be blocked at a later stage (van Hille et al. 1989). Third, a fraction of the NS-1 molecules have recently been found covalently bound to the 5' terminal of the M V M genome and certain replication intermediates (Astell et al. 1983, Cotmore and Tattersall 1988, Gunther and Tattersall 1988). The current model for the replication of the M V M genome proposes the requirement for a site-specific nickase/helicase function to be provided, similar to that of the gene A protein of 0X174, (Astell et al. 1983, Astell et al. 1985, Cotmore and Tattersall 1988). The fact that NS-1 is bound to the viral genome makes it an ideal candidate for this function. In the helper-dependent parvovirus A A V , the rep proteins are analogous to the nonstructural proteins of the autonomous parvoviruses, and have been shown to 8 specifically recognize and bind to the terminal hairpins (Ashktorab and Srivastava 1989, Im and Muzyczka 1989). Finally, Bodnar et al. (1989) have shown that M V M D N A is associated with the nuclear matrix of infected cells through the 5' end and a 60 kDa host encoded nucleolar antigen designated TP (Bodnar et al. 1989, Walton et al. 1989). Presumably, NS-1 is involved in this interaction as well. Bodnar suggests that the nucleoli are the functional sites of viral D N A replication with the M V M D N A being targeted to the nucleolar attachment sites by NS-1 (Walton et al. 1989). Two other functions have been postulated for NS-1. The first is a role in packaging of the viral D N A into the virions. NS-1 was found on the outside of the virions, still covalently bound to the 5' end of the genome (Cotmore and Tattersall 1989, Faust et al. 1989). Removal of the NS-1 molecule by nuclease digestion or protease digestion did not affect the infectivity of the virions, ruling out that NS-1 is involved in receptor recognition (Cotmore and Tattersall 1989). Therefore, Cotmore and Tattersall believe that NS-1 may be directly involved, or at the very least complicate, the packaging mechanism. The second function is as an ATPase. The purine nucleotide binding sequence mentioned previously shows the highest degree of homology to the large T antigen of polyoma viruses and SV40 (Anton and Lane 1986, Astell et al. 1987). This region in SV40 large T antigen has been implicated in an ATP-dependent helicase function, leading to the postulation that M V M NS-1 may have a similar function (Astell et al. 1987). The large size of NS-1 may permit it to be a multi-functional protein, with phosphorylation playing a role in the regulation of these activities. In order to determine the functions of NS-1, several attempts have been made to overexpress NS-1. Attempts at producing mammalian cell lines that constitutively express H - l NS-1 under the control of its own P4 promoter failed. H - l NS-1 under the control of the P38 promoter was constitutively silent, but when induced by exogenous NS-1, the P38/NS-I cell line expressed normal levels of NS-1 transiently prior to cell death (Rhode III 1987). Attempts at producing stable HeLa cell lines capable of expressing the left hand open reading frame of B19 also failed unless the NS gene was mutated to prevent synthesis of the protein 9 product (Ozawa et al. 1988). Similar results were obtained for M V M NS-1 (Moir et al. 1987, Pintel et al., unpublished results, St. Amand and Astell, unpublished results). These results have led to the belief that NS-1 may be the cytotoxic agent responsible for MVM-mediated cell lysis, and that high concentrations of NS-1 impair long-term viability of cell lines expressing NS-1. D. The Baculovirus Expression System The baculovirus expression system developed by Smith et al. (1983) has been used to produce high levels of foreign proteins in insect cells. This helper-independent viral vector system utilizes the strong polyhedrin gene promoter of Autographa californica (multiple nucleocapsid) nuclear polyhedrosis virus (denoted A c N P V or A c M N P V ) to produce functional, post-translationally modified proteins (reviewed by Luckow and Summers 1988b, Miller 1988). The advantages of this system are that it carries out post-translational modifications, does not employ the use of transformed cells or transforming elements, and that the virus has been found to infect only invertebrates and is therefore of relatively low safety hazard. The family Baculoviridae has over 400 known members characterized by an enveloped, rod-shaped nucleocapsid enclosing an 80 - 200 kb double-stranded D N A genome (reviewed by Doerfler and Bohm 1986, Granados and Federici 1986). Subclassification of these viruses is based on the mechanism by which the viral envelope is obtained. The subgroup A viruses (or nuclear polyhedrosis viruses) obtain their lipid envelope by an intranuclear envelopment process and are capable of being occluded into proteinaceous nuclear organelles known as polyhedra. These occlusion bodies contain several virions each (Miller 1988) and will be discussed later with regard to the baculovirus life cycle. Further classification into single nucleocapsid polyhedrosis viruses (SNPVs) or multiple nucleocapsid polyhedrosis viruses (MNPVs) is possible depending on the number of nucleocapsids packaged in each lipid envelope. A member of the latter group, AcNPV, is the best characterized of all the 10 baculoviruses. The nuclear polyhedrosis viruses produce two types of infectious virions, excluded virus (EV) and occluded virus (OV). The E V form is produced first, budding off from infected cells at around eighteen hours post-infection (h.p.i.). This form of the virus is responsible for the systemic infection of the insect via cell to cell transmission through the hemolymph. The OV, on the other hand, is responsible for the horizontal transmission from insect to insect usually via the ingestion of contaminated leaves. Late in infection, OVs are packaged into a paracrystalline matrix which protect the viruses from environmental factors. A highly alkaline environment such as an insect's midgut breaks down the polyhedra and releases the viruses. There, they then infect the new host (Granados and Federici 1986). The major component of the protective polyhedra is a 29 kDa polyhedrin protein (Rohrman 1986). The gene encoding this protein is classified as a 'very late' phase gene, with transcription occurring 18-24 h.p.i. and protein expression being detected from 20 h.p.i. to 70 h.p.i. when cell lysis occurs. In insect cell culture, AcNPV forms greater than 30 polyhedra per nuclei and the polyhedrin protein consists up to 60% of total 'Coomassie blue-stainable' protein (Matsuura et al. 1987, Potter et al. 1976). Most baculovirus expression systems work by the substitution or insertional inactivation of the polyhedrin gene, placing foreign genes under the control of the strong polyhedrin promoter. In this case, only OV production is disrupted, not E V production. Because the polyhedrin gene is non-essential for viral replication and is a late-expressed gene, there is minimal pressure to eliminate foreign genes put in its place. (Miller 1988). A number of A c N P V transfer vectors have been designed for the insertion of foreign genes into the baculovirus genome. They all rely on the in vivo recombination of homologous regions in the vector with the A c N P V genome for the replacement of the polyhedrin gene with the gene of interest. The virus can be grown in insect cell culture in Sf9 cells, a clonal isolate of the IPLB-SF-21 A E cell line of Spodoptera frugiperda (fall army worm) (Summers and Smith 1987). The recombination event occurs during the co-transfection of the vector D N A 11 with viral genomic D N A into uninfected Sf9 cells. Cells infected with recombinant viruses can be selected for based on the lack of occlusion bodies in the nuclei of infected cells (occ phenotype) and the recombinant viruses can then be purified from these cells in the E V form (Summers and Smith 1987). A l l of the transfer vectors contain the polyhedrin flanking sequences including the upstream promoter sequences and the downstream poly-adenylation site, as well as a unique restriction site placed just downstream of the polyhedrin promoter (reviewed by Kang 1988, Luckow and Summers 1988b, Miller 1988). Various vectors differ in the amount of upstream and downstream flanking sequences retained. As with most other expression systems, the level of protein expression of individual foreign genes is highly variable. A number of factors have been found to affect protein expression levels in insect cells; the most well-studied being the upstream and downstream sequences required for high level expression (Luckow and Summers 1988a, Matsuura et al. 1987) . The region from -60 to -1 of the polyhedrin promoter appears to be sufficient for expression. The most popular baculovirus vector, pAc373 , deletes the -7 to +670 region of the polyhedrin gene (Smith et al. 1983), although higher levels of expression have been reported i f the -7 to +1 nucleotides are retained such as D. H . Bishop's p A c Y M l vector or M . D. Summers' new pVL941 vector (Matsuura et al. 1987, Summers 1988). The polyhedrin coding region itself does not appears to contain sequences required for high-level expression. The p A c Y M l vector actually contains a deletion from the +2 nucleotide to a point 13 nucleotides past the polyhedrin termination codon (Matsuura et al. 1987). Other factors found to affect protein expression are the leader and downstream sequences of the inserted gene. The length of the leader and trailing sequences (Kang 1989, pers. comm.), the ribosome binding site, and the sequence immediately upstream of the initiation codon (Miller 1988) appear to play some role in determining the level of expression. Whether or not these are the only factors affecting expression is not known and is being actively investigated. The efficiency of this system has been demonstrated by the very abundant expression of numerous non-invertebrate proteins in the invertebrate host. These diverse proteins range in source from bacteria [E.coli B-galactosidase, (Summers and Smith 1987)] to higher vertebrates [humans B-interferon (Smith et al. 1983)] and in size from 15.5 kDa [human interleukin-2 , (Smith et al. 1985)] to 160 kDa [HIV env, (Hu et al. 1987)]. Correct post-translational modifications have been shown for many of the proteins including phosphorylation, N-glycosylation, O-glycosylation, myristylation, and palmitylation (see reviews by Summers 1989, Kang 1988, Luckow and Summers 1988b, Miller 1988). Although these post-translational modification systems are present in insect cells, whether these systems operate identically to those in mammalian systems has yet to be proven. The Sf9 glycosylation pathway has been shown to process high mannose sugars in a manner similar to the mammalian processing pathway, but not as efficiently nor identically (Jarvis and Summers 1989, Kuroda et al. 1990). Correct proteolytic cleavage of recombinant precursor proteins, and of several mammalian signal sequences has also been seen, as has correct targeting of the proteins to cellular locales. It is therefore not surprising and in fact very encouraging that many of the expressed proteins retain their original antigenicity, immunogenicity, and biological activities (see reviews by Kang 1988, Luckow and Summers 1988b, Miller 1988). E . Project Purpose Prior to the beginning of this project, G. Brown constructed the pUC19/D plasmid (Figure 2), which contains the PstI D fragment of M V M cloned into the PstI site of pUC19 (Brown 1987). This fragment, spanning nucleotides 1349 - 2125 of the M V M p genome, codes for a fragment of NS-1 as well as partially overlapping exon 2 of the NS-2 gene. Due to the reading frame shift required for NS-2, the PstI D fragment is specific for NS-1 i f the correct NS-1 reading frame is maintained. Transformation of E.coli JM83 with the pUC19/D plasmid results in the expression of a 33 kDa B-galactosidase-NS-1 fusion protein NS-1[ \Z3 M V M reading frame 1 M V M reading frame 2 H I lacZ encoded amino acids Figure 2 The construction of pUC19/D. The sequences encoding NS-1 and NS-2 are shown above the line diagram of the M V M viral D N A strand illustrating the PstI sites in the genome. The reading frame of each expressed part of the molecule is shade-coded. Below the line diagram shows the position of the PstI D fragment into the unique PstI site within the multiple cloning site of pUC19 just downstream of the lacZ promoter. designated /acZ/NS-1 (Brown 1987). Brown purified this fusion protein from SDS-polyacrylamide gels and used it to immunize Balb/C mice for the production of monoclonal antibodies against /acZ/NS-1 (Yeung et al. 1990, manuscript in prep.) Six positive colonies secreting antibodies against lacZ/NS-l were isolated, two of which were subcloned to monoclonality by C. R. Astell. The other four colonies were frozen away at the primary subcloning stage. Concurrently, initial work on the construction and isolation of a NS-1 encoding baculovirus was carried out by R. Russnak of this laboratory. During the course of my work, the baculovirus project was continued by W. Chen and G. Wilson in this laboratory (Wilson et al. 1990, manuscript in prep.) This thesis will describe the characterization of the six monoclonal antibodies and demonstrate their use in the detection of M V M NS-1. As well, use of the antibodies in the characterization of a NS-1 fusion protein and of a full-length NS-1 protein, expressed in insect cells, wil l be described. One of the monoclonal antibodies (CE10) was purified from mouse ascitic fluid, coupled to cyanogen bromide-activated Sepharose 4B, and used in the immunoaffinity purification of both proteins from recombinant baculovirus-infected Sf9 cells. 15 VIII . Materials and Methods A . Materials A l l chemicals were analytical or reagent grade and were either from B D H Inc., Fisher Scientific, or Sigma Chemical Co. unless otherwise specified. A l l polyacrylamide gel electrophoresis reagents and protein molecular standards were obtained from Bio-Rad Laboratories; except for Ultrapure SDS, Ultrapure Tris, pre-stained High Molecular Weight Standards, and unstained High Molecular Weight Standards which were from Bethesda Research Laboratories; and Dalton Mark VII-L unstained molecular weight markers and SDS-6H high molecular weight standard mixture from the Sigma Chemical Co. E. M . grade paraformaldehyde was obtained from J B E M . Bactotryptone, yeast extract, and bactoagar were purchased from Difco Laboratories. Penbritin-1000 (ampicillin) was from Ayerst Laboratories. A l l cell culture reagents and chemicals were Sigma Chemical Co. tissue culture grade unless otherwise specified. Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle's Medium (DMEM), M e f D M E M , TC-100 medium, and T N M - F H medium were obtained from Gibco Canada Inc. as was the fetal bovine serum (FCS). D M S O was from the Eastman Kodak Co. Guinea pig anti-TP polyclonal sera was the kind gift of Dr. J. W. Bodnar (Northeastern University). The anti-rhodopsin mouse monoclonal antibody 2B2 was the generous gift of Dr. R. S. Molday (University of British Columbia). The mouse monoclonal sub-isotyping kit was from Hyclone Laboratories and secondary antibodies were purchased from Bethesda Research Laboratories or from Jackson Immunoresearch through Promega Biotec. Nitrocellulose and Optibind N C were obtained from Schleicher & Schuell. The 5-bromo-4-chloro-3-indoyl phosphate / nitro blue tetrazolium (BCIP/NBT) alkaline phosphatase development system was purchased from Bethesda Research Laboratories. Falcon MicroTest III flexible assay plates were from Becton-Dickson. [ 3 5S]-Methionine (1100 C i/mmol), cell labelling grade [35S]-Methionine (600 C i/mmol), 16 and [cc 3 2P]-dATP (3000 c i / m mol) were obtained from N E N Research Products. Deoxynucleotide triphosphates and a-phosphorothioate deoxynucleotide triphosphates were from Pharmacia P-L Biochemicals. Restriction enzymes, React buffers, RNase T i , and T4 D N A Ligase were obtained from Bethesda Research Laboratories. D N A Polymerase I Klenow fragment and Exonuclease III were purchased from Promega Biotec and SI nuclease from Pharmacia P-L Biochemicals. Sequenase, sequencing mixes, and sequencing reagents were from the United States Biochemical Corp. Ultrapure Agarose was also from Bethesda Research Laboratories. Sep-Pak Cis cartridges were obtained from the Millipore Corp., and acetonitrile was from the Aldrich Chemical Co. Geneclean was purchased from Bio lOl or Promega Biotec. Curix RP-1 film was from Agfa-Gevaert. DEAE-Sephacel, CNBr-activated Sepharose 4B, Protein A Sepharose CL-4B, and Protein G Sepharose 4 FF were from Pharmacia P-L Biochemicals. X - O M A T A R film was obtained from the Eastman Kodak Co. B . Bacterial Strains, Cells, Viruses, and Media 1. Plasmids Plasmid pUC19/D, was obtained from Grant Brown (this laboratory) and its construction has been described elsewhere (Brown 1987). Plasmids pAcRP6/AX and CPss /NSl /pAcYMl were constructed by Dr. Roland Russnak (this laboratory). Schematic diagrams of the construction of these two baculovirus transfer vectors, and the p A c N S l construct to be described later are given in Appendix I. For the construction of pAcRP6/AX, the 2.3 kb Hgal fragment of M V M was first isolated from a Hgal digest of pMM984, an infectious clone containing the entire genome of M V M (Merchlinsky et al. 1983). The ends of this fragment were filled-in with T4 D N A Polymerase and blunt-end cloned into the filled-in BamHl site of the baculovirus transfer vector, pAcRP6 (Matsuura et 17 al. 1986, Possee 1986) producing p A c N S l . Finally, this construct was digested with Xhol , filled-in with T4 D N A Polymerase, and religated creating a frame-shift mutation at the unique Xho l site in the NS-1 gene (refer to Appendix la). In order to construct CPss /NSl /pAcYMl , the 2.3 kb Hgal fragment of M V M was obtained by a Hgal digest of pMM984. The fragment's ends were filled-in with T4 D N A Polymerase and blunt-end cloned into the Smal site of pGEM-4Z producing NSl /pGEM-4Z. The preceruloplasmin signal peptide, encoding region was purified from the clone XhCPl (Koschinsky et al. 1986) in the following manner. First, the 1.2 kb EcoRI cDNA was cut out of the clone and then re-ligated back in the opposite orientation allowing purification of a 150 bp HincII/SstI fragment. This D N A piece contains the upstream regulatory sequences as well as the sequence encoding the first 47 aa of preceruloplasmin. The 150 bp fragment was used to replace the 161 bp Sstl/EcoRV fragment in NSl /pGEM-4Z. To move the new CPss/NSl construct into the transfer vector, p A c Y M l (Matsuura et al. 1987), the 2.3 kb Ssfl/ Hindl l l fragment was cut out and the ends filled-in with T4 D N A Polymerase allowing the fragment to be cloned into the Smal site of pUC1813 (Kay and McPherson 1987). This step was the equivalent of placing BamHI linkers onto the ends, facilitating cloning of the BarnHI CPss/NSl fragment into the BamHI site of p A c Y M l (refer to Appendix lb). This final vector was expected to encode a chimeric protein consisting of the preceruloplasmin signal sequence fused to NS-1 that is missing the first 41 aa of its amino-terminal. In vitro transcription and translation demonstrated synthesis of a protein slightly larger than full length M V M NS-1 (R. Russnak, pers. comm.). The plasmid pAcYMl /NS-1 was constructed by Dr. Wei Chen of this laboratory. Briefly, the 2.3 kb Hgal fragment of M V M was isolated from a Hgal digest of pMM984. BamHI linkers were ligated to this fragment and the resulting fragment was then cloned into the BamHI site of pUC19. The NS-1 encoding fragment was then transferred to p A c Y M l by excision using BamHI and ligation at the unique BamHI site in the baculovirus transfer vector (refer to Appendix Ic). 