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Molecular characterization of potato virus S and genetic engineering of virus resistant plants MacKenzie, Donald J. 1990

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MOLECULAR CHARACTERIZATION OF POTATO VIRUS S AND GENETIC ENGINEERING OF VIRUS RESISTANT PLANTS by DONALD J. M A C K E N Z I E B.Sc. University of British Columbia, 1977 M.Sc. University of British Columbia, 1980 Thesis submitted in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN T H E FACULTY OF GRADUATE STUDIES (BIOCHEMISTRY) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA Copyright © 1990, Donald }. MacKenzie 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 writ ten permission. The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT The sequence of 3553 nucleotides corresponding to the 3'-terminal region of potato virus S (PVS) has been determined from cloned cDNA. The sequence obtained contains six open reading frames with the potential to encode proteins of Mr 10,734, Mr 32,515, Mr 7,222, Mr 11,802, Mr 25,092 and at least Mr 41,052. The amino acid sequence of the 33K ORF has been confirmed to be that of the viral coat protein gene. The nucleotide sequence of this ORF was obtained from expression plasmids which were isolated by binding with a specific monoclonal antibody to PVS, and the expression of coat protein fusion products was verified by Western blots of bacterial cell lysates. The deduced amino acid sequence of a 70 amino acid portion from the central region of the PVS coat protein was 59% identical to the analogous region of potato virus X. In addition, the 7K, 12K and 25K ORF's displayed significant sequence homology with similar sized ORF's from a number of potexviruses. The partial 41K ORF was homologous with the C-terminal portion of the viral replicase proteins of potato virus X and white clover mosaic virus. While the biological functions of the 12K and 25K non-structural proteins coded for by PVS and members of the potexvirus group remain unknown, the 12K protein displays a hydropathicity profile consistent with a membrane associated protein and the 25K protein contains a conserved sequence motif found in a number of nucleoside triphosphate binding proteins. Members of the carlavirus i i group are distinguished from the potexviruses by the presence of a small [11K (PVS, potato virus M) - 16K (lily symptomless virus)] 3' terminal ORF which appears to contain a sequence motif similar to the 'zinc-finger' domain found in many nucleic acid binding proteins. The coat protein gene from potato virus S (PVS) was introduced into Nicotiana debneyii tobacco as well as a commercial potato cultivar, 'Russet Burbank', by leaf disc transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral coat protein were highly resistant to subsequent infection following mechanical inoculation with the Andean or M E strains of PVS as indicated by a lack of accumulation of virus in the upper leaves. The coat protein mediated protection afforded by these transgenic plants was sufficient to prevent the accumulation of virus in the tissues of non-transformed 'Russet Burbank' shoots which had been grafted onto transgenic plants inoculated with PVS, and in reciprocal grafts, transgenic shoots accumulated less than 2% (6 weeks after grafting) of the concentration of PVS found in non-transformed shoots similarly grafted onto plants systemically infected with PVS. These transgenic plants also displayed a measure of resistance to inoculation with a related carlavirus from potato, potato virus M . In agreement with previous reports for plants expressing PVX coat protein, plants expressing PVS coat protein were also protected from inoculation with PVS RNA. These results provide further evidence that coat protein mediated protection for these two groups of viruses, which share similar genome iii organizations, may involve inhibition of some early event in infection, other than, or in addition to, virus uncoating. Specific monoclonal antibodies were prepared against a C-terminal derived 18 kDa portion of the 25K protein of PVS expressed as an in-frame chimeric fusion protein with the glutathione S-transferase gene. The in vivo expression of this non-structural protein in virus infected tissue, as well as tissue from transgenic tobacco (var Xanthi-nc) engineered to contain the entire 25K gene, was verified by Western immunoblot labelling. iv TABLE OF CONTENTS Abstract ii Table Of Contents v List Of Figures vii List of Tables ix List of Abbreviations x Acknowledgements xii Aim and Scope of This Work xiii 1.0 Genome Organization and Interviral Homologies of Potato Virus S RNA 1 1.1 Introduction 1 1.2 Materials and Methods 4 1.2.1 Virus Purification and Preparation of Viral RNA 4 1.2.2 Radioiodination of Antibody Reagents 4 1.2.3 Preparation of Competent Cells, Plasmid Transformations and Preparation of Plasmid DNA 5 1.2.4 Preparation of cDNA and Isolation of Recombinant Plasmids Expressing Portions of the PVS Coat Protein Gene 6 1.2.5 Preparation of Random-Primed [ 3 2P]-Labelled cDNA Probes 9 1.2.6 Western Blot Analysis of Expressed Fusion Proteins 10 1.2.7 Immunoprecipitation of PVS RNA in vitro Translation Products 11 1.2.8 Nucleotide Sequence Determination 12 1.3 Results . 14 1.3.1 Western Blot Analysis of Expressed Fusion Proteins 14 1.3.2 Nucleotide Sequence Determination 14 1.3.3 Organization of the 3'-Terminal Region of PVS 15 1.4 Discussion 29 2.0 Genetic Engineering of Plants Resistant to PVS Infection 37 2.1 Introduction 37 2.1.1 Mechanisms of Agrobacterium tumefaciens Mediated Plant Cell Transformation 38 2.1.2 Cross-Protection As a Mechanism For Modulating Virus Infection 44 2.1.3 Genetically Engineered Resistance To Viral Infection 46 2.2 Transgenic Expression of PVS Coat Protein in Nicotiana debneyii 50 2.2.1 Materials and Methods 50 2.2.1.1 Construction of Plasmid pVS153 50 2.2.1.2 Tri-Parental Mating Procedure 54 2.2.1.3 Transformation and Regeneration of Nicotiana debneyii . . 55 2.2.1.4 Western Blot Analysis of PVS Coat Protein Expression in Transgenic Nicotiana Debneyii 56 v 2.2.1.5 Purification of Rabbit Anti-PVS Ig 57 2.2.1.6 Preparation of Enzyme-Antibody Conjugates 58 2.2.1.7 Evaluation of PVS Resistance In Transgenic Nicotiana debneyii 58 2.2.2 Results 61 2.2.2.1 Transformation of N. debneyii and Expression of PVS Coat Protein 61 2.2.2.2 Resistance of Transgenic N. debneyii To PVS Infection . . . 61 2.3 Transgenic Expression of PVS Coat Protein in 'Russet Burbank' Potato . . . . . 67 2.3.1 Materials and Methods 67 2.3.1.1 Transformation and Regeneration 67 2.3.1.2 Enzyme-Linked Immunosorbent Assay (ELISA) 68 2.3.1.3 Virus Inoculation of Potato Plants 70 2.3.2 Results 71 2.3.2.1 Regeneration and transformation 71 2.3.2.2 Expression of PVS coat protein 72 2.3.2.3 Susceptibility of Transgenic 'Russet Burbank' Potato to Infection with PVS or Viral RNA 73 2.3.2.4 Susceptibility to Infection with PVM 82 2.4 Discussion 84 3.0 Characterization of PVS Nonstructural Proteins 90 3.1 Introduction 90 3.2 Materials and Methods 93 3.2.1 Expression of a Portion of the PVS 25K Protein in pGEX-2T 93 3.2.2 Expression of a Portion of the PVS Viral Polymerase in pGEX-1 95 3.2.3 SDS-PAGE Analysis of Chimeric Fusion Proteins 95 3.2.4 Purification of pGEX Based Fusion Proteins 96 3.2.5 Immunization and Cell Fusion 96 3.2.6 ELISA 97 3.2.7 Transgenic Expression of the PVS 25K Protein in Transformed Tobacco 98 3.3 Results 101 3.3.1 Preparation and Purification of pVS631 and pVS707 Fusion Proteins 101 3.3.2 Cell Fusion and MAb Production 102 3.3.3 Expression of the PVS 25K protein in virus infected and transgenic tobacco plants 103 3.4 Discussion 105 Future Prospects 107 References 110 vi LIST OF FIGURES Fig.No. Description Page Strategy used for preparing recombinant complementary DNA (cDNA) clones for PVS RNA. Western blot analysis of E. coli cell extracts from clones expressing different sized /acZ-PVS coat protein fusion products and immunoprecipitation of in vitro translation products of PVS RNA. The genome organization and restriction endonuclease map of the 3'-terminal region of PVS RNA. The sequence of 3553 nucleotides corresponding to the 3' terminus of PVS RNA. Amino acid homology between portions of the coat proteins of PVS, two related cariaviruses, lily symptomless virus (LSV) and potato virus M (PVM), and five members of the potexvirus group, potato virus X (PVX), papaya mosaic virus (PMV), narcissus mosaic virus (NMV), white clover mosaic virus (WCIMV) and potato aucuba virus (PAMV). Alignment of the amino acid sequence of the PVS 7K ORF with the similarly sized ORFs from PVX, PVM, LSV and potato aucuba virus (PAMV), and comparison of the homologies between the 12K ORF of PVS and the 14K ORF from BSMV RNA 2B as well as similarly sized ORFs from PVM, PVX, narcissus mosaic virus (NMV) and WCIMV. Alignment of the 25K protein from PVS with the 25K protein from PVM and the 25K and 26K proteins from PVX and WCIMV. Comparison between a 136 amino acid portion of the extreme 5'-end partial 41K ORF of PVS and similar regions of the 166K protein of PVX and the 147K protein of WCIMV, and a comparison between the amino acid sequences of the 3' terminal 11K ORFs of PVS and PVM. Comparison of the hydropathicity profiles of the 12K proteins from PVS and PVX. Comparison of the genome organization of PVS and PVX. Plant cell transformation by A. tumefaciens. Schematic representation of the method used for integration of the PVS coat protein gene into the intermediate co-integrate vector, pCDX-1. Schematic representation of the tri-parental mating procedure used for the integration of the T-DNA region of pVS153 into the resident disarmed Ti plasmid, pTiB6S3SE, carried by A. tumefaciens GV3111SE, via homologous recombination. Immunoblot analysis of transgenic N. debneyii, line J3, expressing PVS coat protein. vii LIST OF FIGURES Fig.No. Description Page Comparison of symptom development on control, non-transformed, N. 63 debneyii plants compared with the J3 line of transgenic N. debneyii after inoculation with purified PVS, strain ME. Accumulation of PVS coat protein antigen in the upper leaves of non- 64 transformed N. debneyii and J3 transgenic N. debneyii after inoculation with PVS strain ME, at various days post inoculation. DAS-ELISA detection of PVS in J3 transgenic N. debneyii and non- 65 transformed N. debneyii following inoculation with purified PVS RNA. Western blot analysis of PVS coat protein expression in transgenic 72 'Russet Burbank' potato plants. Accumulation of PVS coat protein antigen in the upper leaves of non- 74 transformed, and RB58 transgenic 'Russet Burbank' potato after inoculation with either intact PVS particles, or PVS RNA. Concentration of PVS coat protein antigen in the tissues of systemically 80 infected plants containing shoot grafts from either transgenic (RB41, RB58), PVM infected or non-transformed plants. Concentrations of PVS coat protein antigen in the leaf or stem tissues of 81 plants systemically infected with PVS containing double grafts of transgenic RB58 tissue and upper non-transformed tissue. Accumulation of PVM coat protein antigen in the upper leaves of either 82 non-transformed, or RB52 and RB41 transgenic 'Russet Burbank' potato after inoculation with intact PVM particles, at various days post inoculation. Schematic representation of protocol used for the expression of a portion 94 of the PVS 25K ORF in pGEX-2T as an in-frame fusion protein. Coomassie Blue staining patterns of E. coli cell extracts and purified 101 fusion proteins separated by SDS-PAGE. Binding of MAb's 2E3 and 3E2 to fusion proteins pVS631 and pVS707 by 102 ELISA. Western immunoblot analysis of PVS 25K expression in either PVS 103 infected tissue, or in tissue from transgenic Xanthi tobacco plants engineered to express this protein. viii LIST OF TABLES Table No. Description Page Comparison of amino acid sequence similarities between the capsid 20 protein of PVS and other members of the carlavirus and potexvirus group Concentration of PVS coat protein antigen in transgenic and normal 65 Nicotiana debneyii 45 days after inoculation with either intact PVS or viral RNA. Concentration of PVS and PVX coat protein antigen in transgenic J3 and 66 non-transformed Nicotiana debneyii following inoculation with PVS (ME strain), PVX (severe strain) and a mixture of PVS and PVX. IV Composition of various media used for the regeneration of transformed 68 'Russet Burbank' potato plants. V Concentration of PVS coat protein antigen in transgenic and non- 75 transformed 'Russet Burbank' potato following inoculation with PVS or PVS RNA. VI Accumulation of PVS in non-transformed or transgenic potato tissue 79 following grafting onto transgenic or virus infected host plants. ix LIST OF ABBREVIATIONS A1MV alfalfa mosaic virus (ilarvirus) A M P adenosine monophosphate ATP adenosine triphosphate BA benzyladenine BNYVV beet necrotic yellow vein virus (furovirus) BSA bovine serum albumin BSMV barley stripe mosaic virus (hordeivirus) CaLV carnation latent virus (carlavirus type member) CaMV cauliflower mosaic virus (caulimovirus) cDNA complementary deoxyribonucleic acid dATP deoxyadenosine triphosphate DMSO dimethylsulfoxide D N A deoxyribonucleic acid dNTP deoxynucleotide triphosphate ds double stranded Dt deoxythymidine DTT dithiothreitol E. coli Escherichia coli EDTA ethylenediamine tetraacetic acid ELISA enzyme-linked immunosorbent assay FCS fetal calf serum GUS fi-glucuronidase HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid IAA indole acetic acid Ig immunoglobulin IgG immunoglobulin subtype G IPTG isopropyl-fi-D-thiogalactopyranoside kb kilo base Kda kilo Dalton LSV lily symptomless virus (carlavirus) Mab monoclonal antibody MES 2-(N-morpholino)-ethanesulfonic acid M r relative molecular weight MS Murashige & Skoog salts for plant tissue culture N A A naphthalene acetic acid N M V narcissus mosaic virus (potexvirus) ORF open reading frame PAGE polyacrylamide gel electrophoresis P A M V potato aucuba mosaic virus (potexvirus) X LIST OF ABBREVIATIONS PBS phosphate buffered saline (0.2 g/1 K H 2 P 0 4 / 0.2 g/1 KC1, 8 g/1 NaCl, 2.27 g/1 Na 2 HP0 4 .7H 2 0) PMSF phenylmethylsulfonyl fluoride P M V papaya mosaic virus (potexvirus) PVS potato virus S (carlavirus) P V M potato virus M (carlavirus) PVP polyvinyl pyrrolidine PVX potato virus X (potexvirus) PVY potato virus Y (potyvirus) R N A ribonucleic acid rpm revolutions per minute SDS sodium dodecyl sulfate ss single stranded TCA trichloroacetic acid T-DNA transfer deoxyribonucleic acid Ti tumour inducing (Ti plasmid) TMV tobacco mosaic virus (tobamovirus) Tris Tris(hydroxymethyl)aminomethane Wclmv white clover mosaic virus (potexvirus) xi ACKNOWLEDGEMENTS I am indebted to Dr. J. McPherson of the Department of Plant Science, University of British Columbia for providing the Agrobacterium tumefaciens (GV3111SE/pTiB6S3SE) strain and the pCDX-1 vector. I also thank M . Elder and A . Wiezcorek of the Vancouver Research Station, Agriculture Canada, for providing the ME strain of PVS and specific monoclonal antibodies to PVS, P V M and PVX, respectively, and K. Turner for his many helpful discussions and suggestions regarding potato tissue culture. xii AIM AND SCOPE OF THIS WORK Viruses belonging to the carlavirus group, which is composed of more than 25 different members, many of which are serologically related, have been only poorly characterized to date. While in many cases, much is known of the distribution, host ranges and physical characteristics of these latent viruses (particle morphology, sedimentation coefficients, nucleic acid type and capsid molecular weights) no information regarding their genome organization and relationship with other viruses and virus groups has previously been reported. In this regard the initial goal of this project was to clone and sequence a portion of the genome from potato virus S (PVS), a carlavirus which has been reported to be endemic in many potato cultivars worldwide. This nucleotide sequence information was important in revealing a significant, and hitherto unsuspected, relationship between PVS and members of the potexvirus group which had previously been believed to be (on the basis of particle morphology, modes of transmission and serology) quite distinct. In recent years the ability to genetically engineer transgenic plants expressing new and novel gene products has generated much excitement and many promising results for the prospect of utilizing this approach for control of a number of viral pathogens. The second primary objective of this work was to produce transgenic plants which constitutively expressed the coat protein gene from PVS with the ultimate aim of introducing genetically engineered xiii resistance to viral infection into one of the most important commercial cultivars of potato, 'Russet Burbank'. xiv CHAPTER 1 1.0 GENOME ORGANIZATION AND INTERVIRAL HOMOLOGIES OF POTATO VIRUS S R N A . 