18 2. Bacteria A K-12 derivative, E. coli strain JM83 [ara, A(lac-proAB), rpsL(= strA), <f)80, lacZAM15 ] (Yannisch-Perron et al. 1985) was used to maximize expression of lacZ fusion proteins encoded by the pUC19 plasmid derivatives (Brown 1987). This strain was propagated in Y T media (8 S/i bactotryptone, 5 8/i yeast extract, and 5 S/\ NaCl) at 37 °C with vigorous shaking, or on Y T plates (YT media plus 15 8/i bactoagar) incubated 16 hours at 37 °C. Bacteria containing pUC19 plasmids were selected using ampicillin (Amp) in the plates or media at a concentration of 50 Mg/mi. 3. Hybridomas Six Balb/c splenocyte:NS-l hybridoma cell lines secreting antibodies against the lacZfNS-l fusion product encoded by the pUC19/D construct were isolated by G . Brown according to a modified method of Kohler and Milstein (Campbell 1984, Kohler and Milstein 1975). Two of the lines (CE10 and BE2) had been subcloned three times by limiting dilution while the other four (AC6, EA2 , CH10, and BC4) had not been subcloned. A l l were supplied in 0.5 ml aliquots stored under liquid nitrogen ( N 2 ) . Cells were thawed in a 37 °C water bath and the contents transferred to a 15 ml conical tube containing 10 ml of (IMDM). Cells were spun down [300 x g, 5 min, room temperature (RT)], the supernatant removed, and the cell pellet resuspended in 5 ml of I M D M + 5% FCS with Balb/c splenocyte feeder cells (see following). Hybridomas were then grown in plates at 37 °C and 5% C O 2 in a humidified incubator. Splitting of the cells was done by dilution using I M D M + 10% FCS without feeder cells. Spleen feeder cells were prepared using the methods of Campbell (1984). Using sterilized instruments an 8 - 12 week old Balb/c mouse was killed by cervical dislocation, washed in 70% ethanol, and its spleen removed to a sterile 10 cm petri dish. Once in the sterile hood, the spleen was washed twice with I M D M . In 5 ml of fresh media, the spleen was teased apart with two 18-G needles. The cell suspension was drawn into a sterile 5 ml syringe, passed one-way through a 20V2-G needle four times, and twice through a 22-G 19 needle with the cell suspension ending up in a sterile 15 ml conical tube. Feeder cells were spun for 5 min at 300 x g and then washed once with I M D M . Finally, the cell pellet was resuspended in 50 ml of I M D M + 10% FCS and incubated at 37 °C in a humidified CO2 incubator. The conditioned media and feeder cells were used either fresh or up to a maximum of seven days after preparation. The four uncloned hybridoma lines were subcloned three times by limiting dilution to assure monoclonality. The day before the cloning, 100 pi of feeder cell suspension were laid down per well in 96-well plates. On the day of the cloning, the hybridomas were counted using a Nebauer haemocytometer and diluted to 10 4 c e l l s / m l in I M D M . This dilution was then serially diluted down to 10 c e l l s / m i , 5.0 c e l l s /mb and 2.5 c e l l s / m i using I M D M + 20% FCS. Each of these dilutions were plated out at 100 ^ / w e i i in the 96-well plates containing the feeder cells and then incubated at 37 °C under normal growing conditions for 7 - 1 2 days. Wells with a single visible colony per well were assayed by ELISA or Western blot against lacZ/NS-l (see Materials and Methods sections E, F & G). Positive clones were gradually expanded up to >107 cells and reduced down to a 10% FCS concentration requirement. At this point the cells were subcloned again and/or frozen away. Hybridomas were frozen in I M D M + 20% FCS + 20% dimethyl sulfoxide in 0.5 ml aliquots at a concentration of 5 x 10 6 to 5 x 10 7 c e l l s / m i . Aliquots were then placed at -20 °C for 1 hour, -70 °C overnight, and then transferred to liquid N2 (-185 °C). 4. M V M and A9 cells The prototype strain of M V M was grown in a continuous mouse L-cell derivative, both originally supplied by Dr. Peter Tattersall (Yale University). These cells are A9 ouar/l 1 cells, a ouabain resistant derivative of the HGPRT- mouse fibroblast cell line A9 (Tattersall and Bratton 1983). Cells were grown in Dulbecco's Modified Eagles Medium (DMEM) + 5% FCS in a water jacketed incubator set to 37 °C and 5% CO2. Continuous cultures were split approximately once a week by trypsinization and dilution in D M E M + 5% FCS. The A 9 cells were grown asynchronously, or else synchronized by the double block 20 method of Cotmore and Tattersall (1987) as follows. The cells are grown in isoleucine deficient (He-) D M E M + 5% dialyzed FCS for 48 hours causing the cells to accumulate in the G 0 phase of the cell cycle. The cells are released from the isoleucine block while being exposed simultaneously to aphidicolin at a concentration of 5 V%/mi in D M E M + 5% FCS. At this point the cells can also be infected with virus at the appropriate multiplicity of infection (MOI) for each experiment. The result of this second block is that the cells, still somewhat asynchronous, leave G 0 and accumulate at the Gi -S boundary due to the inhibition of D N A Polymerase alpha by aphidicolin. Virus is taken up by the cells but not replicated because of the requirement for the cell to be in S phase. After 20 hours the cells are released from the second block by three successive 5 minute washes with D M E M . The cells are then returned to normal growing conditions as stated above. The start of the first wash is referred to as t = 0. Asynchronous cells were infected by a different method. In this case, infection occurs by overlaying the cells with virus at an appropriate MOI in the minimum volume necessary. The virus is allowed to attach to the cells for 1 hour at 37 °C with occasional rocking. At the end of this time, the overlay is aspirated off and the cells returned to normal growing conditions. The time of infection here refers to the point when the virus was first added. 5. Baculovirus and Sf9 cells. Sf9 cells (ATCC# CRL1711) are derived from a clonal isolate of Spodoptera frugiperda IPLB Sf21 A E cells (Summers and Smith 1987). These cells were the generous gift of Dr. C. Yong Kang (University of Ottawa) and of Dr. Frank Graham (McMaster University). The media used was either TC-100, or T N M - F H . A l l were supplemented with 10% FCS. Cells were propagated in closed tissue culture flasks or in plates placed inside a closed, humidified container. Both types of closed vessels were kept inside a 27 °C incubator. Monolayer cultures were split every two to three days as described by Summers and Smith (1987), except that they were split 1:3 in media without FCS and were allowed to attach to the plate or flask surface in the absence of FCS for 1 - 1V2 hours. Alternatively, spinner cultures were 21 maintained for large scale infections as described by Summers and Smith (1987) with two exceptions. First, TC-100 media was used instead of T N M - F H and second, cells were split 1:3 or 4 instead of 1:5. The wild type baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV) was the kind gift of Dr. C. Yong Kang (University of Ottawa). The recombinant baculoviruses AcSec and AcAXho were constructed by Dr. Roland Russnak (this laboratory), and AcNS-1 by Gary Wilson (this laboratory). AcSec, AcAXho, and Ac NS-1 were produced by homologous recombination of the wild type virus D N A with the plasmids C P s s / N S l / p A c Y M l , pAcRP6/AX, and pAcYMl /NS-1 respectively. Infection of Sf9 cells was similar to that described for M V M infection of A9 cells. Because there is no requirement of AcNPV for the cells to be in any specific phase of the cell cycle, the cells were always grown asynchronously. For infection of plates, the media was removed by aspiration and virus added for at least IV2 hours at 27 °C. For time course studies the virus overlay was then removed and the cells returned to normal media; otherwise, fresh media was added directly to the viral inoculum. Infection of spinner cultures was as described by Summers and Smith, 1987. The time of the addition of the virus is termed t = 0. Viral stocks were made by infecting plates or spinner cultures of Sf9 cells with the appropriate virus at an MOI of 0.1 - 1.0. After 72 h.p.i. the extracellular virus was harvested by collecting the media in conical tubes. Cells and cellular debris were removed by centrifugation for 10 min at 500 x g. Techniques for plaque assaying the virus stocks are given elsewhere (Summers and Smith 1987). For the labelling of Sf9 cells with [35S]-methionine, approximately 1 x 10 7 cells in 60 mm tissue culture dishes were infected as described above. Prior to labelling, the infected cells were gently pelleted (300 x g, 5 min, RT), washed once with Met" T N M - F H media, pelleted again, and resuspended in 2.5 ml Met" T N M - F H + 10% dialyzed FCS. Cell-labelling grade [35S]-methionine (>600 ^ C i / m mol) w a s added to a final concentration of 150 ^ C i /ml . After a 22 3 hr labelling period at 27 °C, the cells were gently pelleted as before, resuspended in TC-100 +10% FCS, and incubated at 27 "C. The time of the labelling post-infection is given in the Figure legends. C . Purification of tecZ/NS-1 1. Mini-lysates The lacZ/NS-l fusion proteins were expressed in E. coli JM83 as described (Brown 1987) except that only 1.0 ml of the 5.0 ml overnight culture was used to prepare the mini-lysates. 2. Large scale purification Large scale purification of the fusion protein is also detailed by Brown, 1987 and was employed with the following modifications. First, the pooled cell pellet was washed once with STE buffer (10 m M Tris-Cl pH 8.0, 100 m M NaCl, 1 m M EDTA) before being resuspended in lysis buffer. Second, after homogenization, the volume was increased to 10 ml with lysis buffer before collection by centrifugation. Third, after resuspension in sample buffer, the aliquots were incubated at 100 °C for 5 min and then stored at -20 °C. When needed, these aliquots were thawed and run on a 16 cm x 16 cm x 1.5 mm preparative, 4%/12% discontinuous SDS polyacrylamide gel at 100V for 18 - 20 hours at 4 °C. Fourth, after the /acZ/NS-1 band was excised, the gel piece was cut into four pieces, placed inside four 2" x 3/4" dialysis bags containing gel running buffer (123.8 m M Tris, 96 m M glycine), submerged in running buffer in a B R L Mini-Sub D N A Electrophoresis Cell, and electroeluted. The protein was subject to electroelution for 3 hours at 100V in a 4 °C cold room. The eluate was then concentrated in Centricon-10 microconcentrators at room temperature according to the manufacturer's instructions. Washes were performed with K B S buffer [3 m M KC1, 1.5 m M KH2PO4, 137 m M NaCl, 8.1 m M N a 2 H P 0 4 , 0.05% ( v/ v) Tween 20, 0.02% ( w/ v) NaN3]. The yield of protein was quantified as described by Brown, 1987. D. Production and Purification of Monoclonal Antibodies 23 1. Production of monoclonal antibodies Monoclonal antibodies were collected either as tissue culture supernatants or as ascitic fluid by the methods of Harlow and Lane, 1988. Ascitic fluid was stored at -70 °C until ready for use. 2. Sub-tvping of monoclonal antibodies Monoclonal antibodies were sub-typed by ELISA using a mouse monoclonal sub-isotyping kit according to the supplier's instructions. 3. Purification of monoclonal antibodies Purification of the monoclonal antibodies was performed in a two-step process involving ammonium sulfate [ ( N H ^ S C ^ ] precipitation and DEAE-Sephacel chromatography. A l l steps in the (NH4)2S04 precipitation were done at 4 °C as described by Harlow and Lane (1988) with the following alterations. First, ascitic fluid was thawed and diluted 1:3 with PBS buffer (KBS without Tween 20 and without NaN3) to reduce the viscosity of the solution. Secondly, the final pellet was resuspended in PBS buffer, but dialyzed against three changes of 0.01M sodium phosphate buffer (pH 8.0) overnight instead of PBS buffer. Ion exchange chromatography of the dialyzate was done according to the method of Zola (1987) using DEAE-Sephacel. On later purifications a 20 - 250 m M NaCl gradient was used rather than a 0 - 500 m M gradient. One tenth column volume fractions were collected and then analyzed by A280> SDS polyacrylamide gel electrophoresis, and ELISA (see Materials and Methods sections E & F). Salt concentrations in the fractions were determined by conductivity measurements. Monoclonal antibody containing fractions were pooled and the protein concentration determined using a Bio-Rad microassay based the method of Bradford (1976) modified by Bio-Rad laboratories. The pooled fraction was then either coupled to CNBr-activated Sepharose 4B as described later (see Materials and Methods section K) or lyophilized after a small sample had been tested to show that the mAb was in fact stable to lyophilization. DEAE-Sephacel was regenerated through sequential 0.5 N H Q and 0.5 N NaOH washes and then stored at 4 °C under 20% ethanol. E . E L I S A s For the ELISAs, 50 u.1 of lacZ/NS-l at 1 u g / m i in K B S buffer was added to the wells of a Falcon 96-well MicroTest III flexible plate and dried down for 2 hours at 65 °C. The plate was then washed once with distilled water. Wells were blocked with 200 pi of KBS + 5% FCS per well unless the antibody solution used was a tissue culture supernatant. If this was the case, blocking was not required due to the presence of FCS in the supernatant. Following one washing with K B S , 50 u.1 of antibody solution was added and incubated for 1 hour at room temperature. After washing the plate three times with K B S buffer, 50 pi of alkaline phosphatase-linked rabbit anti-mouse immunoglobulin (ramlg) was added, at the manufacturer's recommended dilution, for 1 hour. The plate was then washed as before and developed with 100 pi of p-nitrophenyl phosphate (p-NPP) solution [1 m g / m i in 10 m M diethanolamine, 0.5 m M MgCl2, 0.02%(w/ v) NaN3]. Color development was stopped after 45 min by the addition of 50 pi of 2 M NaOH. The A405 was read in a Titertek microtitre plate reader using the p-NPP solution with 2 M NaOH as the blank. For titration ELISAs, the procedure was the same except that dilutions were done in the wells. To do this, 100 pi of antibody solution was added to the first well of a row and 50 pi of blocking solution in the rest of the wells of the row. Serial doubling dilutions were made by removing 50 pi of solution from the first well, mixing it with the contents of the next well, and repeating this process across the row, finally removing 50 pi from the last well of the row. For competition ELISAs, the monoclonal antibody solutions were used at the dilution that gave 80% binding efficiency, as determined by the titration ELISA. One pi of the competitor at 50X final competitor concentration was added and mixed into the wells immediately after the monoclonal antibody solution. A l l steps following the addition of the primary antibody and the competitor were the same as for the regular ELISAs. Peptide 1 ( H 3 N - P L A S D L E D L A L E P W S T P N T P V A G T A - C O O H ) and peptide 2 ( H 3 N - 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 - C O O H ) were the generous gift of Dr. Ian 25 Clark-Lewis (Biomedical Research Centre, University of British Columbia). F. SDS Polyacrylamide Gel Electrophoresis SDS-polyacrylamide gel electrophoresis was performed using a Bio-Rad Mini-Protean II gel apparatus according to the manufacturer's instructions based on the method of Laemmli (1970). Gels were 0.75 mm thick with a 12% separating gel and a 4% stacking gel, and run at 200V for 45 - 60 minutes at room temperature. Alternatively, 8 cm x 14 cm x 0.75 mm gels were run on a Hoeffer SE600 apparatus at 30 mA constant current for 4 hrs. Proteins gels were stained and destained using Coomassie brilliant blue, and then dried down at 80 °C for 1 - 3 hours. G. Immunoblots Samples were subjected to SDS-polyacrylamide gel electrophoresis as described previously and transferred onto nitrocellulose paper, or Optibind by electrophoretic elution (Towbin et al. 1979) using a Bio-Rad Mini-Trans Blot transfer cell or a Bio-Rad Trans Blot II transfer cell according to the supplied instructions. The transfer buffer used was 25 m M Tris, 192 m M glycine, and 20% (v/v) methanol. Transfers were done at 150 mA constant current for 1 hour for mini-gels or 100 mA constant current for 2V2 hrs for the large gels. Blots were immunostained as described by Harlow and Lane, 1988. Blocking solution was 5% FCS in K B S buffer. The primary antibodies used were unlabeled tissue culture supernatants. The secondary antibody was either alkaline phosphatase-conjugated rocmlg or biotinylated goat anti-mouse immunoglobulin, diluted as instructed by the manufacturer. If the biotinylated secondary antibody was employed, an extra 30 min incubation with streptavidin-alkaline phospatase, according to the supplier's instructions, was included followed by washing with 5 changes of K B S . Development of the immunoblot was done using 5-bromo-4-chloro-3-indoyl phosphate and nitro blue tetrazolium. H . Immunofluorescence For immunofluorescence studies, cells had to be grown on or mounted on coverslips. 26 Acid-etched, 22 mm, circular coverslips were prepared by boiling in 1 M HC1 for 10 minutes, washing thoroughly with 3 changes of distilled water, and finally storing under 95% ethanol until needed. When required, the coverslips were moved to the tissue culture hood, dried and sterilized by flaming, and placed inside sterile, 35 mm tissue culture dishes. For M V M and A9 cell studies, 1.0 x 10 5 to 1.5 x 10 s cells were seeded per dish and incubated for 18 - 24 hours at 37 °C. Cells were then infected, or synchronized and infected, at an MOI of 0.1 to 1.0 P f u/cell- Parallel mock-infections were done using media without serum as the inoculum. For baculovirus and Sf9 cell studies, 1.0 x 10 6 to 1.5 x 10 6 cells were seeded and incubated for approximately one cell division. Again, cells were then infected at a MOI of 0.1 to 1.0 P f u/ Cell or mock-infected using media without FCS. Infection was allowed to proceed for the length of time stated in the Figure legends. At the appropriate time point, cells were fixed, permeabilized, and immunostained. Dishes were rinsed once with PBS-C (PBS buffer with 0.7 m M CaCl2, 0.5 m M MgCl2) and then fixed with 3.68% ( w / v ) E M grade paraformaldehyde in PBS for 10 minutes. Cells were rinsed with three changes of PBS-C followed by the standard wash protocol consisting of three changes of wash buffer (PBS-C +1% BSA) for 5 min each. Coverslips with the cells attached could be stored under wash buffer at 4 °C at this stage. The cells were permeabilized using 0.25% ( w / v ) Saponin in wash buffer. For permeabilization, the Saponin solution was left on top of the cells at room temperature for 45 minutes followed by the standard wash. Primary antibody in the form of a tissue culture supernatant was added for 1 hr at room temperature, again followed by the standard wash. Immunofluorescent staining of the cells was performed in one of two ways. The first method used was a one-step process with a rhodamine-conjugated goat anti-mouse IgG, diluted 1:99 parts PBS-C after it had been centrifuged (5 min, 12,000 x g, RT) to remove unconjugated rhodamine particles. This secondary antibody was incubated for 1 hour at room temperature and unbound antibody was removed using the standard wash protocol. The second method was a two-step process involving the use of a biotinylated goat anti-mouse 27 IgG diluted according to the supplier's instructions. This antibody was incubated the same way as the rhodamine-conjugated antibody in the one-step method, washed, then incubated for 30 min with Texas Red-conjugated streptavidin, and washed again. The coverslips were inverted and mounted on depression slides containing wash buffer. Coverslips were firmly seated onto the slide using a small amount of silicon grease, taking care not to trap air bubbles under the slide. Phase contrast light microscopy and fluorescence microscopy were done on a Zeiss Universal microscope equipped with an epi-fluorescence head. I. Epitope Mapping 1. Basic Molecular Cloning Techniques Many of the techniques used can be found in Maniatis et al. (1982) or Sambrook et al. (1989). These include methods used for: 1) isolation of single bacterial colonies, 2) growth, maintenance and preservation of bacterial strains, 3) small scale plasmid D N A isolation by alkaline lysis, 4) large scale plasmid D N A isolation by alkaline lysis, 5) purification of closed circular D N A by equilibrium centrifugation in cesium chloride-ethidium bromide gradients, 6) extraction of ethidium bromide with organic solvents, 7) preparation of frozen competent and calcium chloride competent E. coli, 8) electrophoresis of D N A in agarose and acrylamide gels, and 9) phenolxhloroform [50:50 ( v / v ) l extractions. The plasmid D N A isolations had the following modifications added. For the small scale isolations, two phenohchloroform extractions were included (Sambrook et al. 1989). In the large scale isolation (Maniatis et al. 1982), chloramphenicol and lysozyme were not used, the centrifugation after the potassium acetate addition was done in a Beckman Ti 45 rotor (30,000 rpm, 5 °C, 1 hr), and the final centrifugation was done in a Beckman T i 70.1 rotor (60,000 rpm, 20 °C, 16 hrs). D N A was precipitated using ethanol (Maniatis et al. 1982). A Vioth vol of 3M sodium acetate was added, i f necessary, to optimize the salt concentration of the D N A solution. This 28 was followed by 2 volumes of 95% ethanol. Precipitation was allowed to occur at 0 °C for 10 min after which the sample was spun down (12,000 x g, 7 min, 4 °C) and washed with 70% ethanol. The supernatant was discarded and the tube inverted on a paper towel to remove as much of the ethanol as possible. Finally, the D N A was dried in a vacuum desiccator for 5 minutes. Restriction enzyme digests were performed according to the supplier's instructions. Reactions were stopped using an appropriate amount of Ficoll gel loading buffer (Sambrook et al. 1989). D N A bands were resolved on 1% agarose gels in T B E buffer (Sambrook et al. 1989). 2. Attachment of the Termination Oligomer Two partially complementary synthetic oligomers were synthesized by Tom Atkinson (University of British Columbia) on an Applied Biosystems D N A Synthesizer. Oligomer # 1 was a 16-mer with the sequence, 5 ' -p -GATCCTAAGTAATTAG-3 ' . Oligomer # 2 was also a 16-mer, but with the sequence, 5 ' -AATTCTAATTACTTAG-3 ' . Crude oligomers were resuspended separately in 1 ml of TE buffer (10 m M Tris-Cl pH 8.0, 1 m M EDTA) + 500 m M NaCl. Two Sep-Pak Cis cartridges were equilibrated with 10 ml of acetonitrile followed by 10 ml of autoclaved, distilled water (dH20). Each oligomer was applied to one of the two columns using a 5 ml syringe over a period of 3 - 5 min, washed with 5 ml of 6H2O, and then eluted with 5 ml of 20% ( v/ v) acetonitrile. One ml fractions were collected. Concentration of the oligomers was determined by A260- Cuvettes were cleaned beforehand using hydrogen peroxide for 15 min, followed by a thorough washing with (IH2O. The molar extinction coefficients were calculated based on the sequence (Wallace and Miyata 1987) allowing spectrophotometric determination of oligomer concentrations. Fractions containing the oligomers were evaporated to dryness on a Savant Speed-Vac Concentrator for 2 hrs and stored at -20 °C. Both oligomers were resuspended separately in 6H2O at a concentration of 1 mM. Ten pi of purified oligomer # 1 and 10 pi of purified oligomer # 2 were added to 78.5 pi of ( I H 2 0 , 1 pi of 29 1 M Tris-Cl (pH 8.0), and 0.5 pi of 200 m M EDTA, in a 500 pi Eppendorf tube. The solution was vortexed and placed in a 70 °C water bath for 5 min. The tube was left in the water bath as it was turned off. Both the tube and water bath were allowed to slowly cool down to room temperature after which 20 pi aliquots of the annealed 'termination linkers' were stored at -20 °C. In order to replace the BamHl/EcoRl fragment in pUC19/D with the termination linker, 3.75 pg of pUC19/D was subjected to a double digestion with 20 U of BamHl and 20 U of E c o R l . After 2 hrs at 37 "C, the reaction mixture was heat-inactivated for 3 min at 68 °C and run on an 1% agarose T B E gel. The 3441 bp band was excised and the D N A was isolated using Geneclean following the supplier's instructions. The fragment was resuspended in 20 pi of dH20. A n aliquot of the annealed termination oligomers was thawed slowly at 4 °C to prevent strand separation, and ligated to the 3441 bp E c o R l / BamHl pUC19/D fragment using 2 pi of the fragment (-0.21 pmol), 2 pi of linkers (-20 pmol), 2 pi of 10 m M ATP, 2 pi of 150 m M DTT, 2 pi of 10X linker kinase buffer [660 m M Tris-Cl pH 7.6, 10 m M spermidine, 100 m M M g C l 2 , 2 m g / m i BSA] , 9 u.1 of d H 2 0 , and 1 Weiss Unit of T4 D N A ligase. The ligation was allowed to proceed for 4 hrs at room temperature. Frozen competent E. coli JM83 were transformed with 2 pi of the ligation mixture and plated on YT/Amp (50 u g / m i ampicillin). The plasmid D N A from the transformants was isolated and subject to restriction enzyme analysis to test for the loss of specific restriction enzyme sites. Positive confirmation of plasmids containing the termination linker was obtained by sequencing of the clones (see Materials and Methods section 1.4), now named pUC19/D.t 3. Nested Deletions Unidirectional deletions were made in pUC19/D.t from the end encoding the carboxy-terminal of /acZ/NS-1. Because there were no convenient restriction sites that would leave a exonuclease III resistant 5' overhang, unidirectional deletions were made by protecting one end with an a-phosporothioate deoxynucleotide analog (Guo and Wu 1982, Putney et al. 1981) as follows. Fifteen pg of pUC19/D.t was cut with 140 U of BamHl for 2 hrs at 37 °C, 30 extracted once with phenohchloroform, and precipitated with ethanol. The D N A was resuspended in 43.5 pi of (IH2O and the BamHI site was partially filled in using 5 pi of 10X NT buffer [500 m M Tris-Cl pH 7.2, 100 m M M g S 0 4 , 1 m M DTT, 500 M/mi B S A Pentax Fraction V] , 1 pi of 8.46 m M a-phosporothioate dATP, 1 pi of 10 m M dGTP, and 4.5 U of D N A Polymerase I Klenow fragment for 30 min at 37 °C. The reaction was terminated by the addition of 1 ul of 0.5 M E D T A . The volume was increased to 150 pi using TE buffer (100 m M Tris-Cl pH 8.0, 10 m M EDTA), extracted twice with phenohchloroform, and precipitated with ethanol. For the second restriction enzyme digest, the D N A was resuspended in 100 pi of I X React 3 and digested with 50 U of Sal I for 2 hrs at 37 "C. The enzyme was heat inactivated for 20 min at 68 °C, extracted twice with phenohchloroform, and precipitated with ethanol. Following incorporation of a-phosporothioate dATP, deletions were made using exonuclease III. The D N A was resuspended in 54 pi of CIH2O and 6 pi of 10X Exoin buffer [660 m M Tris-Cl pH 8.0, 770 m M NaCl, 50 m M M g C l 2 , 100 m M DTT]. Eight 500 pi microfuge tubes were prepared containing 32 pi of Exo l l l stop buffer [200 m M NaCl, 5 m M E D T A (pH 8.0)]. A total of 126 U of exonuclease i n was then added to the D N A solution and 8 pi aliquots were removed at either 1 min or 30 sec intervals depending on the length of the deletion required. Aliquots were heat inactivated for 10 min at 68 °C, then precipitated with ethanol. The D N A was then blunt-ended using SI nuclease and D N A Polymerase I Klenow fragment. Aliquots were resuspended in 50 pi of SI cocktail (250 m M NaCl, 30 m M K O A c pH 4.6, 1 m M ZnSC>4, 5% glycerol, 67 u / m i SI nuclease) and incubated for 30 min at room temperature. To each, 6 pi of SI stop buffer (500 m M Tris-Cl pH 8.0, 125 m M EDTA) was added, followed by one extraction with phenohchloroform, and precipitation with ethanol. Samples were resuspended in 20 pi of TE buffer, of which, 4 pi were subject to restriction digest to check the extent of the deletions, 6 pi was stored at -20 °C, and 10 pi was filled-in using D N A Polymerase I Klenow fragment as follows. A 10 pi volume of SI nuclease-31 treated D N A was added to 2.5 ul of 1 OX NT buffer, 1.0 ul of 2.5 m M dNTP mix, and 11.5 ul of (IH2O. This mixture was incubated with 4.5 U of D N A Polymerase I Klenow fragment for 30 min at 37 °C, and then heat inactivated for 15 min at 68 °C. Eleven pi of the blunt-ended D N A was added to 3.0 ul of 5X ligase buffer and 1 Weiss Unit of T4 D N A ligase, and ligation was allowed to proceed for 5 hrs at room temperature. A 1.4 pi aliquot of each of the time point's ligated D N A was used to transform frozen competent E. coli JM83 cells. A l l of the transformation mixture was plated on YT/Amp (see Materials and Methods section B2). Colonies from each time point were subject to small scale plasmid isolation and restriction enzyme digest to roughly determine the deletion size. Accurate deletion sizing was obtained by sequencing of the individual plasmids as outlined below. E. coli mini-lysates were made, run on a SDS-polyacrylamide gel, and immunoblotted against the six mAbs. 4. Sequencing The source of D N A for sequencing was either cesium chloride gradient purified D N A or D N A from small scale plasmid isolations. The latter had to be treated with 10 U of RNase TI for V2 - 2 hrs at 37 °C followed by extraction with phenohchloroform and ethanol precipitation before sequencing. Cesium chloride gradient-purified D N A was sequenced without pre-treatment. Double-stranded plasmids were alkaline denatured and sequenced using Sequenase according to the protocol of Gatermann et al. (1988). The primer used was a 17-mer M13mpl8 Universal Forward Primer. This method is basically the same as that recommended by the suppliers, except that the labelling/extension step was omitted allowing sequencing closer to the primer. The sequencing mixes were run on a 38 cm x 18 cm x 0.4 cm, 8% polyacrylamide gel containing 8M urea at 32W constant power for ~ l 3 / 4 hrs until the bromophenol blue front was near the bottom of the gel. Autoradiography was done using Curix RP-1 film after the gel was dried down. 32 J . Immunoprecipitations Most immunoprecipitations were done using Protein-A Sepharose CL-4B. Due to the poor affinity of Protein A for mouse IgGi antibodies (Harlow and Lane, 1988), immunoprecipitations done using mouse monoclonal antibodies of this class were performed using rocmlg coupled to Protein-A Sepharose CL-4B, and later using Protein G Sepharose 4 FF. Both Protein-A Sepharose CL-4B and Protein G Sepharose 4 FF were prepared according to the suppliers instructions, with the final resuspension being in K B S buffer (see Materials and Methods section C.2). The suspension was stored at 4 °C until needed. For coupling of the anti-mouse Ig antibody, 2.0 mg of ramlg was added to 1.0 ml of swollen Protein-A Sepharose CL-4B beads and rotated end-over-end overnight at 4 °C. The coupled beads were then washed with K B S buffer and stored in that buffer at 4 °C until required. Cell lysates for immunoprecipitations were prepared as follows. Cells were washed three times with cold PBS-C buffer, transferred to a 1.5 ml Eppendorf tube (scrapping if necessary), and then gently pelleted (300 x g, 5 min, 4 °C). The pellet was resuspended in 1 ml of cold RIPA buffer [10 m M Tris-Cl(pH 7.4), 150 m M NaCl, 1% ( v/ v) Nonidet P-40 (NP-40), 1% ( w / v ) sodium deoxycholate] followed by incubation for 30 min on ice. The lysate was cleared by centrifugation at 12,000 x g for 10 min at 4 °C, and then divided into 100 pi aliquots. The samples could be used immediately or quick-frozen using dry ice and stored at -20 °C. For immunoprecipitations, lysate aliquots were pre-cleared with 100 pi of swollen beads. The pre-cleared supernatants and 100 pi of the mAb-containing tissue culture supernatants were then incubated for 4 - 1 2 hrs at 4 °C with end-over-end rotation. Following this, 50 pi of swollen beads were added and the mixture was incubated for a further 4 - 1 2 hrs. Immune complexes were pelleted by centrifugation (12,000 x g, 15 sec, RT), washed three times with 200 pi cold RIPA buffer, and then disrupted by boiling for 5 min in 10 - 25 pi of Laemmli sample buffer. Samples were subject to SDS-polyaerylamide gel 33 electrophoresis as described before, dried down and autoradiographed on X - O M A T A R film. For double immunoprecipitations, immune complexes were disrupted by boiling in 200 \il of RIPA buffer + 0.05% SDS + 5 m M B-Mercaptoethanol. Disrupted beads were removed by centrifuging for 12,000 x g at RT for 15 sec, with the supernatant being transferred to a fresh tube. This clearing step was repeated once more, after which the supernatant was subject to the second round of immunoprecipitation following the procedure given previously for the single immunoprecipitation. K. Immunoaffinity column preparation CNBr-activated Sepharose 4B was prepared according to the manufacturer's instructions. The pooled, purified antibody was dialyzed versus three changes of 0.5 M sodium phosphate buffer (pH 7.5) before coupling to the CNBr-activated beads according to the procedure of Harlow and Lane (1988). L. Immunoaffinity Purification of CP/NS-1 expressed in Sf9 cells The CE10 mAb column and the pre-column, containing glycine blocked CNBr-activated Sepharose C L 4B, were pre-equilibrated with Buffer I [50 m M Tris-Cl(pH 7.8), 100 m M NaCl, 1 m M DTT, 1 m M EDTA, 10% ( v/ v) glycerol, 50 M8/ml PMSF] without NaCl for 1 hr at a flow rate of 150 m l / h r . 