1.1 INTRODUCTION Potato virus S (PVS) was first described in potato almost 40 years ago in Holland (De Bruyn & Maria, 1951), and has since been found in all temperate climates worldwide. PVS is a member of the carlavirus group and is transmitted by aphids in a non-persistent manner to members of the Solanaceae and Chenopodiaceae. The carlavirus group is composed of more than 25 different viruses, many of which are serologically interrelated. Viruses within this group are composed of slightly flexuous particles with average dimensions of 610 to 700 nm long and 12 to 15 nm diameter. The particles sediment at 147 to 176 S, contain 5 to 7% R N A by weight and have an average molecular weight of 50 to 60 x 106. Virus capsid is composed of a single type of protein subunit generally of 31 to 34 K. The typical carlavirus genome consists of a single molecule of single-stranded R N A of 2.3 to 3,0 x 106 molecular weight, and little is known of the genome organization. Most carlaviruses have restricted host ranges, but the different viruses occur in a wide range of monocotyledonous and dicotyledonous hosts. While some viruses within this group cause serious 1 disease, many of them are symptomless, at least in certain hosts, as is true for carnation latent virus (CaLV), from which the group name is derived. The cytopathology of many carlavirses has been examined with the general conclusion that infected cells do not contain any virus specific inclusion bodies, nor are specific cytopathological effects obvious by which such infections can be recognized (Martelli & Russo, 1977). Virus particles tend to occur singly or in masses within the cytoplasm of infected cells and have not been observed within cell organelles such as chloroplasts, nuclei or mitochondria. Because it is nearly symptomless in potato, PVS has been a difficult virus to control and can occur in seed potatoes at levels of 80% or more. In combination with potato virus X, it can cause significant reduction in tuber yields (Wright, 1977). Elite seed potato stocks in Canada are produced from virus-free material by heat therapy and meristem tip culture (Wright, 1987), but such stocks become rapidly reinfected with PVS once grown in the field (Hahm et al, 1981). Contributing to this is the fact that many potato viruses, including PVS, overwinter in tubers missed in the previous harvest, and volunteer plants emerging from such tubers are a primary source of inoculum and play a significant role in virus epidemiology. PVS RNA is encapsidated in a Mr 32,000 to Mr 34,000 (33K) coat protein species into slightly flexuous filamentous particles of dimensions 650 nm x 12 nm (Koenig, 1982). The viral genome consists of one single-stranded, positive 2 sense, R N A molecule with an estimated Mr=2.39 x 106 which contains a 3' terminal poly-adenylated region (Monis et ah, 1987). Translation of PVS R N A in vitro using a rabbit reticulocyte lysate has been reported to yield primarily four products of Mr= 124K, 112K, 98K and 36K (Monis et al, 1987). While the molecular mechanisms of PVS disassembly/assembly in infected cells is unknown, it is believed that the viral coat protein is translated from an encapsidated subgenomic R N A species of approximately 1.3 kb (Foster & Mills, 1990). The presence of encapsidated subgenomic RNAs encoding the viral coat protein has also been reported for at least two members of the potexvirus group, white clover mosaic virus (Forster et ah, 1987) and narcissus mosaic virus (Short & Da vies, 1983). This chapter describes the preparation of complementary D N A (cDNA) clones to the viral R N A and determination of the nucleotide sequence corresponding to 3553 nucleotides from the 3'- terminus of the Andean strain of PVS. Amino acid sequence homologies between the coat protein gene, and five other predicted open reading frames, with similar regions from potato virus X (PVX) and other members of the potexvirus group are discussed. 3 1.2 MATERIALS AND METHODS 1.2.1 Virus Purification and Preparation of Viral RNA. The Peruvian (Andean) strain of PVS (Hinostroza-Orihuela, 1973) was purified from systemically infected Chenopodiwn quinoa. Leaf tissue was homogenized in a Waring blender with 3 volumes of ice cold 0.1 M sodium borate p H 8.2, 0.1M EDTA containing 2%(w/v) PVP-44 and 0.1% 2-mercaptoethanol. The homogenate was expressed through nylon cloth and the sap was centrifuged at 10,000 x g for 20 min (Sorval GSA rotor). The supernatant was centrifuged at 111,000 x g for 90 min in a Ti 50.2 rotor and the pellets were resuspended in 0.01 M sodium phosphate p H 7.5. Following a low speed clarification, caesium chloride was added to a final concentration of 40% (w/v) and the solution was centrifuged at 252,000 x g for 60 hr in a Ti 70.1 rotor. The opalescent band was collected and dialysed against 0.01 M sodium phosphate p H 7.5. Virus yields were estimated from the absorbance at 260 nm assuming E 2 6 0 i mg/mi — 3.0 . Viral RNA was prepared from alkaline SDS treated particles by multiple phenol-chloroform extractions and ethanol precipitation (MacKenzie & Tremaine, 1988). 1.2.2 Radioiodination of Antibody Reagents. Purified goat anti-mouse immunoglobulin (Ig) was radioiodinated by using the chloramine T method (Hunter & Greenwood, 1962). Approximately 4 0.5 mCi of carrier free Na[125I] was added to 200 ug of protein in 0.1 ml of PBS and the reaction initiated by addition of 5 (il of chloramine T (5 mg/ml in water). After a 10-15 min incubation at room temperature, the iodinated protein was separated from free [125I]iodide and other reactants by diluting the reaction mixture with 0.5 ml of PBS and centrifuging this solution through 0.5 g of AG1X10-C1 ion-exchange resin (Bio-RAD) placed in the reservoir of a 0.2\i microfiltration device (BioAnalytical Systems) (Molday & MacKenzie, 1985). Iodinated protein was stored at 4 °C in the presence of 0.1% (w/v) N a N 3 until used. 1.2.3 Preparation of Competent Cells, Plasmid Transformations and Preparation of Plasmid DNA. In general, and unless otherwise specified, plasmid D N A was transfected into E. coli DH5a cells which had been rendered competent by the CaCl 2 method of Hanahan, 1983. For transformation, aliquots of competent cells (100 (il) were incubated in the presence of 10-50 ng (1-5 jllI) of plasmid D N A for 30 min on ice, then cells were heat shocked (42 °C, 42 seconds) and placed back on ice (5 min). Transformed cells were then cultured for 1 hr at 37 °C with 1.0 ml of antibiotic free LB medium (10 g/1 tryptone, 5 g/1 yeast extract, 5 g/1 NaCl), washed l x by centrifugation (4000 g x 5 min), resuspended with 0.5 ml fresh LB and 100 ul aliquots plated out onto selective LB/agar plates and cultured overnight (37 °C) in the presence of appropriate antibiotics. 5 Plasmid D N A was routinely prepared from 1.5 ml bacterial cultures using a modification of the alkaline lysis protocol of Birnboim and Doly, 1979. Plasmid D N A prepared in this manner was found suitable for further manipulation, restriction endonuclease digestions, generation of specific deletion mutants, subcloning and dideoxynucleotide sequencing. Where large quantities of plasmid D N A were required, this was accomplished by equilibrium density gradient centrifugation (296,000 x g for 20 hrs, Ti80 rotor) using CsCl (1.55 g/ml final density). 1.2.4 Preparation of cDNA and Isolation of Recombinant Plasmids Expressing Portions of the PVS Coat Protein Gene. Unless otherwise indicated all manipulations were performed essentially as described by Maniatis et al., 1982. First strand cDNA was prepared from purified viral template R N A using cloned Molony murine leukemia virus (M-MLV) reverse transcriptase and oligo-dT primer (D'Alessio et al., 1987). Second strand synthesis was accomplished with Escherichia coli D N A polymerase I in the presence of RNaseH. For first strand synthesis, purified PVS R N A (10 15 ug) was heat denatured (65 °C, 5 min) in the presence of 2.5 fig oligo-dT primer and cooled on ice. To this mixture was added 5 [il dNTPs (5 m M each), 10 ul of 5X first strand buffer (250 m M Tris-HCl pH8.3, 375 m M KC1,15 m M MgCl 2 , 50 m M dithiothreitol), 20 u,l H 20 and 2.5 ui (200 U/uI) of M - M L V reverse transcriptase. The reaction mixture was incubated for 60 min at 37 °C and then quenched on ice. Second strand cDNA synthesis was initiated by 6 addition of 263 ul H 2 0 , 7.5 ul dNTPs (20 m M each), 30 | i l 5X first strand buffer, 36.2 ul 1M KC1,1.85 ul 1M MgCl 2 ,10 ul E. coli D N A polymerase I (10 U/ul) and 1.75 ul RNaseH (2 U/ul) . After a 2 hr incubation at 16 °C, the reaction was terminated by addition of 12.5 ul 0.5 M EDTA and the nucleic acid was extracted IX with phenol and IX with chlorofornv.isoamyl alcohol (24:1) and ethanol precipitated. Viral RNA S m a I, BAP Eco Rl Sst I Smal.Xmal w Bam HI P Xba I Sal I, Acc I, Hnc II Pst I Hind III | (A )n 3 ' ' o l i g o d(T) M-MLV Reverse Transcr ip tase DNA Po lymerase I RNase H T 4 D N A Po lymerase Sepharose 4 B ch romatography T 4 D N A Ligase Transformat ion of DH5-a lpha cel ls to Ampici l l in resistance. Clone Selection Fig. 1. Strategy used for preparing recombinant complementary DNA (cDNA) clones for PVS RNA. Purified viral RNA was used as template, together with oligo d(T) primer, for first strand cDNA synthesis using cloned Molony murine leukemia virus (M-MLV) reverse transcriptase. Following second strand synthesis, cDNA was blunt-ended by treatment with T4 DNA polymerase and cloned into the Sma\ site of pUC13. Recombinant plasmids were screened for expression of PVS coat protein sequences, as in-frame fusions with the lacZ alpha peptide, by colony hybridization using a monoclonal antibody specific for the viral coat protein. The double stranded cDNA was rendered blunt ended by treatment with T4 D N A polymerase and was size fractionated by chromatography over Sepharose-4B(CL). The pellet of PVS cDNA was resuspended in 25 ul of IX T4 buffer (70 m M Tris-HCl p H 7.5, 10 m M MgCl 2 , 5 m M DTT) containing 25 uM 7 each of dNTPs. The reaction was initiated by addition of 1.0 u,l (3.7 U) T4 D N A polymerase and continued for 10-15 min at 37 °C and then quenched by addition of 1 ul of 0.5 M EDTA and placed on ice. Following phenol/chloroform extraction, 2 ul of a saturated solution of bromophenol blue was added and the mixture was loaded onto a Sepharose-4B(CL) column (0.25 x 4 cm) which had been equilibrated with 10/1 TE (10 m M Tris-HCl pH7.5, 1 m M EDTA) buffer containing 94 m M ammonium acetate. Eluted fractions were dried in a Speed-Vac, resuspended with 15 u,l H 2 0 and 3 | i l aliquots removed for analysis by alkaline agarose gel electrophoresis. Material eluting in the void volume of the column was blunt-end ligated into Sma\ digested, bacterial alkaline phosphatase treated pUC13 (Pharmacia) using T4 D N A ligase and subsequently used to transform competent DH5a cells to ampicillin resistance (Fig. 1). Recombinant plasmids expressing an in-frame fusion product of the alpha peptide of lacZ and portions of the PVS coat protein were detected by immuno-screening using a specific monoclonal antibody (MAb 1G11 kindly provided by A . Wieczorek, Agriculture Canada, Vancouver Research Station) and [125I]-labelled goat anti-mouse Ig. Bacterial, colonies were replicated onto 82 mm diameter circles of nitrocellulose membrane and grown at 37 °C until the average colony size was approximately 0.2-0.4 mm (8-10 hours). The colonies were lysed by placing the filters for 10-15 min in a closed container on a piece of Whatman 3 M M paper saturated with chloroform. The filters were then 8 incubated in a solution of blocking buffer (10 m M Tris-HCl pH7.4, 0.15 M NaCl, 5 m M MgCl 2 , 0.05% (v/v) Tween-20,1.0 % (w/v) bovine serum albumin) containing 0.1 mg/ml lysozyme and 50 | ig /ml DNase for 1-2 hrs at room temperature with gentle agitation. The initial incubation with M A b 1G11 was performed using hybridoma culture supernatant (diluted 1/100 in blocking buffer) for 60-90 min followed by several washes with PBS/Tween (Dulbecco's phosphate buffered saline plus 0.1% (v/v) Tween-20). Bound antibody was detected by incubation with [125I]-goat anti-mouse Ig (1.5 x 106 cpm/ug, diluted to 1 x 106 cpm/ml in blocking buffer, for 60 min followed by extensive washing with PBS/Tween and overnight autoradiography (-70 °C). 1.2.5 Preparation of Random-Primed f 2P]-Labelled cDNA Probes. Additional PVS specific cDNA clones were identified by colony hybridization with a random primed [32P]-labelled cDNA probe prepared against a PstI restriction fragment obtained from clone pVS61 (which had been previously identified by colony hybridization with a specific monoclonal antibody to the PVS capsid protein). Briefly, 25-50 ng of D N A in 5-20 ul of 10/1 TE buffer was heat denatured (5 min 95 °C) and quenched on ice prior to the addition of 2 ul each of dCTP, dTTP and dGTP (0.5 m M each), 5 \il (approximately 50 uGi, 3000 Ci/mmol) of [a-3 2P]dATP and 15 ui of random primers buffer mixture (0.67 M HEPES, 0.17 M Tris-HCl pH6.8, 17 m M MgCl 2 , 33 m M 2-mercaptoethanol, 1.33 mg/ml BSA, 18 O D 2 6 0 units/ml 9 oligodeoxyribonucleotide primers (hexamer fraction; Bethesda Research Laboratories)). Reaction was initiated by the addition of 1 ul (3 U/ | i l ) of the Klenow fragment of D N A polymerase I and continued for 60-90 min at 25 °C. The reaction was quenched by the addition of 5 ul of 0.2 M EDTA and the incorporation of acid precipitable radioactivity was determined following treatment of a small aliquot (5 ul of a 1/1000 dilution in 10/1 TE buffer) of labelled probe with 10% (w/v) TCA and filtration through a glass fibre filter disk. In general, labelled probes (average specific activity 1-2 x 106 cpm/ng) were not further purified prior to use. 1.2.6 Western Blot Analysis of Expressed Fusion Proteins. Bacterial colonies which were positive for the expression of PVS coat protein fusion products were further analyzed by SDS-PAGE and Western blotting using M A b 1G11. Overnight cultures (0.5 ml) were harvested by centrifugation at 5,000 x g for 10 min in an Eppendorf microcentrifuge and the cell pellets were resuspended with 50 ul of SDS-PAGE sample buffer (4% (w/v) SDS, 125 m M Tris-HCl p H 6.8,10% (v/v) 2-mercaptoethanol, 0.4% (w/v) bromophenol blue, 20% (v/v) glycerol) and then incubated for 10 min at 95 °C. Samples were centrifuged at 13,000 g x 5 min and 5 ul aliquots were electrophoresed in a 10% polyacrylamide gel using the buffer system of Laemmli (1970). Separated proteins were electroblotted onto Immobilon membranes (Millipore) in a buffer composed of 25 m M Tris, 192 m M glycine, 10 20% (v/v) methanol pH8.3 at 100 V (0.25 A) for 60 min at 4 °C. The transfer blots were treated with blocking buffer for 60 min and incubated with diluted hybridoma culture supernatant and [125I]-labelled goat anti-mouse Ig as described above. 1.2.7 Immunoprecipitation of PVS RNA in vitro Translation Products. Purified PVS R N A (2 ug) was translated in a mixture of 50 uCi of [^S] methionine, 1 ul of essential amino acid mixture (1 m M each, lacking methionine) and 40 ul of nuclease-treated rabbit reticulocyte lysate (Promega, Madison, Wis., U.S.A.). After 90 min at 29 °C the translation mixture was 'cleared' by incubation (15 min) with 25 ul of a 50% (v/v) suspension of Sepharose 2B conjugated with goat anti-mouse immunoglobulin, prepared by the CNBr activation method described by Cuatrecasas, (1970). The mixture was centrifuged through a 0.2 u filter to remove the Sepharose beads. Samples (8 ul) of the resulting mixture were incubated with 100 ul of M A b 1G11 hybridoma culture supernatant for 60 min and then for a further 30 to 60 min after the addition of 25 ul of goat anti-mouse immunoglobulin-Sepharose (50% (v/v) suspension). The beads were washed three times by centrifugation in TST (10 m M Tris-HCl pH7.4, 0.15 M NaCl, 0.05% (v/v) Tween-20) and bound protein was extracted by incubation with SDS-PAGE sample buffer for 5 min at 95 °C. Samples (10 ul) of total in vitro translation mixture and the specific immunoprecipitates were electrophoresed in a 12% polyacrylamide gel in the 11 buffer system of Laemmli (1970). The gel was then fixed in 25% (v/v) isopropanol: 10% (v/v) acetic acid (30 min), infiltrated with Enlightening (New England Nuclear) and processed for fluorography. 1.2.8 Nucleotide Sequence Determination. Supercoiled double stranded plasmid DNAs were alkali denatured and used as sequencing templates via the dideoxy chain termination method (Sanger et al, 1977) using [<x32P]dATP (3000 Ci/mmol) and either the Klenow fragment from E. coli D N A polymerase I or modified T7 D N A polymerase (Toneguzzo et al, 1988). Aliquots (15 ul, 5 (ig) of purified plasmid D N A were mixed with 1.5 ul of 2N NaOH, 2 m M EDTA and heated to 70 °C for 5 min. Samples were then chilled on ice, neutralized with 6 ul of 2 M ammonium acetate p H 5.4 and precipitated by addition of 65 ul 95% ethanol. Pellets were washed with 70% ethanol and resuspended with 6 ul H 2 0 and 2 ul of 10X Sequenase buffer (280 m M Tris-HCl pH7.5,100 m M MgCl 2 , 350 m M NaCl). After addition of appropriate sequencing primer, samples were heated to 85 °C for 5 min and then allowed to cool slowly to room temperature over a period of 30-45 min. Samples were then stored frozen at -20 °C until required. Sequencing reactions were performed using the modified T7 polymerase, Sequenase (United States Biochemicals), according to the manufacturers recommendations. Samples were 12 then electrophoresed on 6% polyacrylamide gels (3-4 hrs), and vacuum dried gels were then subjected to overnight autoradiography (24 °C). 13 1.3 RESULTS 1.3.1 Western Blot Analysis of Expressed Fusion Proteins. Twelve recombinant plasmids expressing different portions of the PVS coat protein were isolated by immuno-screening with M A b 1G11. Western blots of bacterial cell lysates from four of these clones, pVS45,-57,-65 and -61, labelled with 1G11 M A b and [125I]-labelled goat anti-mouse Ig are shown in Fig. 2. SDS-PAGE analysis of the viral coat protein (Fig. 2 lane 1) showed a predominant band of Mr=33K as well as 2-3 lower molecular weight species which presumably are proteolytic degradation products. The 1G11 M A b reacted strongly with all of these components (Fig. 2 lane 3). Plasmid pVS45, which contained a 1.4 kb insert, expressed the largest (Mr=40K) fusion protein (Fig. 2 lane 7). The apparent molecular weight of this fusion protein was significantly greater than the Mr=33K viral coat protein, even after allowing for the sixteen amino acids (mol wt 1703) contributed by the N-terminal lacZ alpha peptide sequence. 