2.2 x 10 8 Sf9 cells grown in 100 mm tissue culture dishes were infected with AcSec at an MOI of ~10. At 48 h.p.i. the cells were harvested as follows. The cells were resuspended in 10 ml of PBS-C by gentle pipetting and then pelleted (300 x g, 5 min, RT). The rest of the steps were performed at 4 °C. Cells were washed three times with cold PBS-C buffer and resuspended in 6 ml of hypotonic lysis buffer [10 m M Tris-Cl (pH 7.4), 10 m M NaCl, 1.5 m M MgCl2] for 15 minutes on ice. Lysis was obtained by 30-60 passes with a tight-fitting Dounce homogenizer until 95% disruption occurred, as judged by phase microscopy. Nuclei and cellular debris were pelleted by centrifugation at 300 x g for 10 min at 4 °C and the supernatant transferred to a fresh tube. After the supernatant was adjusted to Buffer I 34 conditions, it was circulated through the pre-column four times. The cleared lysate was applied to the CE10 mAb column and recirculated through the column for 1 hr at a flow rate of 100 m l/hr to allow complete binding. The first wash consisted of 150 ml of Buffer I at a flow rate of 100 m l/hr. The second wash consisted of 100 ml of Buffer II [50 m M Tris-Cl (pH 9.0), 1 m M DTT, 10% ( v/ v) glycerol, 50 " g / m l PMSF] at the same flow rate. Elution was achieved under strong alkaline conditions using one column volume of Buffer III [20 m M triethanolamine, 10% ( v/ v) glycerol, 50 ug/mi PMSF] followed by 100 ml of Buffer I. Fractions of 180 pi were collected in 20 pi of Buffer IV [500 m M Tris-Cl (pH 7.8), 10 m M DTT, 10% ( v/ v) glycerol] and analyzed by SDS polyacrylamide gel electrophoresis and immunoblot against the CE10 mAb. Fractions collected at various steps during the purification procedure were included in the analyses. M . Immunoaffinity Purification of N S - 1 A C expressed in Sf9 cells 1. Elution using peptide and alkali The procedure used for the purification of NS-1 was similar to that of CP/NS-1 with the following exceptions. First, the Sf9 cells were grown and infected with AcNS-1 in a 100 ml spinner culture flask. Second, the volume of hypotonic lysis buffer the cell pellet was resuspended in was 8.5 ml instead of 6.0 ml and the final volume of the cell lysate after adjusting to Buffer I conditions was 10 ml. Third, application of the lysate to the pre-column matrix and to the CE10 mAb column matrix was done in batch suspension (end-over-end rotation at 4 °C for 1 hr) instead of in a column. After absorption to the pre-column matrix, the mixture was centrifuged at 900 x g for 3 min at RT and the supernatant added to the CE10 mAb matrix for binding in batch suspension as before. The beads were pelleted, washed once with Buffer I, poured into the column, and then washed with 150 ml of Buffer I as described before. Fourth, elution was first attempted with a two column volume 0 - 300 m S / m l peptide 1 gradient in Buffer II followed by 100 ml of Buffer II. Twenty-five 400 pi fractions were collected. After analysis of these fractions by SDS polyacrylamide gel electrophoresis, the 35 remaining bound material was eluted with alkali as described previously. 2. Elution using 6 M guanidinium chloride A l l steps were performed as for the peptide elution with the following alterations based on that of Tratner et al. (1990). First, PMSF was included in the hypotonic lysis buffer at a concentration of 50 V%lm\. Second, Buffer la (Buffer I with 500 m M L i C l instead of 100 mM NaCl) was substituted for Buffer I, and Buffer Ha [10 m M piperazine-N-N'-bis(2-ethanesulfonic acid) (pH 7.4), 5 m M NaCl, 1 m M EDTA, 1 m M DTT, 10% ( v/ v) glycerol, 50 ^ / m \ PMSF] for Buffer II. Third, elution was performed using 1 column volume of 6 M guanidinium chloride (~pH 6.2) followed by buffer la. Fractions (500 pi each) were dialyzed versus six changes of T M buffer [50 m M Tris-Cl (pH 7.9), 12.5 m M MgCl2, 1 m M E D T A , 1 m M DTT, 20% ( v/ v) glycerol] before analysis. 3. Elution using acid A l l steps were performed as for the peptide elution with the following alterations suggested by Harlow and Lane (1988). First, PMSF was included in the hypotonic lysis buffer at a concentration of 50 Mg/mi. Second, Buffer lb [10 m M phosphate (pH 8.7), 1 m M DTT, 1 m M E D T A , 10% ( v/ v) glycerol, 50 ug/ m i PMSF] was used in place of Buffer I, and Buffer l ib [10 m M phosphate (pH 6.8), 10% ( v/ v) glycerol, 50 u g / m l PMSF] in place of Buffer II. Third, elution was achieved using one column volume of Buffer Illb [100 m M glycine (pH 2.5), 10% ( v/ v) glycerol, 50 u g / m l PMSF] followed by 100 ml of Buffer lb. Fractions of 450 pi were collected in 50 pi of Buffer IVb [1 M phosphate (pH 8.0), 10% ( v/ v) glycerol]. 36 I X . Results A . Characterization of the mAbs Attempting to generate monoclonal antibodies against the /acZ/NS-1 fusion protein, G. Brown identified six positive hybridomas secreting antibodies against the antigen after the first fusion. These were named CE10, AC6, BE2, EA2, CH10, and BC4 based on the plate letter and well number. The CE10 and BE2 mAb hybridomas were subcloned three times by C. Astell and the remaining four hybridoma cell lines were subcloned three times by the method of limiting dilution (see Materials and Methods section B.3) by P. Tarn and myself. At this point, the mAb secreting cell lines were assumed to be monoclonal. Before these antibodies could be used in the detection of M V M NS-1, they had to be characterized with respect to iso-subtype in order to determine the route of production and purification. Testing of the iso-subtype of the six monoclonal antibodies was done using a commercially available ELISA kit. Mouse monoclonal antibodies were subtyped based on the reaction of specific anti-heavy chain antibodies against each mAb. Of the six antibodies, CE10, AC6 , BE2, EA2, and CH10 mAbs were found to be of the IgGi class. The sixth mAb, BC4, was found to be of the IgG2a class. Since each mAb reacted with only one anti-iso-subtype antibody each hybridoma cell line was assumed to be monoclonal, although the possibility that two or more antibody cell lines of the same subtype being present cannot be ruled out. Next, the reactivity of the mAbs was determined. E. coli JM83 were transformed with pUC19 or pUC19/D. Total cell lysates of the transformed bacteria were prepared from liquid cultures and run on a discontinuous SDS-polyacrylamide gel. Figure 3 shows the Coomassie blue-stained gel and immunoblots of the lysates with the six mAbs. The /acZ/NS-1 band is visible in Figure 3A as a 30 kDa band in the pUC19/D lane only. On the immunoblots, all six mAbs react specifically against a 30 kDa band in the pUC19/D lanes (Figure 3B). Also visible in the same lane is an unidentified cross-reacting band at approximately 40 kDa. B. Q cn cn O O 3 3 € g s s ~3 - » Q cn cn Q Q cn cn cn cn Q Q cn cn cn cn 1 ^ o o o o o o o o 3 3 3 3 3 = 3 3 3 Q. S. Q. Q- j. CI Q. Q. Q. ,_ . I S S I S S S S I I S S S S I ! o o 3 3 Q. CL cn cn O O 3 3 Q. Q. co CO CO CO 2 5 205- — CE-10 AC-6 BE-2 EA-2 CH-10 BC-4 Figure 3: Coomassie blue-stained gel of /acZ/NS-1 and immunoblots using the six  monoclonal antibodies. One ml aliquots of pUC19- and pUC19/D-transformed E. coli JM83 were lysed by boiling in Laemmli sample buffer. Five uls samples were run through a 4%/12% discontinuous SDS polyacrylamide gel and (A) stained with Coomassie-blue or (B) electrophoretically transferred to nitrocellulose. The blots were reacted with one of the six monoclonal antibodies (indicated below each blot) and developed. For further details refer to the Materials and Methods section. 38 Smearing of the bands is due to the presence of D N A in the total cellular lysates with the non-uniform concentration of the D N A in the sample producing the smearing in some of the blots while not in others. The mAbs were tested for their ability to detect M V M NS-1. Asynchronous mouse fibroblast cells were infected with M V M or mock-infected. Cells were harvested at 24 h.p.i., total cellular lysates prepared, and aliquots run on a discontinuous SDS-polyacrylamide gel. Figure 4 shows a Coomassie blue-stained gel and immunoblots using all of the mAbs. Although overloaded, the stained gel shows two distinct bands in the MVM-infected lane not present in the mock-infected lysate at 83 kDa and 66 kDa. These two bands correspond to the expected sizes and proportions of the VP-1 and VP-2 proteins, respectively. On the immunoblots all mAbs reacted against an 83 kDa band present in the MVM-infected cells only. The CE10, A C 6 , and BE2 immunoblots show a cross-reacting 'smear' centered at 40 kDa in the MVM-infected cells only, likely due to degradation products of the 83 kDa band. As the antibodies were raised against an lacZ/NS-l fusion protein and they recognize an 83 kDa band found in MVM-infected cells only, it was assumed that the antibodies recognized NS-1. In order to test whether these six antibodies react against a more native form of the M V M NS-1 protein, the mAbs were used in indirect immunofluorescence studies of M V M -infected L A 9 cells. A time-course immunofluorescence test was first done using only the CE10 antibody, to test the specificity of the CE10 mAb and determine the optimum time post-aphidicolin release for use in the immunofluorescence studies using the other mAbs. Synchronous L A 9 cells were grown on glass coverslips, MVM-infected or mock-infected, and then fixed with para-formaldehyde at t = 2, 4, 6, 8, 10, and 12 hours post-aphidicolin release (h.p.r.). Cells were permeabilized with Saponin, incubated with the CE10 mAb, and detected with a Texas-Red fluorescence system. In this test the mock-infected cells showed no fluorescence (data not shown). In MVM-infected cells, fluorescence was found to be restricted to the nuclear region of infected cells, fluorescence was first visible at 2 h.p.r. and 39 A . B. | 8 > | o > | o > ^ o > ^ o > ^ o > o 5 2 2 43-29-CE-10 AC-6 BE-2 EA-2 CH-10 BC-4 Figure 4: Coomassie blue-stained gel of mock- and MVM-infected L A 9 cell lvsates. and immunoblots using the six monoclonal antibodies. Asynchronous LA9 cells were mock- or MVM-infected at an M.O.I, of 10 and harvested at 24 h.p.i. The cell pellet was washed with PBS-C and then lysed in 100 pi of Laemmli sample buffer by incubation at lOO'C for 5 minutes. Ten ul aliquots were run on a discontinuous SDS polyacrylamide gel and (A) stained with Coomassie-blue or (B) electrophoreucally transferred to nitrocellulose. The blots were reacted with one of the six monoclonal antibodies (indicated below each blot) and developed. For further details refer to the Materials and Methods section. 40 increased in intensity in discrete foci in the nuclei through 4, 6, and 8 h.p.r. (see Figure 5). At 10 and 12 h.p.r., the fluorescence collected at the edge of the nuclear region while still increasing in intensity. From this experiment, the t = 8 h.p.r. time was chosen for the future study because of the ease of detection of the discrete foci. The same procedure as before was used to test whether the other five mAbs could detect fluorescent nuclear foci in the MVM-infected cells fixed at t = 8 h.p.r. For the CE10, AC6, BE2. and E A 2 mAbs, the results were as expected in that there was no fluorescence in the mock-infected cells (data not shown), and the nuclear foci were visible in the MVM-infected cells, albeit at differing intensities (Figure 6). With the other two mAbs, CH10 and BC4, a faint background fluorescence was detected in the mock-infected cells (data not shown). The fluorescent foci were still detectable against the background in the MVM-infected cells using the CH10 antibody, but not with the BC4 antibody. B . Mapping of the epitopes During the course of the characterization studies, R. Russnak isolated the recombinant baculovirus AcAXho using the pAcRP6/AX construct. This recombinant virus was expected to produce a fusion protein containing 604 aa of NS-1 (Metl - Leu604) fused to 71 aa of NS-2 caused by the reading frame shift at the filled-in Xhol site. On an immunoblot, he found that the CE10 mAb did not react against this protein, but that a polyclonal anti-NS-2 serum kindly provided by S.F. Cotmore and P. Tattersall (Yale University) did. Russnak interpreted this result to indicate that the CE10 mAb epitope resides in the 68 carboxy-terminal amino acids of NS-1 not present in the NS-l/NS-2 fusion protein. Of these 68 residues, only 18 were present in the /acZ/NS-1 fusion protein against which the CE10 mAb had been raised. It was concluded that the epitope of the CE10 mAb lay in these 18 residues (Russnak and Astell, unpublished results). In order to test this hypothesis more fully, a set of carboxy-terminal deleted lacZ/NS-1 proteins were made by creating a set of nested deletions in pUC19/D and transforming E. coli 41 t - 8 h p l t - 10 hp* t - f 2 h p l Figure 5: Immunofluorescence time-course study of MVM-infected. synchronous L A 9 cells  using the CE10 monoclonal antibody. Synchronous LA9 cells grown on acid-etched, glass coverslips were mock- or MVM-infected at an M.O.I, of 10 and fixed with paraformaldehyde at 2, 4, 6, 8, & 10 h.p.r. Cells were permeabilized with Saponin, and then reacted with CE10. Detection was obtained with a biotinylated secondary antibody and a Texas Red-streptavidin conjugate. The top-most picture shows a bright field microscopy picture of LA9 cells at 10 h.p.r. under a 63X objective magnification. The lower pictures show the immunofluorescence observed in the MVM-infected cells at the various times post-release under the same magnification. For further details refer to the Materials and Methods section. 42 EA-2 CH-10 Figure 6: Immunofluorescence in MVM-infected L A 9 cells using the CE10. AC6 . BE2.  EA2. and CHIP monoclonal antibodies. Refer to the Figure 5 legend. Cells were fixed with paraformaldehyde at 8 h.p.r. Detection was performed with the CE10, AC6, BE2, EA2, or CH10 mAbs as indicated below each picture. Development was obtained as described previously. 43 JM83 with these deleted plasmids. First, a termination linker containing stop codons in all three reading frames was placed at the carboxy-terminal encoding end of pUC19/D so deletions that changed the reading frame of lacZ/NS-l would still have a termination codon at the deletion junction. Unidirectional deletions were made with exonuclease III utilizing an a-phosporothioate dATP-protected BamHI site and an unprotected Sal I site (refer to Figure 7). The resulting D N A was religated and used to transform E. coli producing /acZ/NS-1 fusion proteins that had truncated carboxy-terminal ends. Recombinant clones were first screened by restriction enzyme digest to identify appropriate sized deletions. Forty-eight suitable clones were identified and sequenced. From the D N A sequence at the deletion junction, the amino acid sequence of the fusion proteins was predicted, allowing calculation of the size of the carboxy-terminal deletion (see Appendix II). Of the forty-eight clones, twenty-two were used to make bacterial protein lysates for immunoblot analyses with each mAb. An example of these results is shown in Figure 8. Clones were scored on whether or not each mAb reacted against the P-galactosidase fusion protein of the predicted size (30 - 23 kDa). Data from all the analyses are compiled in Table 2. The results indicated the presence of four antibody specificity groups. Table 2 shows that the CE10 and A C 6 mAbs have the same recognition specificity, as do BE2 and E A 2 mAbs within the limits of the deletions. CH10 and BC4 have the two other specificities. In all cases, once a particular region was deleted, antibody binding never occurred in the further deletions. Because the mAbs were raised against a heat-inactivated /acZ/NS-1 protein eluted from a SDS-polyacrylamide gel, it was assumed that the epitopes of the mAbs would be linear determinants rather than conformation-dependent determinants. Therefore, the deletion analysis would allow mapping of the carboxy-terminal end of the epitope. Using this reasoning, the locations of the epitopes relative to lacZ/NS-l and M V M NS-1 were predicted (see Figure 9). The A , B, and C epitopes are clustered in a region at the carboxy-terminal 44 • EcoRl 3060 • BamHl 3044 Sal I 3032 PstI 3026 PstI 2250 HinDIII 2237 lac Z coding region rj-ri M V M PstI D fragment encoding a.a. 354 - 623 of NS-1 termination oligomer GATCCTAAGTAATTAG GATTCATTAATCTTAA Figure 7: The construction of pUC19/D.t and its unidirectional deleted clones. For the construction of pUC19/D.t, the EcoRl - BamHl fragment of pUC19/D was replaced with the termination oligomer To create unidirectional deletions in pUC19/D.t, the plasmid was cut with BamHl, protected by filling-in with thea-phosporothioate dATP analog, and then cut with Sal I. Deletions were then made using Exonuclease III, blunt-ended using S1 nuclease, filled-in with D N A polymerase I(Klenow fragment), and finally religated using T4 D N A ligase. (see Materials and Methods) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 97.4 68.0 43.0 9 2 9 ° - AN M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 97.4 68.0 43.0 29.0 — 5 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 10 13 14 X u 97.4 68.0 43.0 29.0 2 Figure 8: Immunoblots of the six monoclonal antibodies against thirteen deletion clones  ofpUC19/D.t. Cellular lysates were made from overnight cultures of E. coli JM83 transformed with one of thirteen pUC19/D.t deletion clones. Aliquots of the lysates were separated by SDS-polyacrylamide gel electrophoresis, transferred electrophoretically to nitrocellulose, and probed with one of the six monoclonal antibodies as indicated beside each blot (refer to Materials and Methods). Deletion clones used were 1) M0,2) A52,3) A81,4) A90,5) A109, 6) A121, 7) A157, 8) A170, 9) A184, 10) A191, 11) A219, 12) A230, 13) A250, and 14) undeleted pUC19/D.t (refer to Appendix 2 and the Materials and Methods for further details) 4 6 Table 2: Immunoblot reaction of carboxy-terminal deleted lacZI NS-1 proteins versus  the monoclonal antibodies.8 Clone D N A deleted last lacZI NS-1 a.a CE10 A C 6 BE2 E A 2 CHO BC4 last NS-1 a.a. w.t. - 273 + + + + + + 623 1.13a A13 273 + + + + + + 623 0.6a A23 269 + + + + + + 622 1.14a A40 263 + + + + + + 616 1.2a A46 261 + + + + + + 614 1.5a A51 259 - - + + + + 612 1.3a A52 259 - - + + + + 612 2.5a A61 256 - - + + + + 609 1.8a A71 253 - - - - + + 606 V2.15 A81 249 - - - - - + 602 V2.13 A90 246 - - - - - + 599 2.4a A102 242 - - - - - + 595 2.6a A102 242 - - - - - + 595 V2.6 A109 240 - - - - - + 593 V2.7 A121 236 - - - - - + 589 1.14 A157 224 - - - - - + 577 1.6 A170 219 - - - - - + 573 1.18 A184 215 - - - - - + 569 1.7 A191 210 - - - - - + 564 1.8 A219 203 - - - - - + 557 1V2.30 A230 199 - - - - - - 553 1V2.32 A250 193 - - - - - - 547 see legends for Figure 5 and Appendix 1. This table is a compilation of the sequence analysis presented in Appendix II and the immunoblot analyses of 22 of the deletion clones using the six mAbs, including the data presented in Figure 8. 