1.3.2 Nucleotide Sequence Determination. Sequence information was obtained from several independent recombinant plasmids following the strategy depicted in Fig. 3. The coat protein specific inserts from plasmids pVS45,-64,-57,-41,- 66,-65 and -61 formed a nested set of 5' deletions all of which were 3' co-terminal. The remaining 14 clones (pVS49,-30,-20,-92,-52,-59,-91,-44 and -58) were isolated following colony hybridization of the PVS cDNA library with a random primed [32P]-labelled restriction fragment obtained by PstI digestion of pVS61. A l l of these clones contained sequences derived from the extreme 3' terminus of PVS R N A including variable lengths of a poly(A) tract. Additional subclones were generated either by specific restriction endonuclease digestion or by sequential digestions with exonuclease i n followed by mung bean nuclease treatment and re-ligation. 1.3.3 Organization of the 3'~Terminai Region of PVS. The sequence of 3553 nucleotides corresponding to the 3' terminal region of PVS R N A is shown in Fig. 4. Six potential open reading frames (ORF's) have been identified and the predicted amino acid sequence of each of these is shown above the D N A sequence in Fig. 4. The 5' terminus of the pVS45 insert was found to be located at position 2136. Because of the size of the fusion product produced by this clone it was likely that the 5' terminus of the pVS45 insert would be located on the 5' side of the coat protein initiation codon, and in the same frame. From the nucleotide sequence there are two possible ATG initiation codons for the coat protein cistron. Translation from the A T G codon at position 2045 would yield a 375 amino acid polypeptide of molecular weight 41.5 kDa which is considerably larger than the 33K size estimated by SDS-PAGE of purified viral coat protein. However, translation from the initiation codon at position 2291 would yield a 293 amino acid 15 polypeptide with a molecular weight of 32.5 kDa which agrees more closely with the experimentally determined value. Therefore, it is likely that this represents the true start of the coat protein ORF. The predicted amino acid composition of this 33K ORF was in close agreement with that previously published for the PVS coat protein (Tremaine & Goldsack, 1968). In addition to the 33K coat protein, small amounts (approx. 5%) of a 42K product could be specifically immunoprecipitated from a mixture of t3^]methionine labelled in vitro translation products of PVS RNA (Fig. 2 lane 9). 16 I 2 3 4 5 6 7 8 9 Fig. 2. Western blot analysis of E. coli cell extracts from clones expressing different sized /acZ-PVS coat protein fusion products and immunoprecipitation of in vitro translation products of PVS RNA. Lanes 1 and 2 respectively show the Coomassie Brilliant Blue staining patterns of purified PVS coat protein and total cell extract from clone pVS61. Immunoblots of purified PVS coat protein (lane 3) or lysates of cells containing pVS61 (lane 4), pVS65 (lane 5), pVS57 (lane 6) or pVS45 (lane 7) were incubated with MAb 1G11 followed by [ 1 2 5 l]-labelled goat anti-mouse Ig. Cell-free translation products of PVS RNA (lane 8) were immunoprecipitated with MAb 1G11 (lane 9) and separated by SDS-PAGE followed by fluorography. Mr standards x 10"3 are indicated. 17 4000 I 3000 1 2000 | | | M * | 14 3? 1000 1 J l -J JJ T J 4 I i Replicose y -y-25K 12K J Z ^ L 42K 7K 11K 33K Coot Protein | | , --49 --30 --20 --92 --52 --59 --91 --44 --5B --94 --45 --64 --57 •-41 --65 --65 --61 Fig. 3. The genome organization and restriction endonuclease map of the 3'-terminal region of PVS RNA. Plasmids pVS45, -64, -57, -41 , -66, -65 and -61 were isolated following immuno-screening with MAb 1G11 and [ 1 2 5 l]-labelled goat anti-mouse Ig and expressed different sized coat protein fusion products. The remaining clones were identified by colony hybridization using a random primed [ 3 2P]-labelled restriction fragment prepared by Pstt digestion of pVS61. In addition, several subclones were prepared either by specific restriction endonuclease digestion or by sequential digestions with exonuclease III followed by mung bean nuclease treatment and re-ligation. Plasmid numbers are indicated along the right hand margin and the extent and orientation of the sequence obtained from each clone is represented by the arrows below. 18 Fig. 4. The sequence of 3553 nucleotides corresponding to the 3' terminus of PVS RNA. The DNA sequence is shown as the equivalent of the viral plus-strand and the amino acid sequence of the six major ORFs is presented above the nucleotide sequence. The amino acid sequence shown in lowercase letters represents in-frame translation of the nucleotide sequence 5' to the predicted coat protein initiation codon. Termination codons are indicated by an asterisk (*). C a r b o x y l t e r m i n u s o f r e p l l e a s e ORF —> - — > M Y G P F L L K E F L N D V P L K P M H N T R M M A E A K F D F E A G A T G T A T G G G C C A T T T C T A C T T A A A G A A T T C C T C A A C G A T G T G C C A C T T A A A C C T A T G C A C A A C A C G C G C A T G A T G G C T G A A G C G A A G T T T G A C T T C G A G G 102 E K K T Q K S A A T I E N H S N R S C R D W L A D M G M V F S K S Q A G A A G A A A A C C C A G A A A A G T G C A G C A A C C A T T G A G A A T C A T A G T A A T A G G T C T T G T A G G G A T T G G C T G G C C G A C A T G G G C A T G G T G T T T T C A A A G T C T C A A C 204 L C T K F D N R F R D A K A A Q T I V C F Q H S V L C R F A P Y M R T C T G C A C C A A G T T T G A C A A C A G G T T C A G G G A T G C A A A G G C T G C G C A G A C C A T C G T T T G C T T T C A G C A C A G C G T T C T G T G C C G C T T C G C C C C A T A C A T G A G G T 306 Y I E K K L N E V L P A T F Y I H S G K G L E E L N K W V I E S K F A C A T A G A G A A G A A G C T T A A T G A G G T G T T G C C T G C C A C A T T T T A C A T C C A C T C A G G C A A G G G C T T G G A A G A G T T G A A C A A A T G G G T G A T A G A A T C C A A A T T T G 4 08 E G V C T E S D Y E A F D A S Q D Q Y I V A F E L A L M R Y L G L P A G G G A G T G T G T A C A G A G T C T G A T T A T G A A G C T T T T G A T G C T A G C C A A G A T C A G T A C A T T G T G G C G T T T G A A T T A G C G C T A A T G A G G T A C T T G G G C C T G C C C A 510 N D L I E D Y K Y I K T H L G S K L G N F A I M R F S G E A S T F L A T G A T C T C A T A G A G G A C T A C A A G T A C A T C A A G A C A C A T C T T G G C T C T A A A T T G G G A A A T T T T G C A A T A A T G C G C T T C T C T G G T G A G G C A A G C A C A T T C T T A T 612 F N T M A N M L F T F L R Y K L K G D E R I C F A G D D M C A N R A T C A A T A C C A T G G C C A A C A T G C T G T T C A C C T T C T T G A G G T A C A A G T T G A A G G G A G A T G A A A G G A T T T G C T T T G C T G G G G A T G A T A T G T G T G C A A A T A G A G C T C 714 L F I K D T H E G F L K K L K L K A K V D R T N R P S F C G W S L S T G T T C A T C A A G G A T A C G C A T G A G G G C T T C C T C A A G A A G C T C A A G C T G A A G G C C A A G G T G G A T A G A A C A A A C A G A C C G A G C T T C T G C G G G T G G A G T T T G A G C T 816 S D G I Y K K P Q L V F E R L C I A K E T A N L A N C I D N Y A I . E C T G A T G G G A T C T A C A A A A A G C C G C A A C T T G T C T T T G A G A G G C T C T G T A T A G C A A A A G A G A C C G C T A A T T T A G C C A A T T G C A T A G A T A A T T A T G C G A T C G A G G 918 V S Y A Y K L G E R I K E R M S E E E L E A F Y N C V R V I I K H K T G T C C T A T G C C T A C A A G C T T G G G G A G A G G A T C A A A G A G C G T A T G T C A G A G G A G G A A C T A G A G G C T T T C T A C A A C T G C G T G A G G G T T A T C A T C A A A C A C A A G C 1020 2 5 K ORF —> H L L K S E I R S V Y E E V * M D V F L Q V A T C T G C T T A A G T C T G A A A T T C G C A G T G T G T A T G A G G A G G T T T G A T A G C T T A G G T A A T C A G C T T A G T A G T A T T G A A T A T A T A T G G A T G T G T T T T T G C A A G T T T 1122 L N K Y K F E R V S S T L N K P I V V H S V P G A G K S S A I R E L T G A A T A A A T A T A A G T T T G A G C G T G T T A G T A G T A C T C T A A A T A A A C C A A T A G T T G T T C A T A G T G T T C C A G G T G C T G G T A A G A G T T C C G C G A T C A G G G A A T T A C 1224 L K L D S R F E C I T R G R P D I P N L E G A F I K A E R S G E S K T G A A G T T A G A T A G T A G G T T T G A G T G C A T T A C C C G T G G C C G G C C A G A C A T T C C C A A T C T A G A G G G A G C T T T C A T C A A G G C C G A A C G T A G T G G T G A G A G T A A G C 1 3 2 6 L L L V D E Y I E G P I P E D A F A I F A D P L Q S T A V S P H R A T G T T A C T G G T A G A T G A G T A C A T A G A A G G G C C C A T T C C A G A G G A C G C C T T T G C A A T C T T C G C A G A T C C G C T T C A G A G C A C A G C C G T C A G T C C A C A C A G A G C G C 14 28 H F I K T L S H R F G K C T D S . L L R D L G W D V Q A E G Q D S V Q A T T T C A T C A A A A C A C T A A G C C A T C G C T T T G G C A A G T G T A C T G A T T C A C T C T T G A G A G A T T T G G G T T G G G A C G T G C A A G C T G A A G G T C A G G A T T C A G T T C A A A 1530 I A D I F T V D P R E T I V Y F E P E V G E L L R S H G V E A S C I T C G C T G A T A T C T T C A C G G T C G A C C C C A G A G A A A C A A T T G T T T A C T T T G A G C C G G A A G T T G G T G A G T T G C T G A G G A G T C A C G G A G T C G A G G C A A G C T G C A T C G 1632 G E V R G A T F E H V T F V T S E N S P L I D K A S A F Q C L T R H G T G A G G T G C G T G G G G C C A C T T T T G A G C A C G T A A C G T T T G T C A C A T C T G A A A A T A G C C C A T T G A T T G A T A A G G C C T C T G C A T T T C A G T G C T T A A C G A G G C A C A 1734 12K ORF —> M P L T P P P N Y T G L Y I A A A L G V S L A A V T K S L L I L C P D A T Y T A A * C C A A G A G C T T A C T C A T A T T G T G C C C T G A T G C C A C T T A C A C C G C C G C C T A A T T A C A C A G G G T T A T A C A T T G C G G C A G C G C T T G G T G T A T C T C T T G C T G C T G T A 1836 V A L F T R S T L P I V G D S Q H N L P H G G R Y R D G T K A I D Y G T T G C C T T A T T C A C A A G A A G T A C T T T G C C G A T T G T A G G G G A C T C A C A G C A C A A C C T C C C A C A C G G G G G G C G G T A G e G T G A C G G C A C A A A G G C C A T A G A C T A C 1938 F K P T K L N S V E P G N Y W Y T Q P W L L V I L L V A L I C L S G T T C A A G C C C A C A A A A T T G A A T T C T G T G G A G C C G G G C A A T T A C T G G T A C A C T C A A C C T T G G T T G T T G G T T A T A C T T T T G G T A G C G C T C A T C T G T C T A T C C G G G 2040 R H A Q C C P R C N R V H S . A * m l n a a q d a t e c t v l n s v l l g l r s r l v r s e t r k 7K ORF —> M L P K M Q P S A Q C L I V F S L A F V L G W Y V L R P G N C G T C A T G C T C A A T G C T G C C C A A G A T G C A A C C G A G T G C A C A G T G C T T A A T A G T G T T C T C C T T G G C C T T C G T T C T A G G T T G G T A C G T T C T G A G A C C A G G A A A T A 2142 19 y k l r f a n h w g i s q a g q l r a h k r s s g g r a t k a v e t T S C V L L I T G E S V R L V N C E L T K D L V . E A V L L B P L K H C A A G T T G C G T T T T G C T A A T C A C T G G G G A A T C A G T C A G G C T G G T C A A T T G C G A G C T C A C A A A A G A T C T A G T G G A G G C C G T G C T A C T A A G G C C G T T G A A A C f l C C 2244 33K C o a t P r o t e i n ORF —> p l g s q v k a r i y s l t a r M P P K P D P S S S G E A P Q A M Q P L * T T T A G G T T C A C A G G T A A A A G C T C G A A T A T A C A G T C T C A C A G C A A G A A T G C C G C C T A A A C C A G A T C C A T C T A G C T C A G G G G A A G C A C C A C A A G C G A T G C A A C C 234 6 A P P P R A E G H M Y A Q P E G P G Q N E E A M L E Q R L I R L I E T G C A C C A C C A C C G C G C G C A G A A G G G C A C A T G T A T G C G C A A C C A G A A G G G C C A G G G C A A A A C G A G G A A G C C A T G C T G G A G C A A A G A C T C A T C A G G T T G A T T G A 24 48 L M A T K R H N S T L S N I S F E I G R P S L E P T P E M R R N P E G C T C A T G G C T A C G A A G A G G C A C A A C T C G A C A T T G A G C A A C A T C T C C T T T G A A A T A G G T A G G C C C A G T C T A G A A C C G A C C C C T G A G A T G A G G A G G A A T C C G G A 2550 N P Y S R F S I D E L F K M E I R S V S N N M A N T E Q M A Q I T A A A A T C C G T A C T C T C G G T T C T C A A T C G A C G A G C T G T T C A A G A T G G A A A T C C G A T C G G T C T C T A A C A A T A T G G C C A A C A C T G A G C A G A T G G C A C A G A T C A C C G C 2652 D I A G L G V P T E H V A G V I L K V V I M C A S V S S S V Y L D P G G A T A T T G C A G G G C T C G G C G T C C C C A C A G A G C A T G T G G C G G G G G T T A T A C T G A A G G T C G T A A T T A T G T G C G C A A G C G T G A G C A G T T C T G T C T A C T T A G A C C C 2754 A G T V E F P T G A V P L D S I I A I M K N R A G L R K V C R L Y A T G C A G G G A C T G T T G A G T T C C C T A C T G G C G C A G T G C C A C T G G A T T C C A T A A T C G C A A T C A T G A A A A A C C G T G C T G G G T T G A G G A A G G T G T G T A G G T T G T A T G C 2856 P V V W N Y M L V Q N R P P S D W Q A M G F Q W N A R F A A F D T F T C C G G T C G T T T G G A A T T A T A T G C T T G T T C A G A A T A G G C C A C C T T C A G A T T G G C A G G C C A T G G G G T T T C A A T G G A A T G C A C G T T T C G C C G C T T T T G A C A C A T T 2958 D Y V T N G A A I Q P V E G L I R R P T P E E T I A H N A H K S M A T G A T T A T G T G A C T A A C G G C G C T G C A A T C C A G C C T G T T G A G G G G C T C A T C C G T A G G C C G A C G C C T G A A G A G A C A A T A G C T C A T A A C G C T C A C A A G A G C A T G G C 3060 I D K S N R N E R L A N T N V E Y T G G M L G A E I V R N H R N A I T A T T G A T A A G T C G A A C A G A A A T G A A A G G T T G G C T A A C A C C A A C G T T G A G T A T A C T G G G G G C A T G C T C G G T G C T G A G A T T G T G C G T A A T C A T C G G A A T G C A A T 3162 U K O R F — > M K A E R L E M L L L C V Y R L G Y I L P V D V C I K I I S V A N Q * A A A C C A A T G A A A G C G G A A C G T T T A G A A A T G T T A C T G T T G T G T G T T T A C A G G C T G G G T T A T A T T T T A C C A G T C G A T G T G T G T A T T A A A A T A A T A A G C G T A G C G 3264 Q V S V Q G R S T Y S C K R R A R S I G R C W R C Y R V Y P P V C N C A G G T C A G T G T C C A A G G T C G T T C A A C C T A C T C A T G T A A G C G A A G G G C C C G C A G C A T T G G A C G A T G C T G G C G T T G C T A C C G T G T C T A T C C A C C A G T T T G T A A T 33 66 S K C D N R T C R P G I S P N F K V V T F I R G W S N * T C T A A G T G T G A T A A T A G G A C A T G C C G T C C A G G C A T T A G T C C C A A C T T T A A A G T A G T G A C T T T T A T T C G G G G T T G G A G T A A C T G A G G T G A T A C C A C C C G G G A T 34 68 G A A A A G T C T G A G T T T C G C A T A A A G C T T A A A T A A T A T A T A A G T G T G C A A C T A T A A A G A A A A T A T G T T T T T A A A A T A T T T T A G C A T (A) „ 3 5 5 3 Table I: Comparison of amino acid sequence similarities between the capsid protein of PVS and other members of the carlavirus and potexvirus group. Overall Percent Identity Percent Identity in 74 Amino Acid Region Beginning at Position 180 of PVS L S V 6 7 . 0 8 5 . 0 P V M 4 4 . 7 7 3 . 0 P V X 3 0 . 5 5 5 . 0 P M V 2 9 . 6 5 0 . 0 N M V 2 4 . 6 4 1 . 9 W C I M V 2 3 . 4 4 1 . 9 P A M V 2 1 . 2 4 4 . 6 20 Four ORF's 5' to the coat protein cistron were identified. The ORF extending from position 2052 to 2247 encodes a 65 amino acid polypeptide of molecular weight 7.2 kDa. The ORF extending from position 1762 to 2086 encodes a 108 amino acid polypeptide of molecular weight 11.8 kDa. A 25K ORF was identified 5' to the 12K protein, extending from position 1101 to 1782 (Mr 25,092). A fourth, partial ORF beginning 5' to the cloned cDNA sequence and terminating at position 1062 encodes a polypeptide of at least 41.05 kDa. A n additional ORF 3' to the coat protein gene was also identified. This ORF extends from position 3169 to a TGA stop codon at position 3448 and is translated into a 93 amino acid polypeptide of molecular weight 10.7 kDa. Comparisons of the deduced amino acid sequences of PVS ORFs with similar sized ORFs from a number of potexviruses were performed using the progressive sequence alignment algorithms of Feng & Doolittle, (1987), implemented on a DEC Micro V A X II minicomputer. A comparison of the predicted amino acid sequence of the PVS coat protein ORF with the recently published coat protein sequence of two related carlaviruses, potato virus M (PVM; Rupasov et al, 1989) and lily symptomless virus (LSV; Memelink et al, 1990), and the nucleocapsid proteins from a number of potexviruses including potato virus X (PVX; Huisman et al, 1988),white clover mosaic virus (WCIMV; Harbison et al, 1988), papaya mosaic virus (PMV; Short et al, 1986), potato aucuba virus (PAMV; Bundin et al, 1986) and narcissus mosaic virus (NMV; Zuidema et al, 1989) revealed some striking 21 similarities (Fig. 5). Not surprisingly, the nucleocapsid sequences of LSV and P V M showed the greatest degree of similarity to the PVS sequence, with 67.0% and 44.7% of the amino acid residues being identical, respectively. The greatest area of identity between PVS and members of the potexvirus group was found in a 74 amino acid long region beginning at position 180 of the PVS sequence. This region showed varying degrees of similarity ranging from 55% identity with PVX to 41.9% identity with WCIMV, as compared to an 85% identity with LSV and a 73% identity with P V M (Table I). The locations of invariant amino acids in four potyviruses (Morozov et al, 1987) which were identical in PVS and at least two of the potexviruses examined, are indicated by asterisks above the aligned sequences in Fig. 5. Little homology was observed between the extreme N and C terminal portions of the PVS coat protein with those regions from the various potexviruses. 22 PVS H P P X PjD P S S S G l E L S V M Q S IMP A Q E | S G | S PVM M G D S T K | K | A E T A K D E G T S HCLM M A T T T A T TJP P V X M | S | A P A S T T P M V M | S | T P N T N M V P A M V A P Q A M Q E3 ' G ! 