364 I 623 K-texmmvfcZZZZZZZZZZZZZZZZZ NS-1 V///////////77? VA C-rerminal 672 ^ ^ l a c Z / N S - l ^ a i u D CBA B epitope C epitope A epitope . . . L L G S A R S P F T T P K S T P L S O N Y A L T P L A S D L E D L A L E P W S T P N T P V A G T A G / ? L * I peptide 1 I 570 ™ 623 lacZ encoded sequences M V M NS-1 sequences MCS encoded sequences Figure 9: Predicted positions of the epitopes of the six monoclonal antibodies on  /acZ/NS-1 and on M V M NS-1 The top line diagram shows the M V M NS-1 protein. Aligned below that is a representation of the lacZ/NS-1 fusion protein with each expressed part of the molecule shade-coded. The positions of the four epitopes are shown based on the data compiled in Table 2. The A epitope corresponds to that for the CE-10 and AC-6 mAbs, B for the BE-2 and EA-2 mAbs, C for the CH-10 mAb, and D for the BC-4 mAb. A n expanded view of the A , B , and C epitopes is shown on the lowest line using the one letter code for the amino acids of the carboxy-terminal of/acZ/NS-1. Amino acids encoded by the multiple cloning site are denoted by italics and the underlined sequence indicates the region encompassed by peptide 1. 48 end in lacZ/NS-l and the D epitope is slightly more toward the amino-terminus of the protein. When the mAbs were tested on immunoblots versus lysates of AcAXho-infected Sf9 cells, the results were consistent with those obtained in the epitope mapping study. A c N P V - or AcAXho-infected Sf9 cells were harvested 48 h.p.i. Cell lysates were prepared, run on a discontinuous polyacrylamide gel, and subject to immunoblot analyses using the six mAbs. A Coomassie-blue stained gel and the immunoblots using the CH10 and BC4 mAbs are shown in Figure 10. The predicted product of the recombinant AcAXho baculovirus is a NS-l /NS-2 fusion protein containing Met l - Leu604 of NS-1 fused to the NS-2 carboxy-terrninal starting at NS-2 A r g l l 4 with two unrelated amino acids in the joining region. From the epitope map, the fusion protein is expected to contain the D epitope and possibly the C epitope, but not the A or B epitopes. The immunoblots show that the CH10 and BC4 mAbs did react to a 77 kDa protein, the predicted size of NS-l /NS-2, confirming the presence of the C and D epitopes (see Figure 10). The CE10, AC6 , BE2, and E A 2 mAbs did not react (data not shown), indicating that the A and B epitopes were not present as predicted. These results also allowed a finer mapping of the C epitope by showing that the M V M NS-1 Glu605 and Asp606 were not required for binding. Before the epitope mapping work was completed, a 25 aa peptide was synthesized by Dr. I. Clark-Lewis to include the 18 amino acids R. Russnak predicted to contain the CE10 mAb epitope. This peptide corresponds to Pro599 - Ala623 of NS-1 (see Figure 9). Once the epitope mapping had been completed, it became evident that this peptide could be used to confirm the epitopes of four of the six monoclonal antibodies by testing whether the antibodies would bind to the peptide. Fifty millilitres of tissue culture supernatant were collected from each mAb cell line and a titration ELISA was performed on each. The purpose of these experiments was to determine the dilution of the supernatant required so that each would be relatively equal in terms of antibody concentration. Serial doubling dilutions were made of the supernatants and run in an ELISA (see Figure 11). From these results, the 49 A. B. C. Figure 10: Immunoblots of the CHIP and BC4 monoclonal antibodies using a NS1/NS-2 fusion protein. Total cellular lysates were made of AcNPV- and AcAXho- infected Sf9 cells (M.O.I. = 5) harvested at 48 h.p.i. as detailed in the Materials and Methods. Aliquots were run on a discontinuous SDS-polyacrylamide gel and then (A) stained with Coomassie blue, or electrophoretically transferred to Optibind. Blots were reacted against (B) the CH10 mAb, or (C) the BC4 mAb and developed as previously described. Two marker lanes are present in the A ; the first being Sigma SDS6H unstained molecular weight markers and the second being the Bio-Rad pre-stained high molecular weight markers used on the immunoblots. 1 10 100 -I 1000 10000 (Antibody dilution) Figure 11: Titration curves of the six monoclonal antibody solutions used in the  competition ELISAs. Tissue culture supernatants of each mAb secreting hybridoma were titrated using doubling dilutions. For complete details refer to the Materials and Methods. 51 dilution at which 80% of maximal binding occurred was determined. At the 80% binding efficiency point, the amount of antigen coating the ELISA plate wells is theoretically slightly in excess of the antibody. Once this dilution had been determined for each supernatant, competition ELISAs were performed. In the competition ELISAs, a soluble competitor molecule was added to the antibody solution at differing concentrations. The added molecule competes with the surface-bound antigen for binding by the antibody forming soluble antigen-antibody complexes instead of surface-bound complexes. Three competitors were used: 1) lacZ/NS-l, as a positive control competitor, 2) the 25 aa NS-1 peptide (peptide 1), and 3) a 28 aa peptide with no homology to NS-1 (peptide 2), as a negative control competitor. Soluble antibody-competitor complexes are washed from the plate and the ELISA then developed as normal. These results are shown in Figure 12. For all antibodies the /acZ/NS-1 competitor demonstrates positive competition, indicated by the decrease in A405 with increasing competitor concentration, and the negative control no competition, indicated by the lack of change in A405 with increasing competitor. The NS-1 peptide competition is expected to show one or the other. Assuming epitopes consist of seven amino acids (Berzofsky and Berkower 1984,1. Clark-Lewis, pers. comm.), then based on Table 2 the NS-1 peptide would be expected to contain the complete A and B epitopes. The C epitope would be missing one amino acid in the peptide and the D epitope would lie completely outside the peptide (refer to Figure 9). Indeed, Figure 12 shows that the NS-1 peptide competes for the CE10 and AC6 mAbs binding, indicating the presence of the A epitope. Also as expected, the peptide does not compete for the CH10 and BC4 mAbs binding, indicating the absence of the C and D epitopes. Unexpectedly, there was no competition shown for the BE2 and E A 2 mAbs representing the B epitope. A possible explanation for the observed non-competition for the B epitope mAbs might be that the epitope was too near the terminus of the peptide and required more amino acids to the amino-terminal side of the epitope to induce the correct local structure for antibody 52 .0001 .001 .01 .1 1 competitor concentration (uM) 10 .0001 .001 .01 .1 1 competitor concentration (uM) 10 0.3 .0001 .001 .01 .1 1 competitor concentration (uM) .0001 .001 .01 .1 1 competitor concentration (uM) Figure 12: Graphs of the peptide 1 competition ELISAs'showing absorbance at  405 nm versus the competitor concentration in each well Competitors were added at the same time as the antibody. The solid line indicates the competition against the NS-1 peptide 1. The positive competitioncontrol using the antigen used to coat the wells (?) and the negative competition control using a similar length peptide with no homology to NS-1 (j) are shown for each antibody on the respective graph (refer to Materials and Methods) 53 recognition. This could be proven or disproven by synthesizing a peptide with the epitope in the middle of the peptide and testing for competition again. The negative control also shows abnormally high readings for the two highest competitor concentrations due to a chromogenic compound in the peptide preparation. C . Recombinant Baculoviruses Expressing NS-1 1- AcSec In addition to AcAXho, two other recombinant baculoviruses, AcSec and AcNS-1, were made in order to overexpress the NS-1 protein. The AcSec virus was isolated before the Ac NS-1 recombinant and characterization of the protein produced by the AcSec virus was done until the AcNS-1 virus became available. The CPss /NSl /pAcYMl plasmid was used by R. Russnak to generate the recombinant baculovirus named AcSec . After isolation of the virus, a high-titre viral stock was produced and used for protein expression studies. Infection of Sf9 cells by AcSec was expected to produce a 77 kDa CP/NS-1 fusion protein which has 47 aa of the amino-terminal of preceruloplasmin replacing 41 aa at the amino-terminus of the NS-1 protein. The inserted 47 aa include the preceruloplasmin signal sequence, the signal cleavage site, and 28 aa from the ceruloplasmin amino-terminus. The construct was generated in the hope that NS-1 might be secreted from the infected Sf9 cells, minimizing cytotoxic effects due to localization and simplifying purification. Figure 13 shows the Coomassie-blue stained gel of total cellular lysates of AcNPV-and AcSec-infected Sf9 cells harvested 48 h.p.i. The 29 kDa polyhedrin protein is visible in the AcNPV-infected cells even though the polyhedra were not lysed under alkaline conditions. In AcSec-infected cells, an 77 kDa band corresponding to CP/NS-1 is visible. The CP/NS-1 protein appears to make up 30% of the total protein based on this gel. The immunoblot of these lysates using the CE10 mAb revealed a strong-reacting band at 77 kDa in the AcSec-infected cells only. This protein was later shown to migrate slightly faster than M V M NS-1 (data not shown). As well, several minor, smaller cross-reactive bands were 54 > 9 ^ Q. A "5 Z CO rt o 0 0 2 5 < < 1 SO-TS-50-3 9 -2 7 . 17 . Figure 13: Coomassie blue-stained gel of mock-. AcNPV- and AcSec-infected total Sf9 cell lysates. Refer to the Materials and Methods section. Sf9 cells were mock-, AcNPV- or AcSec-infected at an M.O.I, of 10, harvested at 48 h.p.i., and lysed by boiling for 5 minutes in Laemmli sample buffer. Aliquots were run through a discontinuous SDS-polyacrylamide gel and then stained using Coomassie blue. 55 observed most likely representing CP/NS-1 degradation products. A strong cross-reacting band was also noted in both AcNPV- and AcSec-infected cell lysates, running at an apparent molecular weight of 75 kDa; the identity unknown. Having shown that the CE10 mAb binds to a denatured CP/NS-1, indirect immunofluorescence was used to determine the cellular localization and show binding to native CP/NS-1. Previous studies had indicated that CP/NS-1 was not secreted into the extracellular media (R. Russnak, pers. comm.) and therefore was not looked for in that location. Sf9 cells were infected with AcNPV or AcSec, permeabilized, and reacted against the CE10 mAb. Immunofluorescence was examined using a rhodamine conjugated secondary antibody. The results of this study are shown in Figure 14. The AcNPV-infected cells showed no fluorescence whatsoever (data not shown). In the AcSec-infected cells, fluorescence was not visible until 24 h.p.i. The fluorescence was restricted to the cytoplasm of the cells and the intensity of the fluorescence increased as the infection proceeded, reaching a maximum at 48 h.p.i. Fluorescence was clearly visible right through until the last time point at 72 h.p.i., at which time the bright field phase contrast microscopy revealed that the cells were lysed (data not shown). Bright field microscopy also revealed the infected cells to look unhealthy at 48 h.p.i. and have lost structural integrity of the plasma membrane at 60 h.p.i., following the normal course of cytopathy seen in baculovirus infection (G. Wilson, pers. comm.). From this immunofluorescence study, the localization of CP/NS-1 appeared to be cytoplasmic or membrane-associated although in a non-uniform distribution. Exact determination of the localization of CP/NS-1 was complicated by the fact that cells of this lineage have a small amount of cytoplasm relative to the size of the nucleus. That said, CP/NS-1 could be on the plasma membrane, in the cytoplasm, or attached to the outer nuclear membrane, but does not seem to be within the nucleus. Under the assumption that CP/NS-1 was cytoplasmic, a trial run for the CE10 mAb immunoaffinity column was performed (see Figure 15 and later discussion). Cells were t = 24 t = 36 t = 48 t = 60 t = 72 Bright Field Figure 14: Immunofluorescence time course study on AcSec-infected Sf9 cells using  the CE-10 monoclonal antibody. Sf9 cells were grown on acid-etched glass coverslips, AcNPV- or AcSec-infected at an M.O.I. of 0.1, and paraformaldehyde fixed at 12, 24, 36,48,60, and 72 h.p.i. Cells were permeabilized with Saponin, reacted with CE-10, and fluorescently tagged using a rhodamine conjugated secondary antibody as described in the Materials and Methods. Coverslips were mounted and viewed under a 63X objective magnification. The AcSec -infected cells immunofluorescence are shown above at various times post-infection indicated below each. The bright field microscopy photography of AcSec-infected Sf9 cells was taken at 24 Figure 15: Coomassie blue-stained gel and immunoblot using the CE10 monoclonal  antibody, of fractions taken during the immunoaffinity purification of CP/NS-1  expressed in insect cells. The method has been given in the Materials and Methods section L. The fractions presented are as follows: B) cytoplasmic supernatant BP) nuclei and cellular debris pellet, D) unbound flow-through from after application to CE-10 column, E) Buffer I wash flow-through, F) Buffer II wash flow-through, fl...f8) fractions collected after alkaline (pH 11.4) elution, and M) Bio-Rad pre-stained molecular weight markers. The Coomassie blue-stained gel is shown above, and the corresponding immunoblot against the CE10 mAb below. 58 infected with AcSec, harvested 48 h.p.i., pelleted, and then resuspended in a detergent lysis buffer containing 0.5% NP-40. Nuclei and cellular debris were pelleted and the supernatant applied to the immunoaffinity column. The Coomassie blue-stained gel and the corresponding immunoblot analysis against the CE10 mAb revealed that >90% of the CP/NS-1 was found in the nuclear pellet and cellular debris rather than the cytoplasmic fraction (see Figure 15). In order to confirm this result, Sf9 cells were mock-infected, or infected with A c N P V or AcSec, harvested at 48 h.p.i., pelleted, and resuspended in a hypotonic detergent lysis buffer containing 10 m M Tris-Cl (pH 7.6), 1% NP-40, and 1% NaDOC. The cells were allowed to lyse on ice for 30 minutes and then spun down in a microcentrifuge. Equal aliquots of the pellets and supernatants were run on a polyacrylamide gel, transferred to Optibind, and reacted with the CE10 mAb (Figure 16). Again, >90% of CP/NS-1 was found in the pellet fraction. The addition of NaCl to a final 500 m M concentration had little effect on the amount of CP/NS-1 in the pellet. The same was true for the addition of E D T A to a final 5 m M concentration (data not shown). Lysis of the cells in the absence of detergents produced the same results as well. Infected cells were lysed by a method used for the preparation of nuclear extracts (Lee et al. 1988) in which the cells are placed in a hypotonic buffer for fifteen minutes and then forced through a narrow gauge hypodermic needle. The cell suspension was centrifuged yielding a cytoplasmic fraction and a nuclear pellet. A high salt (420 m M NaCl) extract was made of the pellet fraction which was then cleared of debris by centrifugation. Aliquots of the cytoplasmic extract, the nuclear extract, and the resuspended debris pellet were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot using the CE10 mAb (data not shown). CP/NS-1 was present in all three fractions, but >75% was still found in the pellet fraction. 