0' A | S E T P A R  R Q [ E R R E P S L T D I R A L T G S T T S I T Q I S I A H I S I T M A T P S T Q T T D P X | P A H i v D Q K K T E T P Q v v D | A S i H | Y J A O | 7 | E C P | G | E P I T N Y T 0' P R A E G H |  Q T P S D A P R D | | T  N N L P T A A D F E G X D T S E N T D G R A A D A D G T S S T V S V A S P A E T T T K T A | G ] A T P A T A K T S Q A G R Q F L S A P K Q P V S L S V P V M N S N S T I L I S N l i s F E I G R P S L E P T NI L | R N V Al F E I G R P S L E P T A I R V T M C L M E P V X D F P M V D N M V P P A M V P G L E T G R P I jT L E J K L I J P N D V S T A R A I V P T D L K K I A S D V R S R F S I D £ L R F S I D E L F K M E 11R S V F K M | K V G V V H D L A R A F A A S N A V A i Q N L | T | P N R | 7 | S Ij E A | L J S R I K P I A I D V G A S s | K j s | E T N E D [ L | S I K I I | E Q ] Q E O ^ L J K S V S T L M^v] 50s °0 KrYJE S T A Y E V R T T A V A T P A T T S I A S P A G L R X V C R L Y A P V V W N 1 A 7] 2 A A -043 „ A H H D L A R A Y A D V I A I D M A R A Y A I D V R K A V L L D A F T L A N [ G ] I S R A R L A A | A I J P J E I S|7]A Q L A sj^ i V [ X ] A S G T S L R * K J F s N P [ A I T R Q A L A R Q F Y V I N I T P T v l V C R L Y A p [ l V H N Tjv c R L Y A P V ^ J H N M Y F | A ] S I V W N M K [ Y A P V V H M R L A Q L K A G A G I S P M L V M L V M L M L M L T H D T T N L D L [ H L 1 9 9 197 2 1 0 1 1 6 141 lie 1 3 1 154 7 | D | R | S L R G A L H R N E R L N E R L A Q T A A F V K T J T ^ K J A R A Q S N D F [i A S Q V H L F Q A A A E D N N F Y G Q L A R Q R T f ^ M E T 3 F H A 5 L S R E R L Q E G T S I 2 67 2 6 5 2 7 8 1 7 7 2 1 0 1 8 5 2 0 1 2 2 3 pvs JAJN T ^ N J V L E -0 E J V J T L S V G S P V M S S H C L M P V X P M V NKV P P H L K S L T V P A M V T T V A E Y T G G M L G A E I V R N E Y T G G | V Q G A E I V R N H R  G G M | H G P [ A I S L D A A V A S N S A F I Q L N K : J L T | R | O Y V K S N R K L C G H | L G J G Y N N L P A L H H K[7]C Q V T L P L Q. "0 K P T ] R I T G T T T A E A V V T L P P P Q I S G S T P T I Q F L P P P A V S T P C T P L K S L Q N C N R N T S K L K L V C G L P P S 2 93 2 9 1 3 0 4 1 9 0 2 3 7 2 1 0 2 4 0 248 Fig. 5. Amino acid homology between portions of the coat proteins of PVS, two related carlaviruses, lily symptomless virus (LSV) and potato virus M (PVM), and five members of the potexvirus group, potato virus X (PVX), papaya mosaic virus (PMV), narcissus mosaic virus (NMV), white clover mosaic virus (WCIMV) and potato aucuba virus (PAMV). Amino acid residues identical to the PVS sequences are boxed. The 74 amino acid long region from PVS which displays the highest degree of homology with members of the potexvirus group is underlined. Asterisks above the sequences indicate where amino acids conserved among four potyviruses are also common between PVS and at least two of the potexviruses compared here. 23 Recent reports have identified the presence of a small (7K) ORF immediately 5' of the coat protein cistron in a number of potexviruses as well as two carlaviruses, P V M and LSV. A comparison of the amino acid sequence of the PVS 7K ORF with those from P V M (Rupasov et al, 1989), LSV (Memelink et al, 1990), PVX (Huisman et al, 1988) and P A M V (Bundin et al, 1986) revealed some regions of significant homology with 25%-30% identical matches between PVS and each of these proteins (Fig. 6 panel a). Alignment of the 12K ORF with the amino acid sequence of the 12K ORF of PVX, the 13K ORF of WC1MV and the similar sized ORF from N M V revealed an overall 39% identity with each of these proteins (Fig. 6 panel b). Comparison with the 14K ORF from barley stripe mosaic virus (BSMV) R N A 2 (Gustafson & Armour, 1986) revealed a 50% homology from positions 40-64 of BSMV. The 12K protein from LSV was the most similar with the PVS sequence, showing a 63% homology of identical amino acids, while the analogous protein from P V M was 57% identical with PVS. A small region of identical matches between the 12K ORF of PVS and the 13K ORF of beet necrotic yellow vein virus (BNYVV) RNA 1 (Bouzoubaa et al, 1987) is indicated by asterisks above the aligned sequences in Fig. 6 panel b. 24 (a) P V X * P V S ^ P V M L S V P A M V Q P N [ A | I I [ L | V S A Q [ C L I M •0  I V Y V L E A N T Y L | | l I | |  L V V T I I AJV I S I F L VIR1 T E P F S L A F V L G MMIV L R P G N T S G L S A r j c I V Ljjfj L I S Q G Q 5 D R S V | A | L T | L | C A l I A G Y L L V S N L Q N V F S P E V Y R Y L D | C L | L V I M C A V L A I A L L W P N N Y I T G E S I T V L A C V L L I T G E S V R c v | i    V V L £T L V L V N V Q G I N G i l S l G l A E I Q I H N I T G E S V R I T G E s [ l | R K J L J D A E T I R | A | t A D E L T K D L V E A V L R I D G E F G S V L S X NJ^LJS P A H F R | A J I S H A E P N K I I S S I Q S B P L P L P F V E Rl L K [ 7 L  G C G S F Q I G T G L S F (b) Y I A A A L G V S L A 7 | A L ] A I G ^ A ] S | T J A L S Y F J K C G H S H S Y F Y F ^ Y | N R N K L N S V E K L N S V E p [ G | N Y m Y T 3 G S R H T F G R I L N S TI E A R K A P L L G N L G S R v i s L B N G K N P V S L S V P V M P V X NMV H S S T H C L M P N Q H 0 P Q P a P T N H K L S K T L A K S W L L V I L L V A L I M L L H J A I A A F A A Y T A L C A N T T I F ' L L V I vj V V L V L L L V L T L I L G L v j T C L I A L H A S Y G S | S G I V T L B A Q C Q G H S H K L G R P N K Y I S Q R H B T F A Q T R L A A G N R I T S V S I H G L H Y F N N N R R V S S S L H BSMV B P F A Y G N A S S P G M L L P A C a r l e . k L N S v E . - g . . w . . Q P M I J T : jc r I S Y L W R T R D S V L G D : H l l V i l L V a L I . a s g . . r . g . G G H N S A G S G N N A A Q G S L S G G N Q N K G E D C Q G . r B B G R V S G 108 106 107 115 I S E L 116 116 115 Fig. 6. (a) Alignment of the amino acid sequence of the PVS 7K ORF with the similarly sized ORFs from PVX, PVM, lily symptomless virus (LSV) and potato aucuba virus (PAMV). The homologies between the 12K ORF of PVS and the 14K ORF from BSMV RNA 2B as well as similarly sized ORFs from PVM, LSV, PVX, narcissus mosaic virus (NMV) and WCIMV is shown in panel (b). Amino acid residues which are invariant with PVS sequences are boxed. In panel (b), the row labelled "Carla" indicates a consensus sequence in which amino acid residues identical in at least two carla virus sequences are shown in lower case, and residues identical among all carla viruses are indicated in uppercase letters. The underlined sequence motif GD(7 aa)GG-YRDGTK indicates the region of greatest conservation between the PVS 12K protein and similar sized gene products from BSMV, B N Y W , PVM and the potexviruses. The position of the asterisks below the aligned sequences identifies identical matches with a similar region from the 13K ORF from B N Y W RNA 1. The 25K ORF also displayed considerable sequence similarity, primarily at the N and C terminal regions, with the similar sized proteins from LSV, P V M , PVX and WCIMV (Fig. 7). The N terminal homologous domain 25 contained the conserved sequence G-GKSS/T which is also found within the 58K protein of BSMV and the 42K protein of BNYVV R N A 2 (Bouzoubaa et al, 1986) (indicated by asterisks in Fig. 7). P V S L S V P V M P V X H C L M M D V M D V M D V M D M D F L Q V L L S L I V D L I L I S S H I B L L C a r l a M D V . 1 t|p D I J T J N J T ] : E s I V E P D P D B s V P G a G K S S l I R * K V S I R T R G P Y S^IJs N P T ) . 1 . G , I K A [ E [ K | K A R X H Q K : x A | P V S L S V R S G E S K | L J L L V H A S I P R S E F V V F D E Y D E Y I E G PI I jpj I E G I D TlP1 E D P H A F A A F A l i V P V M S G Q Q P E G K F V V L D E Y 1 L_l T L L T E V P P V | F A L P V X P G P I P E G N F A I L D E Y T L D N T T R N S Y Q A L H C L M F A Q F X R G TJTJD I L D E Y G 0 I ' J ^ j l ' 1 D , D S S0E 4 H C L M I T 3 L V T K [ ° ] N I S F G S P Y L [ V D p j v G | I I | L A F Q [ T a D . V Q i . d i y t . D p . g t v V y , 1 r G q T F E P V S L S V P V M N R P V X G Q 5 T H C L M E R B L A F Q C L T R H A F Q C L T R B A Y Q C | M | T R H A F Y N L | F I G £ T K S L L I L C P D A T Y T A R s j s L L I L S P J N I A T Y T A R R   A I T R S 2 2 7 2 2 8 2 2 1 2 2 6 s L L I L | G | P D A | F D S S P 2 3 6 L C T 5 V S T Y V R A G T C a r l a d r A . A f Q C . T R H r l l i l c p . a t y t . Fig. 7. Alignment of the 25K protein from PVS with the analogous 25K proteins from PVM and LSV, and with the 25K and 26K proteins from PVX and WCIMV. The position of the asterisks above the aligned sequences indicates homologous amino acids which are also conserved in the 58K and 42K proteins of BSMV and BNYVV RNA 2 and which comprise a putative mononucleotide binding domain. The row labelled "Carla" indicates a consensus sequence in which amino acid residues identical in at least two carla virus sequences are shown in lower case, and residues identical among all carla viruses are indicated in uppercase letters. Amino acid residues identical to the PVS sequences are boxed. Analysis of a portion of the 5' most partial 41K ORF from PVS with similar regions from the 166K protein of PVX (positions 1243-1384) and the 26 147K protein of WCIMV (positions 1078-1219) showed identities of 32% and 40% respectively (Fig.8 panel a). This homologous domain contains the sequence T/SG—T—NT(22 aa)GDD which is conserved in the R N A replicase components from most plant and animal single stranded R N A viruses (Cornelissen & Van Vloten-Doting, 1988). (a) PVX T A F D Q S Q D G A H L Q F E V L K A K f l f l C I P E E I 7|Q A Y I D PVS E A F D A S O D Q ^ F l v A F E L A L M R Y L G L P N I D I L i E ] D Y K HClMV T A F D Q S Q D G S I L Q F E V I K A K F B N I P E U I I E | G Y I Q I K T I K T N A Q I F L G T L S I M R L T | G E G P T F D A 1 3 0 3 H A K I F L G T L S I M R L S G E G P T F • A 1 1 3 8 PVX PVS HC1MV A N A H I A Y M L F I A Y T K F D I P A G T A Q V Y C F Q V Y F L R Y K L K G | D ] E R I i B T K T N I P C I D I A A PVX G S W P E PVS S WCIMV G D F A E F C G H F C G H F C G H L I T P K s L [ S ] S : T I S P G | V M "H 7 K K P K K P (b) P V S M k A E J R J L E M L LJ P V M M K D V T K V A L L I L S V E A L K » [ R ] T T I V LJ A R A M A G O D I S A L D C V P E V A G D D M A G D D M C A N R A L F I S I D Y V A S V P S F M M I E 1 3 8 4 2 8 0 1 2 1 9 V Y R L G Y A S S G T F V F E L A F S L L S C a r l a m k . . . r . . . l l . . . . C . . s . . . . f p . d . c i I T Q Q K K 1 3 6 3 2 6 3 Q 1 1 9 8 D R I T J N R ] F N J T I Q T < i L ^ 7 J V J D J V | C ' 1 K | T | V F E I T E A F J p j R | D J l | c R D 0 L R R ' r 3 S H I V G L S V A Q V S V Q T G R P L G G G R S G R S G R S S C K A R R A ft R R R A R R A R R A S I G R C s I | A ] R C L o i l G R C G R S r r R R A a I g R C PVS PVM LSV R C Y R V Y P P R C Y R | L WJP P R C Y R V Y P P R [ T C R P G | I | S J P J N J F K | V J V T f ^ R G H S N K H | C [ V L V S L T M C A M R N L L M K E K | T C R P G J L J 7 | I | M J T N [ V J A N x 0 d H G V T E V I P K I S P F L R G Q F Y L R P K 9 3 9 S 1 4 0 R C Y R v y P P - V c . s k C D N X t C r p g Fig. 8. (a) Comparison between a 136 amino acid portion of the extreme 5'-end partial 41K ORF of PVS and similar regions of the 166K protein of PVX and the 147K protein of WCIMV. The position of the asterisks below the aligned sequences indicates the conserved sequence motif S /TG—T—NT(22 amino acids)GDD. Panel (to) shows a comparison between the amino acid sequences of the 3' terminal 11K ORFs of PVS and PVM, and a portion of the 16K ORF from LSV. The row labelled "Carla" indicates a consensus sequence in which amino acid residues identical in at least two carla virus sequences are shown in lower case, and residues identical among all carla viruses are indicated in uppercase letters. The asterisks below the sequence in {b) indicate the positions of Cys residues within a putative "zinc-finger" domain. Amino acid residues identical to the PVS sequences are boxed. 27 Examination of the putative 11K ORF located 3' to the PVS coat protein cistron revealed a 34% correspondence of identical amino acid residues with the similar sized ORF from P V M and a 48% identity with the 16K ORF from LSV, but did not reveal any significant sequence homologies with other published plant viral sequences. 28 1.4 DISCUSSION The cDNA sequence corresponding to the 3'-terminal 3553 nucleotides of PVS R N A has been determined by dideoxynucleotide chain termination sequencing of recombinant plasmids containing viral-specific inserts. The sequence obtained contains six ORF's in the viral (+) sense. Translation of the largest, 42K, potential ORF (positions 2045-3170) from an internal ATG codon (position 2291) would yield a 33K polypeptide, the sequence of which has been confirmed to be that of the coat protein. Evidence for the cell-free translation of the entire 42K ORF was obtained by immunoprecipitation of the [^methionine labelled in vitro translation products of PVS R N A which showed that in addition to the 33K coat protein, small amounts of a 42K protein were also produced. The internal initiation site (position 2291) is in a more optimal context for initiation of translation (Lutcke et al, 1987) which may in part explain the much higher (approx. 20 fold) level of expression of the 33K product relative to the 42K protein. However, the presence of a large proportion of subgenomic RNA's which exclude the first A U G site (position 2045) cannot be ruled out. Whether this 42K protein is also produced in vivo, and what biological significance it has, remains to be demonstrated. The high degree of homology between a 74 amino acid length region in the central portion of the coat protein with similar regions from the potexvirus coat proteins was surprising in so far as PVS as not been shown to be 29 serologically related with these viruses. Mi ld proteolysis of potato virus Y (PVY; Shukla et al, 1988) particles has demonstrated that the N and C termini of its coat protein are exposed on the surface of the virus particle and a similar arrangement has been proposed for PVX from studies on limited proteolysis (Koenig et al, 1978) and by examination of predicted secondary structural features (Sawyer et al, 1987). Therefore, it is likely that the central portion of the PVS coat protein sequence, which contains the regions of highest similarity with the potexviruses, is buried within the interior of the virus particle and thus not immunogenic. This central core sequence, which also contains small blocks of homology with the potyviruses, may be important in maintaining correct tertiary structure of the coat protein and/or play a role in interacting with the viral RNA. The amino acid sequence homologies between the 5' most partial 41K ORF from PVS and the 166K and 147K proteins from PVX and WCIMV and the presence of the conserved sequence motif, T/SG—T—NT(22 aa)GDD, indicates that this ORF represents the C terminal portion of the viral replicase. This and other sequence motifs have been found to be conserved amongst virtually all RNA-dependent D N A polymerases and the RNA-dependent R N A polymerases encoded by plus-, minus- and double-strand R N A viruses (Poch et al, 1989). It is likely that the high degree of conservation among such regions reflects their crucial importance for RNA template recognition and/or polymerase activity. This has been emphasized by recent site directed 30 mutagenesis experiments involving the reverse transcriptase of HIV1 in which mutation of the first invariant Asp residue of the Y M D D motif resulted in complete loss of of polymerase activity (Larder et al, 1987). Similar studies with the replicase of a plus-strand R N A virus, the Q-fi bacteriophage, demonstrated that substitution of the G of the YGDD sequence destroyed activity (Inokuchi & Hirashima, 1987). The 7K, 12K and 25K ORFs located 5' to the 33K coat protein ORF show significant sequence homology with similar sized ORF's which have previously been identified for a number of potexviruses. The 13K, 14K, 13K and 12K ORF's of BNYVV, BSMV, WCIMV and PVX share the greatest degree of sequence homology centred about the sequence motif GD(7-8 aa)GG-Y(R/K)DG(T/S)(K/R) (Huisman et al, 1988). This is also the area of greatest homology with the 12K ORF from PVS (Fig. 6, panel B). The 12K and 13K proteins from PVX and WCIMV have been suggested to be membrane bound proteins (Morozov et al, 1987; Forster et al, 1988). The 12K protein from PVS has a hydropathicity profile similar to those of these two proteins from PVX (Fig. 9) and WCIMV and may also be membrane associated. The conserved sequence motif G-GKSS/T found in the N-terminal regions of the 25K - 26K proteins of PVS, PVX and WCIMV, is also present in the C terminal two-thirds of the 58K and 42K proteins of BSMV and BNYVV. This domain has been observed in a number of ATP- and GTP-binding proteins (Zimmern, 1987) as well as the central portions of the 166K and 147K 31 replicase components from PVX and WCIMV (Huisman et al, 1988; Forster et al, 1988) and has been the subject of a recent review by Gorbalenya & Koonin (1989). Similar nucleoside triphosphate (NTP) binding domains have been found in over 100 viral proteins. A l l dsDNA viruses which have been sequenced to date, and most groups of positive stranded R N A viruses and ssDNA viruses have such proteins. It has been postulated that these proteins are possibly involved in duplex unwinding during D N A and R N A replication, transcription, recombination and repair, and perhaps also mRNA translation. 20 40 60 80 100 Amino Acid Sequence Position Fig. 9. Comparison of the hydropathicity profiles of the 12K proteins from PVS (dashed line) and PVX (solid line). The amino acid sequence positions are numbered relative the N-terminus of the PVX 12K protein. Both of these proteins display significant hydrophobic domains near their N and C termini, leading to speculation that they may be membrane-associated. The role of the 7K, 12K and 25K non-structural proteins encoded by PVS RNA, and their counterparts in the potexviruses, remains unknown, and indeed, direct biochemical evidence for their expression in virus infected tissue 32 has not been demonstrated. It is likely that one of these proteins is involved in the cell-to-cell movement of PVS. Previous work with the LSI strain of tobacco mosaic virus (TMV), which is temperature-sensitive with respect to movement, has quite elegantly shown that this function is mediated by a 30K non-structural protein (Ohno et al, 1983; Meshi et al, 1987). The 30K protein function could be complemented in trans by previous inoculation with PVX (Taliansky et al, 1982). Given the significant sequence homologies observed between the 7K, 12K and 25K proteins of PVS and PVX, it would be of interest to know if PVS could also serve to complement the movement of LSI at the restrictive temperature. The ability to produce specific antiserum directed against cloned sequences from each of these proteins produced in bacterial expression systems will enable the measurement and ultrastructural localization of each of these products in virus infected tissue. The 3' terminal U K ORF of PVS has no counterpart among members of the potexvirus group, however, a similar sized protein has been predicted from the nucleotide sequence of P V M (Rupasov et al, 1989) and the presence of a homologous 16K ORF has been identified from the partial nucleotide sequence of lily symptomless virus (LSV; Memelink et al, 1990). Like the analogous P V M protein, the PVS U K protein contains a relatively high proportion of charged amino acid residues, with a total of 16 positively charged Arg and Lys groups and 4 acidic residues of Glu and Asp. The protein also contains 8 Cys residues, four of which are found in the sequence CWRCYRVYPPCNSKC, 33 beginning at position 53, which resembles a consensus sequence CX ( 2 w 4 ) CX ( 2 . 1 5 ) (C/H)X G ^ ) (C/H) (where X is any amino acid) that has been found in many nucleic acid binding proteins (Berg, 1986). This consensus sequence has been postulated to be a metal ion binding site and a similar 'zinc-finger' nucleic acid-binding domain is present in the U K ORF of P V M and the 16K ORF of LSV, as well as the capsid protein of tobacco streak ilarvirus (Sehnke et al., 1989), the 16K protein from R N A 2 of tobacco rattle virus (Angenent et al., 1986), the 3A protein from R N A 3 of cucumber mosaic virus (Gould & Symons, 1982), the P77-80 protein of carnation mottle virus (Guilley et al., 1985) and the helper component of tobacco vein mottle virus (Domier et al., 1986). While the functional significance of this putative 'zinc-finger' domain within the U K protein remains obscure, it is possible that this protein is required for the aphid transmissibility of PVS, by analogy with the helper component of tobacco vein mottle virus. The absence of an analogous protein within the potexvirus group is consistent with the fact that these viruses are not transmitted by aphids. 34 Potato Virus S (PVS) Replicase I [ 12K CP 33K 11K 1-3 7K 25K 42K Potato Virus X (PVX) 166K Replicase — I f 25K 8K 3 ' 12K CP 25K Fig. 10. Comparison of the genome organization of PVS and PVX. With the exception of the 3' terminal 11K ORF present in PVS and also in PVM, a related carlavirus, the overall genome organization of members of this virus group is remarkably similar to viruses within the potexvirus group. The overall similarity in genome organization and the extent of the amino acid sequence homologies observed between the capsid proteins and three small putative non-structural polypeptides between PVS and members of the potexvirus group, as well as BSMV (hordeivirus) and BNYVV (furovirus) indicate that a significant relationship exists. Since this work was first reported (MacKenzie et al, 1989), the partial nucleotide sequences of two other carlaviruses, P V M (Rupasov et al, 1989) and lily symptomless virus (LSV; Memelink et al, 1990), have been described. Despite differences in particle morphology, virus transmission and the presence of an additional 3' terminal open reading frame (ORF) in the carlavirus genome, these data provide strong arguments for placing the carlaviruses and potexviruses in one taxonomic 35 group. The divergence of the carla and potexvirsuses may reflect the insertion of a unique gene at some point during their evolution, as has been previously suggested by comparison of different members of the tobravirus (Angenent et al, 1986) and coronavirus groups (de Groot et al, 1988). 36 CHAPTER 2 2.0 GENETIC ENGINEERING OF PLANTS RESISTANT TO PVS INFECTION. 2.1 INTRODUCTION Potato is one of the most important crops worldwide, however, because of the tetraploid nature of its genome, classical approaches to potato breeding and selection for improved properties are especially difficult and laborious when compared with other diploid species. Despite these drawbacks, significant progress has been achieved over many years and specific cultivars have been developed with desirable characteristics. The 'Russet Burbank' is one of the oldest and most popular of the potato cultivars grown in the continental United States and Canada. While 'Russet Burbank' has excellent processing characteristics and high yield, it lacks resistance to many plant pathogens including bacteria, nematodes and many plant viruses. Because potato is propagated primarily vegetatively, many of these diseases can be perpetuated over time with disastrous results. Because of its near symptomless morphology in potato, PVS has been a difficult virus to control and can occur in seed lots at levels of 80% or more. In combination with potato virus X, it can cause significant reduction in tuber yields (Wright, 1977). Elite seed potato stocks in Canada are produced from virus-free material by heat therapy and 37 meristem tip culture (Wright, 1987), but such stocks become rapidly reinfected with PVS once grown in the field (Hahm et al, 1981). Contributing to this is the fact that many potato viruses, including PVS, overwinter in tubers missed in the previous harvest, and volunteer plants emerging from such tubers are a primary source of inoculum and play a significant role in virus epidemiology. Since the first report of the expression of bacterial genes in plants (Fraley et al, 1983) the process of genetic transformation and regeneration, as a mechanism for the expression of novel new genes, has become routine for a number of plant species (Gasser & Fraley, 1989). However, the application of these techniques for the genetic engineering of desirable resistance traits into agronomically important potato cultivars has only become feasible in recent years with the advent of efficient tissue culture techniques for the regeneration of transformed potato tissue. 2.1.1 Mechanisms of Agrobacterium tumefaciens Mediated Plant Cell Transformation. Agrobacterium tumefaciens is a soil born phytopathogen which uses genetic engineering processes to subvert the host plant cell's metabolic machinery in order to divert some of its organic carbon and nitrogen supplies in order to produce nutrients (called opines) which can be specifically catabolized by the invading bacteria (Tempe & Schell, 1977). Parasitized cells are also induced to proliferate and the resulting crown gall tumour disease is a direct result of the incorporation of a region of transfer D N A , T-DNA, from a 38 large (150-250 kB) circular Ti (tumour inducing) plasmid, carried by A. tumefaciens, into the host plant genome. The T-DNA contains oncogenicity (one) genes which are involved in the biosynthesis of a number of phytohormones including indoleacetic acid (IAA; an auxin) and isopentenyl-AMP (a cytokinin), which are primarily responsible for the de-differentiated, unregulated, growth characteristics of the crown gall. Besides the one genes, the T-DNA contains the biosynthetic genes for a number of bacterial catabolites termed opines, which are either condensates of an amino acid and a sugar (e.g. octopine, nopaline, leucinopine, agropine) or phosphorylated sugar derivatives (agrocinopines). Agrobacterium Tumefaciens Ti plasmid Transformed Plant Cell Cell Growth Chromosome $j$T Vlr Region Fig. 11. Plant cell transformation by A. tumefaciens. Virulent strains of Agrobacterium tumefaciens containing a Ti plasmid attach to plant cells and transfer a portion of the Ti plasmid, the T-DNA segment, into plant nuclear DNA. Genes essential for the transfer and integration of the T-DNA, are located in a separate segment of the Ti plasmid, the vir region. Expression in transformed plant cells of several T-DNA encoded genes involved in auxin and cytokinin biosynthesis results in stimulation of cell division and proliferation of crown gall tumours. Additional genes present within an approximately 40 kB region of the Ti plasmid, called the virulence region (vir genes), together with other genes on the bacterial chromosomal D N A , chv genes, are involved in tumorigenicity and play an essential role in the in vivo transfer of T-DNA from Agrobacterium to 39 plant cells (Douglas et al, 1985). The product of the chvB gene, a 235K protein, is involved in the formation of a cyclic G-1,2 glucan (Zorreguieta et al, 1988) which is transported to the periplasmic space, perhaps by a transport protein encoded by the chvA gene (Cangelosi et al, 1989), where it may function to aid in the attachment of agrobacteria to plant cell walls (Thomashow et al, 1987). While the exact role of many of vir genes remains to be determined they are essential for T-DNA transfer and their products are only expressed following induction by certain plant factors, including the phenolic compounds acetosyringone and a-hydroxyacetosyringone which are released from plant tissue following wounding (Stachel et al, 1985). The vir region of the octopine type Ti plasmid encompasses 22 genes in 7 operons called virA-virG, which are co-regulated (Stachel & Nester, 1986). Mutations within the vir A, virB, virD or virG loci result in completely avirulent strains, while mutations in the remaining vir loci lead to an attenuation of virulence only. It is believed that the vir A protein, associated with the bacterial inner membrane (Leroux et al, 1987), is activated directly by phenolic inducers (Melchers et al, 1989) and may subsequently act as a kinase to phosphorylate the virG protein thus enabling it to bind to regulatory elements (Powell et al, 1989) within vir gene promoter sequences leading to the induction of synthesis of other vir gene products. The large virB operon, encoding 11 proteins which are exported to the periplasmic space or bacterial inner membrane (Ward et al, 1988), is believed responsible for determining the physical structure of a pore, 40 or pilus, which makes T-DNA transfer possible. The first two genes of the virD operon, virDl and virD2, together determine an endonuclease activity which introduces nicks in T-DNA border sequences, thereby initiating the production of single-stranded T-DNA molecules (T-strands) (Stachel et al, 1987). The virDl protein becomes covalently linked to the 5' end of these T-strands (Young & Nester, 1988) and it is possible that one of the virE proteins, virE2, which has been shown to be a ssDNA binding protein and is able to coat T strands, may act to protect the T-D N A intermediate during transfer (Citovsky et al., 1989). One of the virC proteins has been shown to bind to an "overdrive" sequence (Peralta et al., 1986; Toro et al, 1988) located next to the right border repeat of the T-DNA. This overdrive sequence has been implicated as being necessary for stimulation of T-strand formation and efficient T-DNA transfer. The final vir region locus, virF, consists of a single gene coding for a 22.4 kDa protein which, while its exact function is unknown, appears to play an accessory role in the virulence of Agrobacterium (Melchers et al, 1990). The development of vector systems for the Agrobacterium mediated transfer of genes to plants has been based on the following observations. 1) Foreign D N A sequences contained within the T-DNA region become incorporated into the plant genome. 2) With the exception of particular 25-bp (TL and T R border repeats) sequences which flank the T region, no T-DNA gene products are required for transfer and insertion of the T-DNA into the 41 plant genome (Bevan, 1984; Yadav et al, 1982). In fact, plant cells transformed with wild-type Ti T-DNA are tumorous and cannot easily be regenerated into morphologically normal plants, hence the requirement for using non-oncogenic, or disarmed, Ti plasmids in which the one genes have been deleted. 3) The T-DNA region does not have to be physically linked to the vir genes of Ti plasmids. Any D N A segment flanked by the 25-bp T-DNA border sequences whether carried by Ti plasmids, other plasmids, or even by the bacterial chromosome, can be transferred from the host Agrobacterium to the plant genome, provided the bacteria has functional vir and chv genes. Currently two different vector systems have been developed for the A. tumefaciens mediated transformation of plants: 1) cis systems in which new genes incorporated into the T-DNA, carried by an intermediate plasmid, are integrated into a disarmed resident Ti plasmid within Agrobacterium via homologous recombination between a common region of homology within the octopine region of the T-DNA (Rogers et al, 1986); and 2) trans, or binary, systems consisting of two elements, a helper Ti plasmid from which the whole T region, including border sequences, has been removed and a broad host range plasmid with cloning sites and marker genes for the identification or selection of transformed plants which are flanked by right and left T-region border sequences. In the latter case, the vir region gene products which are required for T-DNA transmission/insertion are supplied in trans by the resident helper Ti plasmid in Agrobacterium (Bevan, 1984). 42 Key in the process of expressing foreign genes in transformed plant cells is the choice of proper transcriptional promoter and polyadenylation signals known to function in plant cells. Early attempts to express various mammalian and bacterial genes under control of the SV40 promoter were largely unsuccessful (Caplan et al, 1983). Since the nucleotide sequences of only very few plant genes were available at this time, and the few that were (storage protein or photosynthetic protein genes) exhibited stringent tissue specific or developmental stage regulation, much of the initial work was performed using the only other "plant" gene which had been sequenced, nopaline synthase (NOS). The NOS gene is encoded by the T-DNA and is one of the most highly transcribed T-DNA genes in tumour tissue and as well had been found to be constitutively expressed not only in callus tissue but in all of the tissues from rare regenerated plants that had lost the phytohormone biosynthetic genes, but retained the NOS gene (Wostemeyer et al, 1983). The use of the NOS promoter and polyadenylation signals was important in the development of Ti vectors with selectable marker genes, such as the bacterial Tn5 neomycin phosphotransferase II (NPT II) gene which confers resistance to the aminoglycoside antibiotics kanamycin, neomycin and G-418, all of which are inactivated following phosphorylation by NPT II (Herrera-Estrella et al, 1983). The other most commonly used promoter element used for transgenic expression of foreign genes is the cauliflower mosaic virus (CaMV) 35S transcript promoter. More recently, Kay et al, 1987, have described the use of a 43 modified Ti vector containing a duplicated CaMV 35S promoter for the high level expression of plant genes. 2.1.2 Cross-Protection As a Mechanism For Modulating Virus Infection. For many years it has been known that plants infected with mild strains of virus or viroids (small naked ssRNA pathogens) display reduced or delayed symptom development when challenged with a second more virulent strain (McKinney, 1929; Fulton, 1986). This phenomenon, commonly referred to as cross protection, has been exploited in order to reduce yield losses caused by tomato mosaic virus (Broadbent, 1976), potato spindle tuber viroid (Fernow, 1967) and citrus tristeza virus (Costa & Muller, 1980) in crops such as tomato, potato and citrus, respectively. In successful cross protection, the replication of the challenge virus can in some cases be totally suppressed, but more commonly there is simply a delay in symptom development for a period of time after which protection is overcome and symptoms develop. It has been postulated that one of the possible mechanisms of cross protection is that the presence of coat protein from the first virus acts to inhibit the uncoating, and thus the replication, of the challenge virus or that it encapsidates the R N A of the challenging strain, thereby preventing its replication. However, experiments with viroids (Niblett et al, 1978) and with coat protein-deficient mutants of tobacco mosaic virus (TMV) (Sarkar & Smitamana, 1981) argue against this mechanism. Nevertheless, other experiments demonstrating that 44 naked cucumber mosaic virus R N A or TMV RNA or TMV R N A packaged in a foreign (brome mosaic virus) coat protein ("trans encapsidated") effectively escaped the protection afforded by previous infection with a mild strain of the relevant virus (Dodds et al., 1985; Sherwood & Fulton, 1982), indicate that inhibition of virus uncoating plays some role in cross protection, at least in these instances. Alternatively, it has been suggested that the sense, or anti-sense, R N A of the first virus hybridizes with R N A of the challenging virus thereby blocking is replication (Palukaitis & Zaitlin, 1984). From a practical point of view, there are several disadvantages to employing classical cross protection for the control of virus spread in field agriculture. Aside from the fact that the protecting virus may in itself cause economically unacceptable yield losses, the possibility exists that it may undergo mutation leading to extensive crop losses, rather than protection. The protecting virus in one crop species may be a severe pathogen in other crop species which could be grown in the same vicinity. Also, the protecting virus may act in synergy with another unrelated virus leading to disease symptoms much more severe than observed for either virus alone. This latter phenomenon has been observed in simultaneous infection of cucumber mosaic virus and TMV (Garces-Orejuela & Pound, 1957). Most, if not all, of these objections could be overcome if protection could be accomplished by the expression of a single gene product within plant cells, rather than by infection with an intact virus. Towards this end, a great deal of work has been done in 45 recent years in studying the introduction of viral genes into plants, primarily using the Agrobacterium mediated transformation system, for the purpose of inducing resistance. 