2. AcNS-1 Studies on the purification of CP/NS-1 polypeptide were set aside when the AcNS-1 baculovirus was isolated by G. Wilson. Construction of the plasmid from which this virus 59 ^ Mock ^ AcNPV AcSec « i rt rt 2 o - c / > 2 Q - C 0 Q - c o 130 75 50 39 27 17 Figure 16: Immunoblot of mock-. AcNPV- . and AcSec-infected Sf9 cell fractionation  study. Sf9 cells were mock-, AcNPV-, or AcSec-infected at an M.O.I, of 10 and harvested at 48 h.p.i. Cells were washed twice with PBS-C buffer (see Materials and Methods), and then pelleted at 300 x g for 5 minutes. The cell pellets were resuspended in 1 ml of 10 mM Tris-Cl (pH 7.6), 1% ( v/ v) NP-40, and 1% ( w/ v) NaDOC. Lysis was allowed to occur on ice for 30 minutes. The cell suspension was spun for 2 min, 12,000 x g, at 4°C. The supernatant was carefully removed and saved. The pellet was resuspended in 1 ml Laemmli buffer. A 80 ul aliquot of the supernatant fraction was added to 20 ul 5X Laemmli buffer. Both the pellet fraction (P) and supernatant fraction (S) were denatured by boiling for 5 minutes. Aliquots of the mock-, AcNPV-, and AcSec-infected cell fractions were electrophoresed through a discontinuous SDS-polyacrylamide gel, transferred to nitrocellulose, and reacted with CE-10. The blot was developed as described in the Materials and Methods. 60 was made is described in the Materials and Methods section B l . The recombinant virus was expected to produce a full length NS-1 protein (designated N S - 1 A C to distinguish it from the M V M NS-1 protein) as opposed to a fusion protein like CP/NS-1. For this reason, studies were switched to the N S - I A C protein expression. Sf9 cells infected with AcNPV, AcNS-1, or mock-infected were harvested at 48 h.p.i., and total cell lysates were prepared. Figure 17A shows the Coomassie-blue stained gel of these lysates. Of interest are the 29 kDa polyhedrin band in the AcNPV-infected cells, and the 84 kDa band in the AcNS-1-infected cells corresponding to the expected mobility of NS-1 Ac- Comparison against M V M NS-1 from MVM-infected mouse cells showed the baculovirus expressed N S - 1 A C running at a slightly higher mobility (data not shown). Transfer of the lysates to Optibind and detection using the CE10 mAb identifies a major band at ~84 kDa and several smaller minor bands in the AcNS-1-infected Sf9 cells only (Figure 17B). These smaller bands likely represent degradation products of the N S - 1 A C protein. In order to see i f this protein was more easily solubilized than CP/NS-1, crude localization studies were done. AcNS-1-infected and AcNPV-infected Sf9 cells were harvested at 48 h.p.i. and subject to lysis in 1% NP-40 at two salt concentrations (N.B. both conditions gave the same results and only the lower salt condition is presented in Figure 18). The cell suspensions were then centrifuged to produce a supernatant containing the solubilized proteins, and a pellet containing the cellular and nuclear debris. Aliquots of each fraction were subject to discontinuous polyacrylamide gel electrophoresis and stained with Coomassie blue (Figure 18A). Identification and quantification of N S - 1 A C in the respective fractions was done by immunoblot analysis using the CE10 mAb. The immunoblot (Figure 18B) shows that >75% of N S - 1 A C is present in the supernatant fraction, although a small amount is found in the pellet fraction. The N S - 1 A C in the pellet may be due to the fact that the pellet fraction was not washed. Confirmation of the cellular location was obtained using indirect immunofluorescence. 61 Figure 17: Coomassie blue-stained gel of mock-. AcNPV- . and AcNS-1-infected Sf9 cell  lysates. and immunoblots using the CE10 monoclonal antibody. Mock-, AcNPV-, or AcNS-1-infected Sf9 cells (M.O.I. = 2) were lysed at 48 h.p.i. by boiling for 5 minutes in Laemmli buffer. Aliquots were electrophoresed through a 4%/12% SDS-polyacrylamide gel, and (A) stained with Coomassie blue or transferred to Optibind and reacted against the CE10 mAb. The blot was developed as described in the Materials and Methods. The arrow indicates the position of the NS-1 Ac band. B. AcNPV AcNS-1 CO 0_ CO CL AcNPV AcNS-1 CO CO Q . 66.0-45.0-36.0-* 29.0-« „ 97.4-68.0-43.0-29.0-Figure 18: AcNPV- and AcNS-1 infected Sf9 cell fractionation study Refer to the legend for Figure 16. The cell resuspension buffer contained 10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% (v/v) NP-40, and 0.2% ( w/ v) NaN3. The Coomassie blue-stained gel (A) and the immunoblot against CE10 (B) are shown. As before, centrifugation yielded a supernatant fraction (S) containing the NP-40 solublized proteins and a pellet fraction (P). The arrow indicates the position of NS-1 Ac-63 A time course study using indirect immunofluorescence was done following the same procedure used for the AcSec study (Figure 14) but with the AcNS-1 virus. Cells were fixed at t = 12, 24, 36, 48, 56, 60, and 72 h.p.i. As before, AcNPV-infected cells showed no fluorescence at any of the time points. In the AcNS-1 infection, fluorescence was not visible until 36 h.p.i. (see Figure 19). At this time point, fluorescence was very intense and showed cytoplasmic localization. After this time point, the intensity decreased and by 60 h.p.i fluorescence was no longer visible. Like the AcSec infection, the Sf9 cells showed the cytopathic effects of baculovirus infection, but the process appears to occur sooner in the AcNS-1 infection. The cells swell to a maximum size by 48 h.p.i. and have completely lysed by 60 h.p.i. The decreasing and eventual loss of fluorescence by 60 h.p.i. in the AcNS-1-infected cells is most likely due to the loss of integrity of the plasma membrane allowing cytoplasmic leakage. D. Immunoprecipitation of [35S]-methionine labelled N S - 1 A C In order to test if the mAbs could be used to purify N S - I A C from insect cells, an immunoprecipitation experiment was performed. Sf9 cells were infected with AcNPV or AcNS-1, labelled with [35S]-methionine for 3 hours at 31 h.p.i., and harvested at 48 h.p.i. Cells were lysed in RIPA buffer, centrifuged and the supernatant aliquoted. Tissue culture supernatants containing each mAb were added to individual aliquots and incubated together overnight. Antigen-antibody complexes were isolated using Protein A-Sepharose CL-4B coupled to a ramlg antibody, washed, and then incubated at 100 °C for 5 minutes to release the precipitated material. Aliquots were subject to SDS-polyacrylamide gel electrophoresis. The autoradiograph of the gel is shown in Figure 20. As a control, an anti-rhodopsin monoclonal antibody 2B2 (MacKenzie et al. 1984) was used to show the non-specific bands immunoprecipitated (lanes 1 & 2). A 40 kDa band is visible in these lanes. Immunoprecipitations using the six mAbs all precipitated two major bands in AcNS-1-infected cellular lysates. The band at 84 kDa corresponds to the expected size of N S - 1 A C 64 t- 36hp* t - 4 8 h p i t - 5 6 h p i Figure 19: Immunofluorescence time course study of AcNS-1 -infected Sf9 cells using the  CE10 monoclonal antibody. AcNPV- or AcNS-l-infected Sf9 cells (M.O.I. = 0.1) grown on acid-etched glass coverslips were fixed in 3.68% paraformaldehyde at 12, 24, 36, 48, 54, 60, and 72 h.p.i. Cells were permeabilized with Saponin, reacted with the CE10 mAb, and detected using a biotinylated secondary antibody and a Texas Red-streptavidin conjugate. Cells were viewed under a 63X objective magnification. Bright field phase microscopy pictures from 24,48, and 60 h.p.i are shown on top. Immunofluorescence microscopy pictures from 36, 48, and 56, h.p.i. are shown below. For further details refer to the Materials and Methods. cx-rho CE-10 AC-6 BE-2 EA-2 > S-1 > 1 > Q_ S-1 CL CO CL Z z Z z z O O o o o < < < < < V > T CO 0- CO z z z o o o < < < > CL CO o o < < CH-10 BC-4 > CL Z o < CO z o < > CL z o < CO z o < 200 •97.4-• 68 • 4 3 • 29 • 18.4-Figure 20: Immunoprecipitation of AcNS-1- infected cell lysates using the six monoclonal  antibodies. 1 x 107 Sf9 cells were AcNPV- or AcNS-1 infected. At 30 h.p.i., the cells were washed with Met" TNM-FH media and resuspended in the same media with 100 ^ C i /ml ^5S-Met and 10% dialyzed FCS. After a 4 hour labelling period, the cells were returned to TC-100/10% FCS. At 48 h.p.i. the cells were harvested, washed 2X with PBS-C buffer, and the cell pellet was resuspended in 1 ml RIPA buffer. After 30 minutes on ice, the cell lysate was cleared by centrifugation (12,000 x g, 10 min, 4'C), and the supernatant divided into lOOul aliquots. AcNPV- and AcNS-l-infected cell lysates were incubated with 50 ul of the various mouse monoclonal, antibodies as indicated above each pair, rotating end-over-end at 4'C for 10 hrs. Immune complexes were precipitated by the addition of 25pl ramlg-ProtA-Sepharose (2m8 r a m I 8 / s w o i ien ProtA-Sepharose) rotating end-over-end at 4°C for 10 hrs. Complexes were washed 3X with RIPA buffer, resuspended in 20ul Laemmli sample buffer, and incubated at 100°C for 5 minutes. Samples were electrophoresed through a discontinuous SDS-polyacrylamide gel. The gel was dried down and subject to autoradiography. 66 and the second band, at 60 kDa, was of unknown origin. As well, two other very faint bands are seen in the immunoprecipitations of N S - 1 A C a t 43 kDa and 40 kDa. Bands of similar mobility, together with a 60 kDa band, were later shown to be immunoprecipitated from the wild-type infected cell lysates. In an attempt to identify the immunoprecipitated bands, the lysates were subject to a double immunoprecipitation. Lysates were first immunoprecipitated as just described above, using the CE10 mAb. To disrupt any immunoprecipitated protein complexes, the beads were boiled in RIPA buffer + 0.5% SDS + 5 mM B-Mercaptoethanol. Sepharose beads were removed by centrifugation, and the supernatant was subject to a second immunoprecipitation with the CE10 mAb or a guinea pig anti-TP polyclonal sera (Chow et al. 1986). Single immunoprecipitations using the anti-TP sera or the CE10 mAb were also performed. A l l are shown in Figure 21. None of the original CE10 mAb immunoprecipitated bands were detected in the second round of immunoprecipitation using the anti-rhodopsin mAb or the anti-TP sera (Figure 21A). Using the CE10 mAb, only the 84 kDa band was detected. The unidentified lower bands in the previous immunoprecipitation were all found in the first round immunoprecipitations of the AcNPV-infected cells (see Figure 2IB). These bands were not detected in the original autoradiogram (Figure 20) because the amounts of AcNPV-infected cell lysates loaded were lower than those of the AcNS-1-infected cell lysates. This does not explain why the anti-rhodopsin antibody did not detect the 60 kDa band in Figure 20 and may indicate that N S - 1 A C is complexed to a 60 kDa protein distinct from the one faintly seen in the Figure 21B immunoprecipitations. The intensity of the 60 kDa band in the CE10 mAb/AcNS-1 lane was stronger than that in the CE10 mAb/AcNPV or anti-rhodopsin mAb/AcNS-1 lanes. The anti-TP sera did not immunoprecipitate any labelled proteins under the conditions used (Figure 21B). The TP protein, a nucleolar antigen found in mouse cells and HeLa cells, would likely require more extensive extraction conditions to dissociate it from the nuclear 67 Figyre 21; Immunoprecipitation of AcNS-1- and AcNPV-infected cell lvsates using the CE10 mAb and an anti-TP sera, and double immunoprecipitation studies on  CE10 mAb immunoprecipitated AcNS-1 infected Sf9 cell lysates using the CE10  mAb and an anti-TP sera. Cells were labelled and harvested as described in the Figure 20 legend. First-round immunoprecipitation was performed using the CE10 mAb. Immune complexes were disrupted as described in the Materials and Methods section J and subject to second-round immunoprecipitation with anti-rhodopsin antibodies, the CE10 mAb, or the anti-TP sera. These complexes were washed, resuspended, denatured, and analyzed as in Figure 20. The first round immunoprecipitations with the CE10 mAb, anti-rhodopsin sera, and the anti-TP sera are shown in B. 68 lamina if it is actually present in the insect cells as well (Walton et al. 1989, Bodnar et al. 1989). E . Purification of NS-1 using a CE10 immunoaffinity column From the studies described above, the CE10 mAb was shown to recognize all forms of NS-1 and NS-1 fusion proteins, and have the highest A405 in the ELISA. Therefore, this antibody was chosen initially to be used in the construction of a NS-1 specific immunoaffinity column. In order to construct the column, large quantities of the mAb had to be produced, purified, and then coupled to a support matrix. A total of twelve spf Balb/C mice were inoculated with the CE10 hybridoma. The first three inoculated mice produced low quantities of ascitic fluid and developed solid tumors at the point of injection. Hybridomas from one of these mice's ascitic fluid was grown in cell culture, and these cells were then used to inoculate the latter mice. The average titre of ascitic fluid obtained from these latter mice was 8.1 mis and solid tumors were no longer detected. The monoclonal antibody was purified from the ascitic fluid by ammonium sulfate precipitation and DEAE-Sephacel chromatography, and coupled to CNBr-activated Sepharose CL-4B at 7 mS/mi swollen beads- A pre-column matrix was prepared by the same method used for coupling the mAb, but without adding the mAb. As mentioned previously, the first test of the immunoaffinity column was performed using the CP/NS-1 protein based on a method used for the immunoaffinity purification of SV40 large T antigen expressed in insect cells (Lanford 1988, Murphy et al. 1988, Simanis and Lane 1985). Aliquots were taken at various points during the purification procedure as outlined in the Figure 15 legend, and separated on a SDS-polyacrylamide gel. CP/NS-1 was detected immunologically by immunoblot analysis using the CE10 mAb. Comparison of lanes B and BP, which were adjusted to be equal with respect to cells per ml, indicates that >90% of CP/NS-1 remained in the first centrifugation pellet and was never applied to the column. Although not easily seen in the Coomassie blue-stained gel, a small amount of CP/NS-1 was 69 solubilized and applied to the column (see immunoblot, lane B). Lane D shows the proteins not bound to the CE10 mAb column and the immunoblot of this fraction showed that not all of the applied CP/NS-1 was bound. The elution of the bound proteins by strong alkaline (pH 11.3) conditions showed that the column material was functional (see lanes f5 - f8) and the immunoblot of the eluted proteins indicates the presence of CP/NS-1. Unfortunately, a large number of proteins appear in the stained gel of the eluted fractions that did not react with the CE10 mAb on the immunoblot. Also, the CP/NS-1 band in the eluted fractions was not clearly visible on the stained gel and recovery of CP/NS-1 from the Sf9 cells was estimated at only 10% at best. At this point, efforts were directed to increasing the proportion of CP/NS-1 solubilized, but because a more efficient method was never found, refinement of the column conditions and immunoaffinity purification of CP/NS-1 was not pursued further. With the isolation of a N S - 1 A C expressing recombinant baculovirus, the column was put to use in attempts at purification of the unfused N S - 1 A C protein. Having proved that peptide l was bound by the CE10 mAb, it was thought that this peptide could be used to elute bound N S - 1 Ac fr°m the CE10 mAb column and provide a very specific elution method. The results of this purification attempt are shown in Figure 22. The immunoblot of the total cellular extract (lane A) shows a large band running at 84 kDa corresponding to N S - 1 A C - There are also several lower molecular weight bands appearing on the immunoblot, including ones that had not been seen previously (c/: Figure 1 7 ) . As there were no protease inhibitors added during the hypotonic lysis, these bands were assumed to be degradation products. Comparison of lane B to lane BP shows that, unlike the CP/NS-1 purification, -90% of the NS-1 Ac protein was solubilized. Lane C shows that pre-column clearing of the sample did not affect any of the immunoreactive bands; the change in intensity on the immunoblot being accounted for by a dilution not adjusted for when the gel samples were made. Not all of the N S - I A C applied to the CE10 mAb matrix was bound to the column (see lane D). Addition of peptide 1 up to a concentration of 300 "Wml had no effect on the bound N S - 1 A C » as only an unidentified -62 kDa protein appeared on the gel and not on the immunoblot. If this protein Figure 22: Coomassie blue-stained gel and immunoblot using the CE10 monoclonal antibody, of fractions taken during the immunoaffinity purification of NS-1 Ac  expressed in insect cells and eluted by peptide and alkali. The method has been given in the Materials and Methods section M . l . The fractions presented are as follows: A) total cellular lysate, B) cytoplasmic supernatant after hypotonic lysis, BP) nuclei and cellular debris pellet after hypotonic lysis, C) cytoplasmic lysate after pre-column clearing, D) unbound flow-through after cytoplasmic lysate application to CE-10 column, F1..F9) fractions collected after elution by a 0 - 300 m S / m i peptide 1 gradient, fl...fl4) fractions collected after elution by alkali (pH 11.4), and M) Sigma SDS-6H molecular weight markers (for gel) or BRL pre-stained high molecular weight markers (for immunoblot). The Coomassie blue-stained gel is shown above, and the corresponding immunoblot against the CE10 mAb below . 