2.1.3 Genetically Engineered Resistance To Viral Infection. While the precise mechanism responsible for cross-protection has yet to be fully elucidated, it was predicted that expression of the inducing molecule in genetically engineered (transgenic) plants could possibly result in protection against virus infection (Hamilton, 1980). Bevan et al, (1985), were the first to transform tobacco plants with the TMV (OM strain) coat protein gene, but the levels of coat protein expression they obtained (<= 0.001% w / w of soluble leaf protein) were insufficient to cause any resistance to TMV "challenge" inocula (Bevan & Harrison, 1986). The first successful demonstration of genetically engineered resistance to a plant virus was reported by Powell-Abel and coworkers (1986), who, using a modified vector, found that transgenic tobacco plants expressing the coat protein gene from TMV displayed a significant delay in symptom development following inoculation with TMV. Using purified TMV coat protein as standards on Western immunoblots, these transgenic tobacco expressed approximately 100 times the level of coat protein as previously reported by Bevan et al, 1985. The degree of protection afforded by these transgenic plants was dependent on the concentration of TMV in the initial inoculum, with low concentrations (less than 0.8 ug/ml) affording long-46 term protection (greater than 15 day delay in symptom development) in a significant proportion of the challenged plants. Resistance could be almost totally overcome by inoculation with significantly higher levels of TMV. This mimicking of the cross-protection response in transgenic plants expressing the viral coat protein has now been demonstrated for a number of different viruses including alfalfa mosaic virus (A1MV; Van Dun et al, 1987; Turner et al, 1987; Loesch-Fries et al., 1987), cucumber mosaic virus (CMV; Cuozzo et ah, 1988), tobacco rattle virus (Van Dun et al, 1987), tobacco streak virus (Van Dun et al., 1988) and PVX (Hemenway et al, 1988; Hoekema et al, 1989). More recently, simultaneous resistance to two viruses, PVX and potato virus Y, has been reported in transgenic potato plants expressing the nucleocapsid proteins from each of these viruses (Lawson et al, 1990). Results obtained with coat protein mediated protection with T M V (Nelson et al, 1987) and A1MV (Van Dun et al, 1987; Loesch-Fries et al, 1987) showed that protection could largely be overcome by inoculation with naked viral RNA. These data support the model that protection is due to the prevention of uncoating of the challenge virus, however, similar experiments with transgenic plants expressing PVX coat protein showed them to be resistant to challenge with viral R N A as well as intact particles (Hemenway et al, 1988). While these latter results do not obviate a role for coat protein in prevention of uncoating, they do suggest that coat protein and/or its R N A transcript can inhibit the initial events during challenge R N A inoculation by 47 some other mechanism. Recently, Osbourn et al, 1989, using transgenic tobacco protoplasts expressing the coat protein from TMV (Ul strain) have shown that transencapsidation of E. coli fi-glucuronidase (GUS) with TMV U l coat protein, and subsequent electroporation of these pseudovirus particles into either control or TMV U l CP-transformed protoplasts, resulted in an approximately 100 fold difference in GUS activity, thus demonstrating that particle uncoating is inhibited in transgenic cells. However, while such U l CP-transformed protoplasts were resistant to infection with the homologous virus, and were susceptible to infection by the cowpea strain of TMV (Cc) as well as unencapsidated Cc or U l RNA, there was no resistance to infection with hybrid virions composed of Cc R N A transencapsidated in vitro with TMV U l coat protein. These data provide further evidence to indicate that some later step in virus replication may be involved in coat protein mediated resistance. That the resistance observed with plants expressing the viral coat protein gene is in fact due to accumulation of capsid protein and not the presence of coat protein R N A sequences (transcripts) has recently been demonstrated by Powell et al, 1990. When tobacco plants which had been transformed with a chimeric gene encoding the TMV coat protein, or a similar construct in which the initiating ATG codon had been removed, only those plants which accumulated coat protein, as opposed to silent transcript, were protected against infection by TMV. 48 Another approach which has been used with some success with at least two plant viruses, PVX (Hemenway et al, 1988) and C M V (Cuozzo et al, 1988), is the transgenic expression of an antisense transcript to the viral coat protein. Mechanistically, it has been proposed that such antisense constructs may interfere with viral replication by hybridization with the opposite sense viral RNA. The efficacy of this approach is uncertain, since double stranded RNAs (dsRNAs) are naturally occurring replicative intermediates for many plant viruses. In general the levels of protection observed in such transgenic plants is only significant at low levels of virus inoculum and does not approach the same degree of protection afforded by coat protein expression. While the coat protein mediated resistance observed in transgenic plants for a number of plant viruses may not operate via the same mechanism as for classical cross-protection, and indeed may well vary amongst different viruses (e.g. TMV and PVX), it is currently the most applicable method for engineering resistance. This chapter describes the production of transgenic plants expressing the nucleocapsid protein of PVS, either Nicotiana debneyii, a tobacco model system for studying PVS infection, or 'Russet Burbank' potato, and their use in studying virus resistance. 49 2.2 TRANSGENIC EXPRESSION OF P V S COAT PROTEIN IN NICOTIANA DEBNEYII. 2.2.1 MATERIALS AND METHODS 2.2.1.1 Construction of Plasmid pVS153. The preparation and characterization of a complementary D N A (cDNA) clone (pVS57), which contains the coding sequence of the entire PVS coat protein gene, as an in-frame fusion with the lacZ oc-peptide, has been previously described (Chapter 1; see Fig. 3). The insert contained in clone pVS57 corresponds to the 3' terminal 1284 nucleotides from PVS R N A and the 5' terminus of this insert is located 16 nucleotides upstream from the initiating A T G codon for the 33K viral coat protein gene. D N A containing a full length copy of the PVS coat protein gene was transferred into the intermediate co-integrate Ti plasmid, pCDX-1. This vector is a derivative of pMON178 which has been modified to contain a duplicated cauliflower mosaic virus 35S promoter sequence upstream from a multiple cloning site and the nopaline synthase (Nos) polyadenylation signal (Kay et al, 1987). In order to obtain compatible restriction enzyme digestion sites, plasmid pVS57 was digested with HindUl and the gel purified fragment ligated into HmdIII digested Bluescript vector. Following transformation, purified plasmid D N A from individual colonies was screened by restriction enzyme digestion with Apal. The pVS57 insert contains an Apal restriction site located 245 50 nucleotides from the 3' terminus. Therefore, clones in which the 5' terminus of the viral coat protein gene was proximal to the Xhol site within Bluescript should yield a large, approximately 1 kb, fragment following Apal digestion. Plasmid D N A from one such clone, pVS136, was double digested with EcoKL and Xhol and the resulting fragment was ligated into similarly digested pCDX-1 vector. Ligated D N A was then used to transform E. coli MM294 cells to spectinomycin resistance. Recombinant clones were then screened for the appropriate PVS coat protein insert by digestion of purified plasmid D N A with Xbal and EcdBl. 51 Genome organization of PVS and the preparation of an intermediate vector, pVS153, for expression of viral coat protein. Fig. 12. Schematic representation of the method used for integration of the PVS coat protein gene into the intermediate co-integrate vector, pCDX-1. A HindW fragment derived from pVS57 was cloned into Bluescript vector and subsequently excised by double digestion with Xho\ and EcoR\ for insertion into Xhol - EcoR\ digested pCDX-1. 52 Co-Integration of Intermediate Plasmid Containing PVS Sequence Into Disarmed Ti Plasmid TL KarV Integrated Into Plant Genomic DNA Fig. 13. Schematic representation of the tri-parental mating procedure used for the integration of the T-DNA region of pVS153 into the resident disarmed Ti plasmid, pTiB6S3SE, carried by A. tumefaciens GV3111SE, via homologous recombination. A culture of E. coli containing plasmid pVS153 was combined with cultures of E. coli, containing the mobilization plasmid pRK2013, and A. tumefaciens by filtration onto a 0.2 micron filter disk and incubated overnight at 29 °C. Resuspended cells were then cultured in the presence of spectinomycin, kanamycin and chloramphenicol for selection of appropriate colonies which had undergone recombination. 53 2.2.1.2 Tri-Parental Mating Procedure. The PVS coat protein construct from plasmid pVS153 was inserted into the resident disarmed octopine type plasmid, pTiB6S3SE, harboured by Agrobacterium tumefaciens strain GV3111SE, using the mobilization plasmid pRK2013 in a triparental mating procedure (Rogers et al., 1986). Colonies of A. tumefaciens GV3111SE/pTiB6S3SE in which the transfer D N A from plasmid pVS153 had been successfully co-integrated into pTiB6S3SE by homologous recombination were isolated by resistance to spectinomycin (100 ug/ml), kanamycin (50 fxg/ml) and chloramphenicol (25 u.g/ml). Overnight cultures of £. coli MM294 containing either plasmid pVS153 or pRK2013 were diluted into fresh Luria Broth (LB) and cultured at 37 °C to mid log phase ( O D ^ ^ = 0.75) in the presence of 100 ug/ml spectinomycin or 50 Ug/ml kanamycin respectively. A culture of A. tumefaciens GV3111SE/pTiB6S3SE was grown to log phase at 29 °C in LB, containing 50 Ug/ml kanamycin and 25 u,g/ml chloramphenicol. For mating, 1 ml aliquots of each culture were combined in a sterile polypropylene tube (100 mm x 17 mm), centrifuged for 5 min at 3,000 x g and resuspended with 2 ml of sterile 10 m M MgS0 4 . The mixture was then transferred to a 10 ml disposable syringe and the cells deposited onto a sterile 25 mm 0.2 micron filter (Millipore type GS) using a Swinnee filter apparatus. The filter disc was then transferred onto a fresh LB agar plate (w/o antibiotics) and cultured overnight at 29 °C. The filter was then removed and vortexed with 2 ml of 10 m M MgS0 4 to 54 resuspend the cells. Aliquots of this cell suspension (0.25 ml) were then plated onto fresh LB agar plates containing chloramphenicol (25 ug/ml), kanamycin (50 ug/ml) and spectinomycin (100 ug/ml). Plates were incubated for 3-4 days at 29 °C and individual antibiotic resistant colonies were isolated and transferred to 3 ml of LB (containing antibiotics as above) and cultured for 24-36 hrs at 29 °C prior to freezing at -70 °C. 2.2.1.3 Transformation and Regeneration of Nicotiana debneyii. Leaf disc explants from Nicotiana debneyii were transformed with A. tumefaciens (GV3111SE/pTiB6S3SE) carrying the PVS coat protein construct using the protocol originally described by Horsch et al., 1985. Leaves (6-8 cm in size) were surface sterilized in a solution of 10% (v/v) bleach (Javex), 0.1% (v/v) Tween-20 for 20 min then rinsed with several changes of distilled water. Leaf discs were punched out using a sterile #8 cork borer and overlaid onto a solution of MS (Murashige & Skoog, 1962) salts containing a 1/50 suspension of an overnight culture of A. tumefaciens, and incubated for 5-10 min with gentle agitation to ensure that all edges were infected. Leaf discs were then blotted dry and plated upside down onto agar plates made up with MS salts, 30 g/1 sucrose, benzyladenine (BA; 1.0 |ig/ml) and naphthalene acetic acid (NAA; 0.1 ug/ml), and incubated for 36-48 hrs at 23 °C. Following co-culture with Agrobacterium, the explants were then rinsed with two changes of sterile MS salts to remove excess agrobacteria and plated 55 onto callus regeneration-shoot initiation medium containing MS salts, 0.5% (w/v) agar (Sigma), sucrose (30 g/1), BA (1.0 ug/ml), N A A (0.1 ug/ml), as well as carbenicillin (Sigma; 500 ug/ml) and kanamycin (Sigma; 250 ug/ml). Plates were sealed with parafilm and incubated in growth chamber at 23 °C set for a 16 hr light cycle (5000 lux). Explants were transferred onto fresh shoot initiation plates every 7-10 days. After 4-6 weeks, transformed shoots that developed were excised and transplanted into phytohormone free medium, containing 100 ug/ml kanamycin and 250 ug/ml carbenicillin, for root initiation. Rooted plantlets were then transferred to soil after 2-3 weeks. 2.2.1.4 Western Blot Analysis of PVS Coat Protein Expression in Transgenic Nicotiana Debneyii. The ability of transgenic N. debneyii to produce PVS coat protein was assessed by Western immunoblotting using a specific monoclonal antibody to the viral capsid protein. Samples of leaf tissue (100 mg) were homogenized with 100 ul of SDS-PAGE sample buffer (4% (w/v) SDS, 125 m M Tris-HCl pH6.8, 10% (v/v) 2-mercaptoethanol, 0.04% (w/v) bromophenol blue, 20% (v/v) glycerol) and incubated at 95 °C for 5 min. Following centrifugation at 13,000 x g for 5 min in an Eppendorf microcentrifuge, 15 ul aliquots were loaded onto a 12% polyacrylamide gel and, after electrophoresis using the buffer system of Laemmli (1970), separated proteins were blotted onto Immobilon membrane (Millipore) in 25 m M Tris, 192 m M glycine, 20% (v/v) 56 methanol pH8.3 at 100V (0.25 A) for 60 min at 4 °C. The transfer blots were treated with blocking buffer (10 m M Tris-HCl pH7.4, 0.15 M NaCl, 1% (w/v) BSA, 0.05% (v/v) Tween-20, 0.1% (w/v) NaN 3) for 60 min and incubated with M A b 1G11 hybridoma culture supernatant (1/50 in blocking buffer) for 60-90 min. Following rinsing with PBS plus 0.1% (v/v) Tween-20, blots were incubated with [125I]-labelled goat anti-mouse IgG (1.5 x 106 cpm/ug; 1.0 x 106 cpm/ml in blocking buffer) for 60-90 min followed by exhaustive rinsing with PBS-Tween and overnight autoradiography (-70 °C). 2.2.1.5 Purification of Rabbit Anti-PVS Ig. The IgG fraction from rabbit polyclonal anti-PVS serum was purified by ammonium sulfate fractionation and ion-exchange chromatography. Approximately 25 ml of serum was diluted 1:1 with Dulbecco's PBS and brought to 50% saturation with (NH 4 ) 2 S0 4 by addition of an equal volume of saturated solution. Precipitated protein was washed 2x by centrifugation with 50% (NH 4) 2S0 4 , resuspended in a minimum volume of distilled H z O and dialysed overnight against 2L of 10 m M Tris-HCl pH7.4. The IgG fraction was further purified by fast liquid protein chromatography (FPLC) on Mono-Q resin (10 x 100 mm; Pharmacia) developed with a linear gradient of NaCl (0-0.3 M) in 10 m M Tris-HCl pH7.4. Purified antibody was concentrated by 50% (NH 4) 2SQ 4 precipitation and extensively dialysed against PBS. 57 2.2.1.6 Preparation of Enzyme-Antibody Conjugates. For use in double antibody sandwich (DAS)-ELISA, specific antibodies were conjugated to alkaline phosphatase using the one-step glutaraldehyde condensation procedure originally introduced by Avrameas, (1969). Briefly, alkaline phosphatase (3.0 mg) and purified IgG (1.0 mg) were mixed together in a total of 0.5 ml of PBS and incubated in the presence of 0.2% (v/v) glutaraldehyde for 2 hrs at room temperature with gentle stirring. The reaction mixture was then diluted by addition of 1.0 ml 10 m M Tris-HCl pH8.0, 0.15 M NaCl , 1 m M MgCl 2 and dialysed against this same buffer (2-3 changes; 1 litre) for 16-24 hrs at 4 °C. Following dialysis, the conjugate was stabilized by addition of BSA to 1% (w/v), diluted with glycerol to 50% (v/v) and stored at -20 °C. In general, further purification of enzyme-antibody conjugates prepared in this manner was not necessary. 2.2.1.7 Evaluation of PVS Resistance In Transgenic Nicotiana debneyii. Three transgenic lines (J3, J5 and J7) which expressed similar quantities of viral coat protein, were used to evaluate resistance to PVS infection. The ME strain of PVS which infects N. debneyii was used in these tests since the Peruvian strain used in the cloning did not readily infect N. debneyii. The ME strain was purified as described previously (MacKenzie et al, 1989) from inoculated leaves of Chenopodium amaranticolor. Groups of F2 progeny plants 58 (12 plants each group), which had been selected by germination on medium containing 200 mg/ml kanamycin, together with equal numbers of non-transformed N. debneyii, were mechanically inoculated with a preparation of purified virus at concentrations of 0.5,1.0, 2.0 and 5.0 ug/ml. Alternatively, plants were inoculated with purified viral R N A (2 ug/ml) prepared from alkaline SDS treated particles by multiple phenol-chloroform extractions and ethanol precipitation. The accumulation of viral antigen in the inoculated and upper leaves was measured 19, 27, 36 and 45 days after inoculation by using a double antibody sandwich (DAS) ELISA. Leaf tissue samples (108 mg) were obtained using a #10 cork borer and processed through a mechanical leaf press irrigated with 0.5 ml of ELISA blocking buffer (10 m M Tris-HCl pH7.4, 0.15 M NaCl, 1% (w/v) bovine serum albumin, 0.05% (v/v) Tween-20, 0.1% (w/v) NaNg). For DAS-ELISA, EIA microtitre plate wells were coated with 100 ul of FPLC purified rabbit anti-PVS Ig (20 ug/ml in 50 m M sodium carbonate pH9.6) overnight at 4 °C. Wells were then rinsed with water, treated with blocking buffer for 60 min and incubated with 100 ul volumes of serially diluted tissue homogenate in blocking buffer overnight at 4 °C. Following rinsing, wells were incubated sequentially with 100 ul of alkaline phosphatase conjugated rabbit anti-PVS Ig (200 ng/ml in blocking buffer; 90min) and p-nitrophenyl phosphate substrate (0.5 mg/ml in 10% (v/v) diethanolamine pH9.8; 2 hrs at 24 °C). The absorbance of each well was measured at 405 nm 59 using a Titertek Multiskan M C (Flow) multiwell plate reader interfaced with an IBM P C / A T microcomputer. Concentrations of PVS in each sample were computed relative to a standard response curve constructed using samples of purified virus (5000 to 9.8 ng/ml) diluted in blocking buffer. Composite samples representing an average of at least four plants were analyzed in triplicate for each time point and for each inoculation level. Control samples consisting of non-inoculated transgenic plant tissue were also included. 60 2.2.2 RESULTS 2.2.2.1 Transformation of N. debneyii and Expression of PVS Coat Protein. Approximately 40% of the transgenic plants obtained expressed detectable levels of PVS coat protein as judged by immunoblotting. The highest levels of expressed coat protein, as illustrated by line J3 (Fig. 14), were on the order of 0.1-0.2% of total SDS-soluble protein. F ig . 14. I m m u n o b l o t analysis of transgenic N. debneyii, line J3, expressing P V S c o a t p r o t e i n . Immunoblots of coat protein (100 ng) from a purified preparation of PVS (lane a) and total SDS soluble protein (10 u.g) extracted from J3 transgenic tissue (lane b) or a non-transformed plant (lane c) were incubated with MAb 1G11 followed by [ 1 2 5 l ] -labelled goat anti-mouse Ig. The migration of PVS coat protein (CP)is indicated. a b c 2.2.2.2 Resistance of Transgenic N. debneyii To PVS Infection. After inoculation all of the control, non-transformed, plants developed characteristic symptoms of vein clearing and chlorosis (Bagnall et al, 1959; Mackinnon & Bagnall, 1972) on the upper uninoculated leaves, with little or no apparent symptoms on the inoculated leaves (Fig. 15 a,c). These symptoms were clearly discernable 3 weeks after inoculation with each of the inoculum 61 concentrations tested. As indicated by DAS-ELISA (Fig. 16), these plants also had accumulated high levels of viral antigen in systemic leaves by 45 days after inoculation, with concentrations ranging from 80 to 110 ng/mg wet weight of tissue. In contrast, the transgenic J3 plants neither exhibited symptoms (Fig. 15 b,d), nor accumulated greater than background levels of coat protein antigen after 45 days, even at the highest inoculum level tested (Fig. 16; Table II). Transgenic lines, J5 and J7, which expressed similar levels of viral coat protein as J3, were equally resistant to infection (Table II). In contrast to previous reports for. plants expressing TMV or A1MV coat protein, inoculation of J3 transgenic plants with PVS R N A did not overcome protection (Fig. 17; Table II). This result is in agreement with other data obtained using transgenic plants expressing PVX coat protein (Hemenway et al, 1988). Not surprisingly, J3 transgenic plants were not protected against inoculation with a severe strain of PVX. Both J3 and non-transformed N. debneyii rapidly accumulated high, levels of PVX viral antigen within 10 days following inoculation (Table HI). Also, transgenic plants which were inoculated with a mixture of PVS and PVX continued to retain resistance to PVS infection, while control, non-transformed, plants accumulated high levels of both PVS and PVX by 41 days after inoculation (Table in). 62 Fig. 15. Comparison of symptom development on control, non-transformed, N. debneyii plants (panels a,c) compared with the J3 line of transgenic N. debneyii (panels b,d) at 36 days after inoculation with 5.0 u.g/ml of purified PVS, strain ME. 63 19 27 36 45 Days After Inoculation Fig. 16. Accumulation of PVS coat protein antigen in the upper leaves of non-transformed N. debneyii (A), and J3 transgenic N. debneyii (B) after inoculation with PVS strain ME at concentrations of 0.5,1.0 and 2.0 ug/ml, at various days post inoculation. The blank bar, (C), shows the background value obtained with uninoculated J3 transgenic plants. Viral coat protein concentrations were determined by DAS-ELISA and are expressed as the log 1 0 of ng virus coat protein / mg wet weight tissue. 64 Table II. Concentration of PVS coat protein antigen in transgenic and normal Nicotiana debneyii 45 days after inoculation with either intact PVS or viral RNA. PVS Concentration (ng/mg tissue) Plant Line Inoculum Level (u.g/ml) Inoculated Leaf Upper Leaf J3 transgenic 0.5 <0.1(a) <0.2 5.0 ' <0.2 <0.2 2.0 (RNA) N.D. <0.1 J5 transgenic 2.0 <0.2 <0.1 J7 transgenic 2.0 <0.1 <0.2 Non-transformed 0.5 30.7 80.3 5.0 47.6 110.2 2.0 (RNA) N.D. 55.3 < a ) Concentrations of PVS coat protein antigen in the range of < 0.1-0.2 ng/mg tissue represent the lower limit of detection by DAS-ELISA and are equal to the background values obtained from non-inoculated transgenic plants. N.D.- not determined. Tissue Homogenate Reciprocal Dilution Fig. 17. DAS-ELISA detection of PVS in J3 transgenic N. debneyii (m) and non-transformed N. debneyii (A) 45 days following inoculation with 2.0 ng/ml purified PVS RNA. Microtitre plate wells, previously coated with rabbit anti-PVS IgG, were incubated with 100 u.l volumes of serially diluted tissue homogenate and bound PVS coat protein antigen was detected following incubation with alkaline phosphatase conjugated rabbit anti-PVS Ig. 65 Table in . Concentration of PVS and PVX coat protein antigen in transgenic J3 and non-transformed Nicotiana debneyii following inoculation with PVS (ME strain), PVX (severe strain) and a mixture of PVS and PVX. Concentration (ng/mg tissue) J3 Transgenic Non-Transformed Inoculum 10 DPI"" 41 DPI 10 DPI 41 DPI PVS (1.0 ug/ml) N.D. ^o.is*' N.D. 85.1 PVX (0.5 ug/ml) 191 N.D. 207.4 N.D. PVS (1.0 ug/ml) + PVX (0.5 ug/ml) PVS antigen N.D. <0.25 N.D. 126.3 PVX antigen 239.4(c) 241 169.2 262.5 Days post inoculation. Concentrations of PVS coat protein antigen in the range of < 0.1-0.2 ng/mg tissue represent the lower limit of detection by DAS-ELISA and are equal to the background values obtained from non-inoculated transgenic plants. < 0 The concentration of PVX coat protein antigen was determined using DAS-ELISA in a similar manner as described for the detection of PVS, with the exception that EIA microtitre plate wells were coated with purified rabbit anti-PVX IgG (5 ug/ml) and bound viral antigen was detected using a specific monoclonal antibody to the capsid protein. N.D.- not determined. 66 2.3 TRANSGENIC EXPRESSION OF P V S COAT PROTEIN IN 'RUSSET B U R B A N K ' POTATO 2.3.1 MATERIALS AND METHODS 2.3.1.1 Transformation and Regeneration. Leaf explants from in vitro grown 'Russet Burbank' potato were wounded and co-cultivated with Agrobacterium tumefaciens strain GV3111SE carrying the pTiB6S3SE-pVS153 Ti plasmid construct and transformed shoots regenerated under kanamycin selection following the procedure of De Block, 1988. Sterile plants of cultivar 'Russet Burbank' were propagated in vitro by transferring stem explants, together with an axillary bud, to RO (Table IV) medium. The shoots were grown at 23 °C with a daylength of 16 hr under 5000 lux light intensity. Leaves (3-8 mm) from 3-4 week old shoots were wounded by cutting at the base and floated upside down on 10 ml of RO medium (w/o kinetin, agar) in 100 mm diameter petri plates, which had been seeded with 50 | i l of a late log phase culture of A. tumefaciens. The plates were then incubated for 48-72 hrs at 23 °C under low light intensity (500 lux). The leaf explants were then washed with R0 medium containing 500 mg/1 carbenicillin, 250 mg/1 cefotaxime (Calbiochem), patted dry on sterile filter paper, and placed upside down on R l medium containing 100 mg/1 kanamycin. Explants were 67 transferred to fresh medium at weekly intervals. After 3-4 weeks in culture, explants were transferred to R2 medium where they were maintained for a further 3 weeks (again with weekly transfer to fresh medium). Regenerated calli were then transferred to R3 medium in 400 ml glass jars where they were maintained for a further 2-3 weeks until shoots appeared. Shoots (0.5-1.0 cm in height) were transferred to individual tubes containing R4 medium for root initiation. After 2-3 weeks, rooted shoots were transferred to soil, or expanded in tissue culture using the normal propagation medium, RO. Table IV. Composition of various media used for the regeneration of transformed 'Russet Burbank' potato plants. Designation Composition RO MS salts pH5.75 (Murashige & Skoog, 1962) with 30 g/l sucrose, 0.04 mg/l kinetin, 0.5% agar (Sigma). R1 MS medium without sucrose and supplemented with 200 mg/l glutamine, 0.5 g/l MES. 0.5 g/l PVP-40, 20 g/l mannitol, 20 g/l glucose, 40 mg/l adenine sulfate, 1 mg/l zeatin riboside, 0.1 mg/l NAA, 500 mg/l carbenicillin, 400 mg/l cefotaxime, 10 mg/l AgN0 3, 0.5% agar, 0.5 g/l casein hydrolysate. R2 R1 medium without NAA, but with 0.1 mg/l IAA and with one-half concentration of antibiotics. R3 R2 medium supplemented with 0.1 mg/l GA3, 200 mg/l carbenicillin. R4 R0 medium supplemented with 2 mg/l IAA, 200 mg/l carbenicillin. 2.3.1.2 Enzyme-Linked Immunosorbent Assay (ELISA). Samples of leaf tissue (100 mg) from kanamycin resistant regenerated 'Russet Burbank' shoots in tissue culture were initially screened for expression 68 of PVS coat protein using a double antibody sandwich (DAS) ELISA. Tissue samples were homogenized with 200 ul of blocking buffer containing 1 m M phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor, using a stainless steel pestle fitted to an Eppendorf microcentrifuge tube (1.5 ml). Samples were tested by ELISA using plates coated with rabbit anti-PVS IgG and alkaline phosphatase conjugated rabbit anti-PVS IgG as previously described. Transgenic plantlets which had been transferred to soil and which tested positive by ELISA were subsequently screened by Western immunoblotting using polyclonal rabbit anti-PVS Ig and [125I]-Protein A using the same protocol described for detection of PVS coat protein in transgenic N. debneyii. A similar procedure was used for the ELISA detection of virus infection in inoculated plants. Duplicate composite samples, consisting of 6 mm diameter leaf discs obtained from at least 6 plants (approx. 108 mg tissue total), were processed through a mechanical leaf press irrigated with 0.5 ml of blocking buffer and antibody coated wells were incubated with 100 ul volumes of serially diluted tissue homogenate in blocking buffer overnight at 4 °C. Concentrations of PVS in each sample were computed relative to a standard response curve constructed using samples of purified virus (5000 to 2.45 ng/ml) diluted in blocking buffer. The concentration of P V M in infected tissue samples was similarly determined by DAS-ELISA, except that microtitre plate wells were initially coated with rabbit anti-PVM IgG (10 ug/ml) and bound virus was detected 69 using a specific monoclonal antibody and alkaline phosphatase conjugated goat anti-mouse Ig. 2.3.1.3 Virus Inoculation of Potato Plants. Transgenic and non-transformed potato plants were propagated in tissue culture and transferred to soil. Plants at the 4-6 leaf stage were dusted with Carborundum (used as an abrasive for leaf wounding) and inoculated with various dilutions (0.5,1.0, 2.0 and 5.0 M-g/ml in 10 m M sodium phosphate buffer pH7.2) of a preparation of the Andean strain of PVS. Duplicate composite samples, consisting of 6 mm diameter leaf discs obtained from at least 6 plants (approx. 108 mg tissue total), were collected at various times after inoculation and assayed for the presence of PVS coat protein antigen as previously described. In separate experiments, plants were also inoculated with purified viral R N A (2.0, 5.0 |ig/ml), obtained from alkaline SDS treated particles by multiple phenol/chloroform extractions and ethanol precipitation, or with a purified preparation of P V M (5.0 ug/ml). 70 2.3.2 RESULTS 2.3.2.1 Regeneration and transformation. The A. tumefaciens mediated transformation and regeneration of 'Russet Burbank' potato, using the method described, is an efficient process and gives rise to rooted transgenic shoots in about 9-12 weeks. Virtually all of the initial leaf explants gave rise to one or more vigorously growing calli and greater than 90% of these ultimately produced shoots. The addition of A g + to the shoot initiation medium had a profound effect on callus formation and proliferation with 'Russet Burbank' leaves. It is known that potato plants in vitro can produce large amounts of ethylene, which is characterized by the formation of brown-yellow, friable callus which is nearly impossible to regenerate (Hussey & Stacey, 1981). As previously reported by De Block, 1988, callus formed in the presence of A g + maintained a robust green colour and was highly regeneratable with multiple shoots initiating from each individual callus. This stimulatory effect of A g + on callus formation has been suggested to be due to its ability to block ethylene action by binding to the ethylene receptor(s) (Aharoni et al, 1979; Beyer et al, 1976). Of the 85 rooted transgenic shoots generated, approximately half were tested for expression of PVS coat protein by ELISA, and 70% of these produced easily detectable levels of viral coat protein. Ten of these coat protein positive transgenic lines, which appeared morphologically normal, were maintained in tissue culture and expanded further. 71 2.3.2.2 Expression of PVS coat protein. Western immunoblot analysis was used to confirm the in vivo expression of PVS coat protein in regenerated transgenic potato plants. Protein extracts from four different transgenic lines, non-transformed 'Russet Burbank' potato, and different concentrations of purified PVS were separated by SDS-PAGE and blots were probed with rabbit anti-PVS IgG and [125I]-labelled Protein-A (Fig. 18). Based on comparisons with PVS standards (Fig. 18 lanes 1,2,3) the level of coat protein expression in three of these lines, RB18, RB41 and RB58 (Fig. 18 lanes 5, 6, 8) was judged to be approximately 0.1-0.2% of the total SDS soluble protein present in tissue extracts, while RB52 plants (Fig. 18, lane 7) produced about 2-5 fold less coat protein. 1 2 3 4 5 6 7 8 66.2-42.7-21.5-Fig. 18. Western blot analysis of PVS coat protein expression in transgenic 'Russet Burbank' potato plants. Immunoblots of coat protein (100, 50, 20 ng) from a purified preparation of PVS (lanes 1,2,3) and total SDS soluble protein (10 ug) extracted from leaf tissue of transgenic lines RB18 (lane 5), RB41 (lane 6), RB52 (lane 7), RB58 (lane 8) or a non-transformed potato plant (lane 4) were incubated with rabbit anti-PVS IgG followed by [ 1 2 Sl]-labelled Protein-A. The migration of PVS coat protein (CP) and Mr standards (x10 3) are indicated. 72 2.3.2.3 Susceptibility of Transgenic 'Russet Burbank' Potato to Infection with PVS or Viral RNA. Transgenic potato plants expressing the PVS coat protein were challenged by inoculation with purified PVS or PVS RNA. Virus replication and spread were monitored in these plants by sampling discs from the upper non-inoculated leaves of each plant at various times after inoculation and quantitating viral antigen levels by DAS-ELISA. By 2-3 weeks following inoculation of non-transformed 'Russet Burbank' potato with a purified preparation of the Andean strain of PVS, measurable quantities of virus could readily be detected, and after 5 weeks the virus titre had reached approximately 20-25 ng/mg wet weight of tissue (Fig. 19, panel a). Plants inoculated with the ME strain of PVS showed a more rapid accumulation of virus and also displayed an approximately 2 fold higher maximum level of virus, as compared with the Andean strain. Non-transformed plants which had been inoculated with PVS RNA, while displaying a slower rate of virus accumulation in systemic leaves when compared with plants inoculated with intact virus particles, did show comparable virus titres by 6 weeks after inoculation (Fig. 19, panel b). In contrast, RB58 transgenic plants failed to accumulate any significant (greater than background) quantities of viral antigen over the course of the experiment (Fig. 19, panel a), even at the highest inoculum level tested (5 (xg/ml). In agreement with previous results with transgenic Nicotiana debneyii expressing PVS coat protein, these PVS CP+ potato plants also failed to show 73 any significant accumulation of virus in the inoculated (Table V) or upper leaves (Fig. 19, panel b) following inoculation with purified viral RNA. The PVS resistance of 4 other transgenic potato lines (RB18, RB41, RB52 and RB81), which expressed similar levels of PVS coat protein, paralleled the results obtained with RB58 plants (Table V). 74 m P V S - A n 0.5 ug/ml 2.0 ug/ml • 5 - ° u 9 / m l P V S - M E II 1.0 ug/ml PVS-An RNA 2.0 ug/ml 5.0 ug/ml 1 0 2 1 3 3 4 1 Days A f te r I n o c u l a t i o n Fig. 19. Accumulation of PVS coat protein antigen in the upper leaves of non-transformed (A), and RB58 transgenic 'Russet Burbank' potato (B) after inoculation with either intact PVS particles, or PVS RNA, at various days post inoculation. Plants were mechanically inoculated with preparations of the Andean strain of PVS (0.5, 2.0, 5.0 ug/ml), the ME strain of PVS (1.0 ug/ml), or PVS-An RNA (2.0, 5.0 ug/ml). Viral coat protein concentrations were determined by DAS-ELISA and are expressed as the log 1 0 of ng virus coat protein / mg wet weight tissue. 75 Table V. Concentration of PVS coat protein antigen in transgenic and non-transformed 'Russet Burbank' potato following inoculation with PVS or PVS RNA. Plant Line Inoculum Level (ug/ml) Days After Inoculation PVS Concentration (ng/mg tissue) RB18 1.0 41 <0.008(a> 10.0 41 <0.004 RB41 1.0 41 <0.003 10.0 41 <0.007 RB52 1.0 41 <0.004 10.0 41 <0.002 RB58 5.0 60 <0.002 5.0 (RNA) 21 ^O.OM0" 5.0 (RNA) 60 <0.003 RB81 10.0 47 <0.009 PVS-ME sap(c) 47 <0.003 Non-transformed 1.0 47 21.49 10.0 47 21.98 5.0 60 53.82 5.0 (RNA) 21 15.73*' 5.0 (RNA) 60 24.35 PVS-ME sap 47 115.3 0 0 Concentrations of PVS coat protein antigen in the range of <0.002-0.030 ng/mg tissue represent the lower limit of detection by DAS-ELISA and are equal to the background values obtained from non-inoculated transgenic plants. Concentration of PVS coat protein antigen in lower inoculated leaves 21 days following inoculation with purified viral RNA. < c ) Plants were inoculated with a sap extract from plants systemically infected with PVS-ME prepared by homogenizing 1.0 g leaf tissue with 10 ml of 10 mM sodium phosphate buffer pH7.4. 