71 were actually competed off the matrix-attached CE10 mAb by the peptide, it would be expected to react on the Western blot. As it did not, this protein was assumed to be non-specifically eluted. After realizing from the SDS-polyaerylamide gel that N S - I A C had not been released from the column, the remaining proteins were eluted by alkaline conditions (lane fl...fl4) and collected in 400 (il fractions one column volume after the application of the elution buffer. The immunoblot of these fractions indicated that N S - 1 A C had been eluted, but as a broad peak centered at fraction 6. Once again, the Coomassie blue-stained gel showed a large number of eluted proteins, only a small number of which react against the CE10 mAb on the Western blot. The eluted proteins (see lane f6) showed a new immunoreactive species migrating at an apparent molecular weight of 68 kDa (cf: lane C) and the five original immunoreactive bands each appeared with a second slightly lower molecular weight band following alkaline elution. This could indicate dephosporylation of N S - 1 A C on serine or threonine residues; phosphserine and phosphothreonine residues being alkaline labile under the pH conditions employed in the elution method. With the failure of the peptide elution method and the probable dephosphorylation caused by the alkaline elution, alternative methods for the release of N S - 1 A C were sought. Elution with 6 M guanidinium chloride has been used to immunoaffinity purify a functional c-fos protein expressed in insect cells (Tratner et al. 1990), hence this method was attempted for N S - 1 A C (see Figure 23). Lane A showed the addition of the serine protease inhibitor, PMSF (50 ^g/mi), to the hypotonic lysis buffer resulted in the loss of the 80 kDa immunoreactive band seen in the previous purifications (cf: Figure 22, lane A) , but had no effect on the four other lower molecular weight bands. The Tratner protocol included 500 m M L i C l in the first wash buffer which results in a large number of proteins being washed off the column including a small proportion of N S - 1 A C 0 a n e E). The guanidinium elution itself caused several undesirable problems. First, the column was seriously denatured, indicated by the two very distinct bands at 50 kDa and 27 kDa in lanes F3...F18. These bands correspond to the heavy Figure 23: Coomassie blue-stained gel and immunoblot using the CE10 monoclonal antibody, of fractions taken during the immunoaffinity purification of NS-1 \r expressed in insect cells and eluted by 6M Guanidinium chloride. The method has been given in the Materials and Methods section M.2. The fractions presented are as follows: A) total cellular lysate, BP) nuclei and cellular debris pellet after hypotonic lysis, C) cytoplasmic lysate after pre-column clearing, D) unbound flow-through after cytoplasmic lysate application to CE-10 column, E) Buffer la wash flow-through, F) Buffer Ha wash flow-through, F1..F25) fractions collected after elution by 6M guanidinium chloride, Ml) BRL pre-stained high molecular weight markers, and M2) BRL high molecular weight markers. The Coomassie blue-stained gel is shown above, and the corresponding immunoblot against the CE10 mAb below. and light chains of the CE10 mAb and appear on the immunoblot due to the cross-reaction with the anti-mouse Ig secondary antibody. Second, the guanidinium was difficult to dialyze away completely, adding an extra day to the purification protocol. The presence of the guanidinium affected the running of the polyacrylamide gel by causing smearing, streaking, and aberrant mobilities in some fractions. A milder elution procedure using acidic conditions (pH 2 . 5 ) was attempted and shown to produce more encouraging results (Figure 2 4 ) . In the hypotonic lysis prior to the column purification, the nuclear pellet was resuspended in RIPA buffer before gel loading (lane BP). This appears to release more N S - 1 A C (c/-' Figure 22, lane BP). Whether this represents a small nuclear population of NS-1AC> or results from incomplete washing of the nuclei or incomplete lysis of the Sf9 cells is yet to be determined. As before, not all of the N S - 1 A C protein was bound to the 3.0 ml CE10 mAb column (lane D), although the washes did not release any of the bound N S - 1 A C (lane E & F). Elution at pH 2 .5 resulted in N S - 1 A C eluting from the column in a major peak centered at fraction 9 and a second minor peak at fraction 18 (lanes fl...f20). The second peak occurs after approximately 2V2 column volumes have been eluted, long after all released proteins would have been expected to elute. This 'tailing effect' may be due to re-absorption and re-elution occurring as the elution buffer is chased through by Buffer lb (Scopes 1987). Comparison of lane A and lane f9 in the immunoblot shows that no additional immunoreactive bands appear during the purification procedure. The gel of fraction 9 shows that several bands in addition to those identified on the blot are eluted; the major band being at 60 kDa that is not seen in any of the other elution methods. This band may be related to the one seen in the immunoprecipitations of N S - 1 A C . The absence of this band in previous purifications is likely due to the different wash protocol used. The N S - 1 A C band was the second major band and recovery of this protein, based on estimation of protein intensities from the Coomassie blue-stained gel, was approximately 18% at best. 74 9 7 4 ~ 68 .0 - — 43.0-29.0-18.4-Figure 24: Coomassie blue-stained gel and immunoblot using the CE10 monoclonal antibody, of fractions taken during the immunoaffinity purification of NS-1 xr expressed in insect cells and eluted by acid (pH 2.5). The method has been given in the Materials and Methods section M.3. The fractions presented are as follows: A) total cellular lysate, BP) nuclei and cellular debris pellet after hypotonic lysis, D) unbound flow-through after cytoplasmic lysate application to CE-10 column, E) Buffer lb wash flow-through, F) Buffer lib wash flow-through, fl..f20) fractions collected after elution by Buffer Illb , M) BRL high molecular weight markers (for gel) or BRL pre-stained high molecular weight markers (for immunoblot). The Coomassie blue-stained gel is shown above, and the corresponding immunoblot against the CE10 mAb below. 75 X. Discussion A. Monoclonal antibodies against MVM NS-1 Previous antibodies against M V M NS-1 have been obtained from polyclonal sera directed against NS-1 specific peptides, bacterial fusion proteins expressing portions of NS-1, or other members of the autonomous parvoviruses (Cotmore et al. 1983, Cotmore and Tattersall 1988). In this study, we describe the first characterization of monoclonal antibodies to NS-1. The six mAbs raised against a fusion protein containing Cys364 to Ala623 of M V M NS-1 were shown to be specific for the /acZ/NS-1 antigen by Western blot analyses and epitope mapping of the antibodies. Immunoblot analyses using the mAbs also showed their ability to detect an 83 kDa protein found only in MVM-infected mouse fibroblast cells. Uninfected cells did not express this protein. Therefore, the 83 kDa protein had to be a viral protein or a virus-induced host protein. Of the M V M proteins, only NS-1 and VP-1 migrate at an apparent mobility of 83 kDa. The predicted epitopes of the mAbs are found in NS-1 and not in VP-1, leading to the belief that the mAbs could be recognizing NS-1. Further evidence that the mAbs were, in fact, NS-1 specific was obtained from the indirect immunofluorescence studies of infected mouse cells. The kinetics of expression of the protein detected by the CE10 mAb in the first indirect immunofluorescence study are consistent with that of NS-1. The immunoreactive protein was detectable at 2 h.p.r. in the nucleus of MVM-infected cells with the intensity of the fluorescence increasing through 4, 6, and 8 h.p.r. The kinetics of NS-1 expression have been investigated previously by Cotmore and Tattersall using Western blot analysis (Cotmore and Tattersall, 1987). They, too, first detected NS-1 at 2 h.p.r. and found that the levels of NS-1 expression increased through 4 and 6 h.p.r. In their study, NS-1 was said to reach maximal levels of expression by 6 h.p.r, but no time points were shown past 6 h.p.r. Secondly, Bodnar et al. (1989) have proposed that the interaction with the nuclear matrix proceeds until all nuclear matrix attachment sites have become saturated and involves the breakdown of the 76 nucleoli. In our study, the fluorescence is seen first as discrete foci, corresponding to the nucleoli, and then on the periphery of the nuclei, indicating nuclear matrix attachment. Unfortunately, this experiment was not extended past 12 h.p.r. as it would be of interest to see the pattern of fluorescence in the final stages of the infection. From the data presented in this study, it is reasonable to assume that the mAbs are NS-1 specific. In the second immunofluorescence study, five of the six mAbs demonstrated nuclear staining in discrete foci. The M V M NS-1 protein is known to localize to the nucleus (Cotmore and Tattersall 1986a) and previous immunofluorescence studies by Walton et al. (1989) showed that M V M D N A and a 60 kDa host nucleolar antigen TP exhibit the same intranuclear organization as that of the protein recognized by the six mAbs. The Bodnar group have shown: 1) the foci to co-localize with nucleolar marker antigens using double-labelling experiments (Walton et al. 1989), and 2) the interaction of the M V M D N A with the nuclear matrix being mediated through the 5' end of the genome and the TP protein (Bodnar et al. 1989). Cotmore and Tattersall (1988) have shown 90-95% of M V M genomic D N A to be covalently bound through the 5' end to NS-1. Therefore, the association with the nuclear matrix is likely to involve a complex containing TP, NS-1, the 5' end of the M V M D N A , and possibly other proteins. The detection of fluorescence in the discrete foci within the nucleus using the mAbs further supports the assumption of NS-1 specificity and demonstrates the presence of NS-1 in the nuclear matrix attachment complex. The epitope mapping studies showed four epitopes (A - D) for the six mAbs; the CE10 and A C 6 mAbs recognize the same A epitope, the BE2 and EA2 mAbs recognize the B epitope, the CH10 mAb recognizes the C eptiope, and the BC4 mAb recognizes the D epitope. Three of the epitopes (A, B, and C) were clustered in a 16 aa region near the carboxy-terminal of lacZ/NS-l. The reason for the high antigenicity of this region is not clear. Keeping in mind that the original antigen had been denatured by heat and SDS, Kyte-Doolittle hydropathy plots showed that this region was only slightly hydrophilic. There are two proline residues which may produce a protruding surface structure that would be 77 antigenic (Berzofsky et al. 1985). The D epitope, on the other hand, does not contain any distinctive structural features or hydropathic characteristics which would account for its antigenicity. The ability of the mAbs to detect M V M NS-1 was not surprizing because the epitopes lay near the carboxy-terminal, and the hydropathy plot of M V M NS-1 predicts this end to be hydrophilic and, therefore, exposed to the aqueous environment. Of the two termini, the carboxy-terminal of M V M NS-1 is expected to be the more accessible, as the covalent linkage to the 5' end of the genome is thought to occur through an alkali-stable ester linkage near the amino-terminal rather than the carboxy-terminal (Cotmore and Tattersall 1988, Cotmore and Tattersall 1989). One disadvantage of these antibodies being specific for carboxy-terminal epitopes is that they would be unable to detect the 60 - 63 kDa, carboxy-terminal deleted forms of NS-1 which have been reported by Cotmore and Tattersall (1986b, 1987, 1988). Out of the six mAbs, only five showed binding to the paraformaldehyde-fixed, M V M -infected cells. The inability of the sixth mAb, BC4, to bind may be attributed to two factors. First, the binding affinity of the BC4 mAb may be weaker than the other five IgGi mAbs. The maximum A405 obtained in the titration ELISAs was 5- to 9-fold lower than that of the other mAbs. Unfortunately, the binding affinities of the mAbs could not be obtained from the competition ELISA data because the concentrations of the mAbs were not accurately known and not necessarily equivalent. Second, the D epitope may be inaccessible in the native protein. The antibodies which recognize epitopes closest to the carboxy-terminal of M V M NS-1 had the highest intensity of fluorescence, reaction on immunoblots, and absorbance readings in ELISAs; the antibodies with the epitopes furthest from the carboxy-terminal had the lowest. The termini of proteins usually have the greatest mobility and are often on the exposed surface of the protein (Lerner et al. 1984). It is possible that the D epitope is buried in the interior of the M V M NS-1 protein making detection of this epitope in the native protein impossible. This theory is not supported by hydropathy plots of the protein, which indicate the D epitope region to be hydrophilic. 78 B . Recombinant baculoviruses In this thesis, three recombinant baculoviruses expressing M V M N S - 1 or portions of N S - 1 have been used. Initial attempts by R. Russnak to express a full length M V M N S - 1 in insect cells using the pAcRP6 vector were unsuccessful, but the introduction of a frame-shift mutation in the NS-1 gene resulted in the isolation of the AcAXho virus. The ease in which AcAXho was isolated seemed to support the idea that NS-1 was cytotoxic to cells. Under this assumption, Russnak attempted to make the insect cells secrete NS-1, by fusing the preceruloplasmin signal sequence onto N S - 1 . The AcSec virus was thus isolated. The N S - 1 A C expressing virus, AcNS-1 was the result of later work by W. Chen and G. Wilson. Of the three recombinant viruses, the AcAXho virus had the lowest level of expression of the inserted gene. The low level of NS-l/NS-2 seen in the Coomassie blue-stained gel may be due to the pAcRP6 vector not containing the -7 to -1 polyhedrin nucleotides. These nucleotides have already been implicated in the high level expression of foreign genes (Matsuura et al. 1987). As this protein was not overexpressed and was not a full length NS-1 protein, further characterization of this protein was not attempted. This protein may be more useful when more is known about the functions of NS-1 or NS-2. The second recombinant virus, AcSec produced the CP/NS-1 protein, containing 47 amino-terminal amino acids from preceruloplasmin in place of the NS-1 amino-terminal. Human ceruloplasmin is a 132 kDa blood plasma protein synthesized in the liver and is the principal copper transport protein in plasma (Owen Jr. 1982). Preceruloplasmin contains a canonical signal sequence; the first 19 amino acids of the preceruloplasmin protein contain an 8 - 9 aa hydrophobic core, and alanine residues at the -1 and -3 positions (von Heijne 1981, von Heijne 1983, von Heijne and Abrahmsen 1989). Cleavage of the 19 amino acids produces ceruloplasmin. Unfortunately, the CP/NS-1 protein was not secreted into the extracellular media as expected. It was reasonable to believe that it would have been as there are several examples of human signal sequences being recognized in the Sf9 cells (Luckow and Summers 79 1988b) and of chimeric proteins containing non-homologous signal sequences being properly secreted (Zerial et al. 1987). The failure of this chimeric protein to be secreted by the insect cells may be due to the preceruloplasmin signal sequence not being recognized by the Lepidopteran protein processing mechanism. It would have been better to employ a signal sequence that was known to be cleaved by the signal recognition mechanism of these cells, although recognition of the signal sequence does not necessarily guarantee proper cleavage. Carbonell et al. (1988) reported the failure of a chimeric human B-interferon signal sequence/scorpion neurotoxin to be properly processed even though human B-interferon has been produced and secreted properly in insect cells (Smith et al. 1983). The problem was attributed to improper protein folding and post-translational modifications. Two other factors that may play a role in signal cleavage are the large size of the fusion protein, and the presence of a nuclear localization signal. Regardless of the failure of the signal sequence to be cleaved, a 77 kDa CP/NS-1 protein was identified on polyacrylamide gels of AcSec-infected Sf9 cell lysates, and on immunoblots using the CE10 mAb. The pattern of immunofluorescence shown by the CP/NS-1 protein indicated that the polypeptide was membrane-bound. Fluorescence appeared in a distinct manner around the circumference of the cell and remained until late in infection, when the cells had presumably lysed and soluble cytoplasmic proteins had been lost. As would be expected of a membrane-bound protein, Dounce homogenization did not release the majority of CP/NS-1, but neither did lysis in the presence of 1% ( v/ v) NP-40. Franke et al. (1981) showed that 0.5% ( v/ v) NP-40 disrupted all cellular membranes leaving an intact nuclear matrix scaffold. If CP/NS-1 were membrane-bound why was it not solubilized? One simple explanation would be that the protein was released from the membrane, but aggregated upon solubilization and remained in the pelleted fraction. Subcellular fractionation will be necessary to confirm the localization of this polypeptide. In searching for NS-1 function, the preceruloplasmin fusion protein had several drawbacks compared with M V M NS-1: 1) the CP/NS-1 protein had an altered cellular 80 location, 2) it contained 47 foreign amino acids, and 3) it did not contain a full length NS-1 protein; missing 41 aa from the amino terminal. Rather than simplifying the purification, the addition of the signal sequence actually complicated isolation of this protein. With all of these differences and purification problems, the use of this protein in determining the function of M V M NS-1 was not apparent. It did, however, serve as a useful test for the CE10 mAb column. With the isolation of the AcNS-1 virus, the further characterization of the CP/NS-1 protein became secondary to N S - 1 A C studies. The final recombinant virus presented in this thesis was the A c N S - l virus which was expected to express a full-length NS-1 protein. Infection of the insect cells with AcNS-1 produces an 84 kDa protein not found in AcNPV-infected cells, and Western blots of the lysates using the CE10 mAbs confirmed identification of the protein as N S - 1 A C - Expression levels for NS-lAc-were found to be intermediate between that seen for NS-l /NS-2 and CP/NS-1; the reasons for the differences in expression not being clear. The start codon sequence in this recombinant gene does not match the Miller consensus sequence for highly expressed baculovirus genes, being A C C A T G as opposed to A A N A T G (Miller, 1987). Inexplicably, site-directed mutagenesis of this sequence to A A C A T G did not result in a noticeable increase in expression levels (W. Chen, pers. comm.). Both the AcSec and the AcNS-1 viruses were isolated using the p A c Y M l vector, which contains the -7 to +1 nucleotides of the polyhedrin gene. Removal of the upstream non-coding regions of the inserted gene has been shown to result in greatly increased levels of expression (C.Y. Kang, pers. comm.) and this is currently in progress for N S - 1 A C to increase levels of expression to that of CP/NS- l or higher. The insect cell-expressed N S - 1 A C l S much more similar to native N S - l than the preceruloplasmin fusion protein. It runs at an apparent molecular weight of 84 kDa and appears to be phosphorylated . M V M NS-1 runs at an apparent molecular weight of 83 kDa, but a highly phosphorylated form of M V M NS-1 has been detected running at 85 - 88 kDa (Cotmore and Tattersall 1986a). For the related autonomous parvoviruses, H - l and PPV, 81 the predominant phosphorylated amino acid in the NS-1 protein was phosphoserine (Paradiso 1984, Molitor et al. 1985). Phosphoester bonds on serine and threonine residues are alkaline labile and alkaline elution of immunoaffinity purified N S - 1 A C resulted in the appearance of lower molecular weight bands that may be the result of such dephosporylation. A n initial attempt at [ 3 2P/]-labelling of baculovirus-encoded N S - 1 A C was unsuccessful although there were several problems with the protocol used that may have caused the failure of the NS-1 protein to be labelled (Yeung and Astell, unpubl. res.) The effect of the higher degree of phosphorylation on NS-1 is unknown, but levels of phosphorylation are known to affect activity and cellular location (Gerace and Blobel 1980, Sevaljevic et al. 1981, Roth et al. 1989). Caution must be taken when making speculations about N S - 1 A C modifications with respect to M V M NS-1, in that it is not known if the post-translational modification mechanisms are similar in insect cells and mammalian cells. A n interesting observation about the insect cell-expressed N S - 1 A C w a s its intracellular location. As mentioned previously, M V M NS-1 is a nuclear protein, but the immunofluorescence studies and the localization studies on the AcNS-1-encoded protein have shown N S - 1 A C to be cytoplasmic. The altered locale may indicate mutations in the nuclear localization signal, improper folding causing masking of the signal, or perhaps, incorrect post-translational processing (Roberts et al. 1987, Hunt 1989). Another intriguing possibility is that NS-1 requires the presence of another viral product (e.g. M V M D N A or NS-2) for proper localization. Though the cellular localization makes purification of this protein easier, the effect on function is yet to be determined. Indirect observations about the cytotoxicity of NS-1 can be inferred from the time-course study of the AcNS-1 infection. In this study, the bright field phase contrast microscopy pictures show that the cytopathic effects are seen earlier in AcNS-1 infection than in AcSec infection or wild-type AcNPV infection. The cytopathic effects (plasma membrane degradation and cytoplasmic release) are noticeable at 48 - 60 h.p.i in AcNS-1 infection but, at 60 - 72 h.p.i. in A c N P V infection. As well, the number of recombinant baculoviruses 82 obtained from the pAcYMl /NS-1 co-transfection was lower than expected (G. Wilson, pers. comm.). In the co-transfection of Sf9 cells with wild-type AcNPV D N A and the transfer vector, expression of foreign genes can be detected by24 hours post-transfection (Summers, pers. comm.). Therefore, a negative selection against recombinant baculoviruses expressing high levels of N S - 1 A C would occur. Possibly, the isolation of a virus with an altered cellular location for N S - 1 A C m a y he a result of this selection pressure. Both observations indirectly support, but in no way prove, the notion that N S - l is cytotoxic to cells in high concentrations. C. Immunoaffinity purifications The main purpose of this project was the isolation and purification of the N S - l protein. The monoclonal antibodies against NS-1 provided a specific probe and a means for its purification. Many references demonstrate the use of immunoaffinity chromatography as a single-step purification method yielding homogeneous and functional protein (Murphy et al. 1988, Simanis and Lane 1985, Tratner et al. 1990) and it was hoped that the CE10 mAb column would yield the same results. Several problems complicate the purification of N S - 1 from MVM-infected L A 9 cells; the main one being the very low level of N S - 1 produced in infected cells. Secondary problems include the tight association of N S - 1 with the nuclear lamina, the abundance of VP-1 which co-migrates with N S - 1 , and the probable cytotoxicity of N S - 1 . Over-production of N S - 1 A C using the baculovirus expression system alleviated the problems of low amounts of starting materials, VP-1 contamination, and N S - 1 cytotoxicity. Although partial purification of the polypeptide was achieved, the immunoaffinity purification of N S - 1 A C did not yield a 100% pure preparation of the protein, and no biochemical activity of the polypeptide has yet been demonstrated. The first problem with the purification procedure was the presence of other immunoreactive bands and non-specific bands that elute with the CE10 mAb-bound proteins. The cross-reactive bands detected by the immunoblot analysis appear to be degradation products of NS-1 Ac as they can be minimized by the addition of PMSF. Other protease 83 inhibitors have not been tried, although it would be wise to include them to see i f the number of lower molecular weight bands could be reduced further. Reduction of the number of proteins non-specifically eluted will require more work on optimizing the wash conditions. A 0.5M L i C l wash buffer removed several of these proteins but also affected the bound N S - 1 A C (Figure 23). Ideally an elution method specific for N S - 1 A C will have t o be found. Peptide elution was attempted in order to elute only molecules bound to the CE10 mAb at its antigen binding site. Even though the CE10 mAb had been shown to bind the peptide, this method did not result in the release of N S - 1 A C from the column. The reason for the failure of the peptide elution is likely due to the respective binding affinities of the mAb for the NS-1 Ac protein and the peptide; the affinity for the protein being higher than that for the peptide. Under the elution conditions used, the peptide was theoretically at a 5-fold molar excess over the antigen binding sites. A second explanation for the failure of the peptide to compete off the bound N S - 1 A C molecules would be that there was not sufficient time for the replacement reaction to occur. Antibodies with strong binding affinities (K a ) have a calculated ti/2 on the order of 23 minutes for the dissociation reaction (Berzofsky and Berkower, 1984). A worthwhile option would be to re-test the peptide elution method using one of the other five mAbs as the immunoaffinity matrix and an appropriate peptide. Of the four elution methods attempted, the acid elution procedure appears to be best; not adversely affecting the post-translational modifications of the NS-1 protein or the CE10 mAb column stability. The effect of the acid elution on the activity of the NS-1 preparation is not known although it has been used in other immunoaffinity purifications yielding functional proteins. A second problem was the recovery of protein. The column had the CE10 mAb bound at a concentration of 7 r n 8 / m i swollen beads- Samples taken before and after binding of the mAb to the CNBr-activated Sepharose 4B indicated a 99.3% coupling efficiency of the mAb to the Sepharose beads, but when the AcNS-1 cellular lysate was added to this column material, not all of the N S - 1 A C was bound even though it would theoretically amount to only Vionth of the binding capacity of the column. The degradation products were not included in this 84 calculation, but would not be expected to account for all of the remaining binding sites. The CNBr method for coupling of antibodies to solid matrices can produce the wrong spatial orientation of the mAbs with the antigen binding site pointed in towards the matrix. This would lead to a reduced binding efficiency for the column (Schneider et al. 1982). Alternative methods for antibody coupling include the use of other chemical linkages, Protein A , or Protein G (Harlow and Lane 1988, Bio-Rad Laboratories 1988) and should be investigated. The work presented in this thesis serves as a starting point for future studies. The mAbs characterized in this study will be of use in the detection of the M V M N S - 1 expressed in infected cells or other systems. With the high degree of antigenic conservation of N S - 1 polypeptides among the parvoviruses, it would be interesting to see if these mAbs could also detect N S - 1 proteins from the other parvoviruses. Monoclonal antibodies have several advantages over the traditional polyclonal sera in terms of supply, homogeneity, and specificity. Already, we have demonstrated the usefulness of the antibodies in the specific purification of NS-1AC> although the results presented here indicate that the purification protocol has not yet been fully optimized. Still, functional studies can be done on the partially purified N S - 1 A C - The expression of N S - 1 in the baculovirus system is the first documented case of the overexpression of this protein and will be of great value once protein activity can be demonstrated. 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Boca Raton: C R C Press Inc. XIII. Apendices EcoRl Appendix la: Construction of pAcRP6/AX. This clone, constructed by R. Russnak, encodes a NS-l /NS-2 fusion protein. The NS-1 gene contains a frameshift mutation as a result of the filling-in of the unique Xhol site within the NS-1 gene, shifting the reading frame to that for NS-2. Hindm Hindi Smal SstI Cut with Hgal, isolate 2.3 kb Hgal fragment, & fill-in ends J pGEM-4Z J Hindi . . . A n , . i .EcoRl EcoRl >»" '>l^ SstI I J l h C P l j Cut with Smal EcoRV SstI Ligate together HindDI Cut out insert with EcoRl, & religate in reverse orientation HincII Hindi EcoRl EcoRl SstI Cut with SstI / EcoRV & replace the fragment with the SstI / Hind fragment of hCPLR Ssd Cut with Hind / SstI & isolate the 150 bp S st / Hind fragment containing the hCP signal sequence Hindm CPss/NSl/pGEM-4Z 96 Appendix lb: Construction of CPss /NS- l /pAcYMl . The strategy used to construct the human preceruloplasmin (hCP) signal sequence - M V M NS-1 fusion protein encoding baculovirus transfer vector is shown above. This was conceived and carried out by R. Russnak as described in the Materials and Methods section B2. 97 EcoRI BamHI Hindffl BamHI Hindffl Cut with Hgal, isolate 2.3 kb Hgal fragment, & fill-in ends Ligate on BamHI linkers BamHI Cut with BamHI & isolate 2.3 kb fragment BamHI polyhedrin Cut with BamHI Ligate BamHI BamHI Appedix Ic: Construction of p A c Y M l / N S - 1 . W. Chen's construction of the baculovirus transfer vector for the expression of an unfused, un-mutated NS-1 is schematically shown above. For the method of construction, refer the Materials and Methods section B2. Appendix II: Nucleotide sequences of the pUC19/D deletions at the junction, predicted  carboxy-terminal protein sequence of the resulting lacZ/NS-l fusion  proteins, number of the last amino acid sharing homology with lacZfNS- 1. and the number of the corresponding amino acid in M V M N S - l a . trivial name delete size coding strand sequence at the deletion junction 0 protein sequence at junction^ last lacZI NS-1 a.a. last NS-1 a.a. 1.13a A13 ACT GCA GGT CGA CTA AGT AAT TAG .. .TAGRLSN* 273 623 2.2a A18 G GGC ACT GCA GCC TAA GTA ATT AG . . .GTAA* 270 623 0.8a A21 G GGC ACT GGA TCC TAA GTA ATT AG ...GTGS* 269 622 0.14a A21 G GGC ACT GGA TCC TAA GTA ATT AG ...GTGS* 269 622 0.6a A23 G C G GGC ACG ATC CTA AGT AAT TAG ...AGTILSN* 269 622 1.14a A40 ACA CCA AAT ATC CTA AGT AAT TAG ...TPNILSN* 263 616 1.2a A46 GG AGC A C A GAT CCT AAG TAA TTA G ...WSTDPK* 261 614 1.5a A51 A GAG CCT T G G ACC TAA GTA ATT AG ...EPWT* 259 612 1.3a A52 TA GAG CCT T G G CCT AAG TAA TTA ...EPWPK* 259 612 2.5a A61 T G GCT T T A GAT CCT AAG TAA TTA G ...ALDPK* 256 609 V2.2 A70 AT CTC GAG GAC CCT AAG TAA TTA . . .LEDPK* 253 606 1.8a A71 GAT C T C GAG GAC CTA AGT AAT TAG . . .DLEDLSN* 253 606 V2.15 A81 T GCA T C G GGA TCC TAA GTA ATT AG ...ASAS* 249 602 V2.13 A90 A ACT CCA CGA TCC TAA GTA ATT AG ...TPRS* 246 599 2.4a A102 G AAC T A T GGA TCC TAA GTA ATT AG ...NYGS* 242 595 V2.3 A103 AGC CAG AAC TAT CTA AGT AAT TAG ...SQNYLSN* 242 595 2.6a A102 G AAC TAT GGA TCC TAA GTA ATT AG ...NYGS* 242 595 V2.6 A109 CT CTC AGC CAG CCT AAG TAA TTA G ...LSQPK* 240 593 V2.4 A121 AA ACT ACG GAT CCT AAG TAA TTA G ...STDPK* 236 589 V2.7 A121 C C G AAA AGT A C G CTA AGT AAT TAG ...PKSTLSN* 236 589 V2-14 A123 A C C G AAA AGT ACC TAA GTA ATT AG ...PKST* 236 589 V2.1 A138 C T C A CCA T T C ACC TAA GTA ATT AG ...SPFT* 230 583 1.14 A157 AAT TTA CTA GGT CTA AGT AAT TAG . . .NLLGLSN* 224 577 1.6 A170 CCA ACT CCT ATC CTA AGT AAT TAG ...PTPILSN* 219 573 1.18 A184 AG CCA A A G GAT CCT AAG TAA TTA G ...PKDPK* 215 569 1.23 A184 C G GAG CCA A A G CCT AAG TAA TTA G .. .EPKPK* 215 569 1.7 A191 C T G C GGA GCC ATC TAA GTA GTT AG . . .NCAAI* 210 564 1.25 A201 T G G TCA GAA ATC CTA AGT AAT TAG ...WSEILSN* 209 563 1.20 A202 GG T C A GAA GAT CCT AAG TAA TTA G ...SEDPK* 209 563 1.8 A219 G GGC AAA GGA TCC TAA GTA ATT AG ...GKGS* 203 557 1V2-30 A230 C T A C T G T GCT AAC TAA GTA ATT AG . . . Y C A N * 199 553 1V2-32 A250 AA TCT A C C GAT CCT AAG TAA TTA G ...STDPK* 193 547 .1V2.16 A251 T A C CAA T C T ACC CTA AGT AAT TAG .. .YQSTLSN* 193 547 1V2-6 A263 AAG AAT GGG ATC CTA AGT AAT TAG ...KNGDPK* 189 543 1V2-13 A276 T T TGT GCT T G G TCT AAG TAA TTA G ...CAWSK* 184 538 1V2-4 A276 TT TGT GCT T G G TCT AAG TAA TTA G ...CAWSK* 184 538 1V2-7 Mil GT GCT T G G GAT CCT AAG TAA TTA G ...AWDPK* 184 538 1V2-3 A298 C AAA AAT GAA TCC TAA GTA ATT AG . . .KNEF* 177 531 1V2-1 A314 TTT GGT TTG ATC CTA AGT AAT TAG .. .FGLILSN* 172 526 1V2-10 A315 GT GAC TTT GGT TCT AAG TAA TTA G .. .DFGWD* 171 525 2V2-4 A316 GGT GAC TTT GGT CTA AGT AAT TAG . . .GDFGLSN* 172 526 1V2-14 A317 GGT GAC TTT GGC CTA AGT AAT TAG . . .GDFGLSN* 171 525 1.15 A355 GAC AGA A T G CTT CTA AGT AAT TAG . . .DRMLLSN* 158 512 2J/2.1 A386 AGA CCA GAG ATC CTA AGT AAT TAG ...RPEILSN* 148 502 2V2-5 A400 GA ATA GGC TGC CCT AAG TAA TTA G ...IGCPK* 143 497 3V2.2 A479 CAA AAA GGG ATC CTA AGT AAT TAG . .QKGMLSN* 117 471 1.15a A970 promoter deleted none 0 0 1.6a A1025 promoter deleted none 0 0 Deletion clones were made as described in the Materials and Methods section and named based on the time point they were taken from, followed by a distinct number for that time point. The 'a' designation indicates an earlier trial. Sequencing of the clones was performed as described in the Materials and Methods, allowing calculation of the size of the D N A deletion and prediction of the carboxy-terminal protein sequence. Amino acids are given by their single letter code with '*' designating a termination codon. This sequence was then compared against that of /acZ/NS-1 to determine amino acid sequences not present in M V M NS-1. Finally, the last amino acid of M V M NS-1 sharing homology with the carboxy-terminal-deleted /acZ/NS-1 proteins was determined from the primary protein sequence of M V M NS-1. M V M sequences are in bold text, and termination linker sequences are given in plain text. 

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