76 The extent of protection offered by transgenic plants expressing T V S coat protein was further evidenced by the lack of accumulation of PVS in the upper leaves of non-transformed 'Russet Burbank' shoots which had been grafted onto RB41 plants inoculated with purified PVS-An (Table VI). In separate experiments, transgenic RB41 or RB58 shoots which had been grafted onto non-transformed potato plants systemically infected with PVS also failed to accumulate any significant levels of PVS by 21 days after grafting. Under the same conditions, the concentration of PVS coat protein antigen in non-transformed, initially virus-free, shoots similarly grafted onto PVS infected parent plants was equivalent to that observed in systemically infected 'Russet Burbank' by 21 days after grafting (Table VI). In a similar manner, the ability of P V M to effectively cross-protect against PVS infection was also demonstrated by the lack of accumulation of PVS in the upper leaves of P V M infected shoots which had been grafted onto PVS infected parent plants (Table VI). The accumulation of PVS (42 days after grafting) in stem or leaf tissues from various parts of these grafted plants is shown in Fig. 20. Tissue samples removed from either the lower or upper stem segments, or the apical leaves of grafts of transgenic (RB41, RB58), or P V M infected shoots onto PVS infected parent plants showed a marked reduction in the level of PVS antigen when compared with similar samples from plants containing non-transformed grafts. For example, stem segment samples removed from transgenic RB41 and RB58, or P V M infected grafts just above the graft union 77 contained 0.28, 0.04 and 0.05 ng PVS/mg tissue, respectively, as compared to an average of 12.93 ng PVS/mg tissue in stem segment samples taken from just below the graft union from these same plants, or 18.17 ng PVS/mg tissue in similar samples taken from plants containing non-transformed shoot grafts. In separate experiments, transgenic RB58 shoots containing a non-transformed apical graft were also grafted onto PVS infected parent plants. By 25 days after grafting no significant concentrations of PVS could be detected by DAS-ELISA in samples of leaf or stem tissue taken from either the transgenic portion of the graft or the upper non-transformed graft (Fig. 21). The cross-protection observed in grafts of P V M infected tissue onto PVS infected host plants was not reciprocal in that PVS infected shoots grafted onto plants systemically infected with P V M contained similar titres of P V M , by 21 days following grafting, as non-infected shoots which had been similarly grafted (results not shown). 78 Table VI. Accumulation of PVS in non-transformed or transgenic potato tissue following grafting onto transgenic or virus infected host plants. Parent Plant Graft DPI/DAG< < 0 PVS Concentration (ng/mg tissue) Non-transformed 42 <0.017<c) RB RB41 42 <0.026 Non-transformed RB (d> (none) 42 15.98 PVS infected RB ( e ) Non-transformed 21 25.45 RB 42 32.33 RB41 21 <0.028 42 0.265 RB58 21 <0.016 42 0.083 PVM infected RB 21 <0.019 42 0.115 (none) - 23.93(0 < a ) Days post inoculation or days after grafting. *' Shoots from either non-transformed or transgenic RB41 'Russet Burbank' were grafted onto transgenic RB41 parent plants. The concentration of PVS coat protein antigen present in the upper leaves of each graft was determined from DAS-ELISA of tissue samples removed 42 days following inoculation of the lower, transgenic, leaves with a purified preparation of the Andean strain of PVS (2.0 ug/ml). ( c ) Concentrations of PVS coat protein antigen in the range of <0.002-0.03 ng/mg tissue represent the lower limit of detection by DAS-ELISA and are equal to the background values obtained from non-inoculated transgenic plants. ( d > Concentration of PVS in the upper leaves of control non-transformed plants 42 days following inoculation with 2.0 ug/ml PVS. ( e > Shoots from either non-transformed, transgenic RB41 or RB58, or from 'Russet Burbank' plants systemically infected with PVM were grafted onto PVS infected parent plants. The level of PVS accumulation in the upper leaves of each graft was determined by DAS-ELISA 21 and 42 days after grafting. < 0 Concentration of PVS in systemically infected parent plant. 79 RB58 RB41 PVM Inf. RB wt G r a f t I d e n t i t y Apica l leaf from graft I I Ap ica l stem sect ion from graft | ^ Graft stem sect ion just above graft union | Stem sec t ion from parent plant Lower leaf from parent plant Fig. 20. Concentration of PVS coat protein antigen in the tissues of systemically infected plants containing shoot grafts from either transgenic (RB41, RB58), PVM infected or non-transformed plants. Tissue samples from either the lower infected leaves or stem segments of the parent plant, as well as stem segments from just above the graft union and apical stem segments or apical leaves from the graft were removed 42 days after grafting and assayed for the presence of PVS by DAS-ELISA. Viral coat protein concentrations are expressed as the log 1 0 of ng virus coat protein / mg wet weight tissue. 80 PVS Concentration Non-Transformed RB Graft RB58 Transgenic Graft PVS Infected Host Plant Fig. 2 1 . Concentrations of PVS coat protein antigen in the leaf or stem tissues of plants systemically infected with PVS containing double grafts of transgenic RB58 tissue and upper non-transformed tissue. Tissue samples from either the lower infected leaves or stem segments of the parent plant, as well as leaf and stem segments from either the transgenic or non-transformed portions of each graft were removed 25 days after grafting and assayed for the presence of PVS by DAS-ELISA. 81 2 0 3 5 4 2 5 2 Days After Inoculation Fig. 22. Accumulation of PVM coat protein antigen in the upper leaves of either non-transformed, or RB52 and RB41 transgenic 'Russet Burbank' potato after inoculation with intact PVM particles, at various days post inoculation. Plants were mechanically inoculated with a purified preparation of PVM diluted to 5 ug/ml in 10 mM sodium phosphate buffer pH7.4. Viral coat protein concentrations were determined by DAS-ELISA using a monoclonal antibody specific for the PVM capsid protein and are expressed as ng virus coat protein / mg wet weight tissue. 2.3.2.4 Susceptibility to Infection with PVM. Transgenic plants expressing the PVS coat protein also displayed a measure of resistance to infection by P V M following mechanical inoculation with purified virus. This resistance was not as complete as was observed following inoculation with PVS in that it could be overcome with high levels of inoculum and appeared to be related to the level of PVS coat protein expression. Transgenic RB41 potato, which expressed approximately 2-4 fold higher levels of PVS coat protein than the RB52 line (Fig. 18), showed a lower titre of P V M (8.36 ng/mg tissue) at 42 days following inoculation with 5 j ig/ml P V M than similarly inoculated RB52 plants (25.2 ng/mg tissue). 82 Control, non-transformed, plants which had been treated under the same conditions, showed a greater than 10 fold higher concentration of P V M (91.8 ng/mg tissue) in systemically infected leaves than the transgenic RB41 plants (Fig. 22). Not surprisingly, these transgenic plants failed to display any significant resistance to PVX infection and showed the same levels and rate of viral antigen accumulation as non-transformed control plants following inoculation with a severe strain of this virus. Also, transgenic plants inoculated with a mixture of PVX and PVS, while showing high titres of PVX (2-3 weeks post inoculation), maintained their resistance to PVS infection (results not shown). 83 2.4 DISCUSSION Resistance to PVS has been successfully introduced into Nicotiana debneyii as well as one of the most important commercial cultivars of potato, 'Russet Burbank'. Transgenic plants, expressing PVS coat protein, regenerated following Agrobacterium tumefaciens mediated transformation, were obtained with high frequency and were morphologically normal in appearance. The use of genetic engineering techniques for the introduction of new and desirable traits into existing cultivars is a highly efficient process when compared with the laborious and time consuming process of traditional plant breeding, especially with crop species such as potato containing tetraploid genomes. Also, unlike traditional breeding approaches in which the gain of each new trait inevitably results in the loss of some old features, genetic engineering is an additive process. As has already been alluded to in the introduction, the extent to which classical cross-protection and the coat protein mediated resistance in transgenic plants are related biological phenomena remains to be determined. It is likely that the mechanisms involved in each case are different, and may in fact vary according to the particular virus involved. The only report of cross-protection amongst different members of the carlavirus group was that of Rose, (1983), who found that inoculation of N. debneyii with the symptomless CE strain of PVS could cross-protect against subsequent inoculation with PVS (O), which 84 normally produces chlorosis of the upper leaves. The results presented here demonstrate that transgenic plants, whether N. debneyii or 'Russet Burbank' potato, which express the coat protein gene from PVS are highly resistant to infection following mechanical inoculation with purified PVS. The fact that such transgenic plants are also protected from infection by PVS RNA, together with similar results obtained with PVX coat protein expressing plants (Hemenway et al., 1988), indicates that some event other than, or in addition to, virus uncoating is being inhibited. In contrast to TMV, in which particle assembly proceeds in a 3'~>5' direction from an origin of assembly (OAS) located 925 nucleotides upstream of the 3' terminus of the R N A (Zimmern, 1977), the assembly of PVX is believed, by analogy with papaya mosaic virus (Lok & Abouhaidar, 1986), to occur in the 5'~>3' direction from an OAS located near the extreme 5' terminus of the RNA. It has been proposed by Hemenway et al, (1988), that PVX coat protein in transgenic plants may bind at or near this 5' proximal OAS of the challenge viral R N A and subsequently prevent translation of the replicase and/or interfere with replication. Such interactions may result in the at least partial recoating of infectious PVX RNA, and would be consistent with the observation that TMV coat protein does not protect against inoculation with TMV RNA, since in the latter case, binding of coat protein subunits to the OAS site would not be expected to interfere with replicase translation or R N A replication, as indicated by cotranslational disassembly experiments (Wilson, 85 1984; 1986). While the mechanism of disassembly/assembly of PVS is not known, the extent to which PVS and PVX share similar genome organizations indicates that analogous processes may be involved. A n alternative explanation for the apparent lack of accumulation of PVS in the inoculated or potentially systemic leaves of transgenic potato plants inoculated with viral R N A must also be considered. It is possible that virus replication did occur to some low level in only those individual cells initially infected with R N A and that the resulting encapsidated progeny virus was unable to infect additional cells because of interference in particle uncoating. The likelihood of this occurring is uncertain since it is not at all clear, for any plant virus, exactly what form (intact virus particles vs some type of ribonucleoprotein complex) of the infectious agent is responsible for either the cell-to-cell or long distance spread of the initial infection. This hypothesis could be readily tested by examining viral replication in transgenic protoplasts transfected with PVS RNA. The extent of this coat protein mediated protection was further emphasized in plant grafting experiments in which non-transformed potato shoots grafted onto transgenic plants, which were subsequently inoculated with PVS, failed to accumulate any significant concentration of PVS. These results further indicate that inhibition of virus replication in primary infected cells also inhibits the systemic spread of the infectious moiety to other susceptible tissues. 86 Also, in reciprocal grafts, transgenic shoots remained essentially virus free by 21 days following grafting onto host plants systemically infected with PVS, and even after 42 days following grafting typically contained less than 2% of the PVS concentration of similarly grafted non-transformed shoots. This resistance to systemic infection by PVS was also mimicked in grafts of P V M infected shoots onto PVS infected plants. While it is likely that the long distance systemic movement of PVS is via the phloem tissues of the vascular system, it is unknown in what form the virus is transported. In the case of TMV it has been found that TMV coat protein, but not necessarily virion assembly is required for efficient long distance spread (Oxelfelt, 1975) and other studies have concluded that long distance spread of TMV is via a viral ribonucleoprotein complex composed of several proteins including coat protein (Dorokhov et al, 1983, 1984). While not conclusive, the results presented here support the notion that long distance transport of PVS may involve some form of the virus other than intact virions since stem tissue taken from transgenic RB58 or RB41 grafts, just above the graft union, contained only 0.2% or 1.5%, respectively, of the level of PVS found in similar tissues from non-transgenic grafts. Also, the lack of accumulation of PVS in the apical non-transformed tissues of double-grafted plants may indicate that the long distance transport of the infectious agent, as well as virus replication, may both be suppressed in transgenic phloem tissue. Wisniewski et al, (1990), have recently described somewhat similar results using transgenic tobacco 87 expressing the coat protein from TMV. In these experiments grafts from coat protein expressing plants caused a delay in the movement of T M V from the inoculated non-transformed rootstock to similar non-transformed tissues above the graft union. While no conclusions regarding the nature of the transport form of TMV could be made, these results did support the hypothesis that the presence of coat protein in the phloem and associated cells interferes with the long-distance transport of TMV and systemic disease development. Transgenic plants expressing the PVS capsid protein were also found to show a measure of resistance to infection with a related carlavirus from potato, P V M . However, the degree of resistance observed following challenge with P V M was not as great as observed following inoculation with the homologous virus, PVS. Transgenic RB41 potato plants which had been inoculated with P V M showed an accumulation of virus which was approximately 10% the level observed for control non-transgenic plants, while other plants from this line showed no detectable accumulation of virus following inoculation with PVS. The relative inefficiency of PVS coat protein expressing plants in inhibiting P V M infection was further emphasized by the observation that PVS infected shoots also failed to significantly cross-protect against systemic spread of P V M when grafted onto P V M infected host plants, while P V M infected shoots grafted onto plants systemically infected with PVS did inhibit systemic spread of PVS into the graft. This type of non-reciprocal cross-protection likely accounts for the observation that infection with both of these viruses is often 88 found under field conditions. It has previously been reported that transgenic tobacco plants expressing the capsid proteins from either tobacco rattle virus (TRV; Van Dun & Bol, 1988) or alfalfa mosaic virus (Van Dun et al., 1987) were resistant to infection with other closely related strains of these viruses. However, in the case of TRV, transgenic plants expressing the coat protein from the T C M strain of TRV were not resistant to infection with the PLB strain of TRV, whose coat protein shares a 39% amino acid sequence identity with the T C M strain. From nucleotide sequence analysis, the deduced amino acid sequences of the coat proteins from PVS and P V M are 44.7% identical, overall, and this level of homology increases to 73% if one examines a 74 amino acid long region beginning at position 180 of the PVS sequence. It is likely that the relatively high degree of homology between PVS and P V M in this region of the coat protein sequence is responsible for the partial resistance to P V M infection in plants expressing PVS coat protein. This central region of the coat protein sequence also contains significant blocks of homology with the coat protein sequences from a number of potexviruses, and may represent an internal domain involved in RNA-protein interactions during capsid assembly. The extent to which PVS resistant transgenic potato cultivars retain their virus resistance properties under field conditions, as well as the other intrinsically desirable characteristics of the 'Russet Burbank' cultivar, will be the subject of future investigation. 89 C H A P T E R 3 3.0 C H A R A C T E R I Z A T I O N OF P V S N O N - S T R U C T U R A L PROTEINS 3.1 INTRODUCTION In addition to the nucleocapsid protein, the genome of PVS potentially encodes four other non-structural gene products of Mr 10734, Mr 7222, Mr 11802 and Mr 25092 in addition to the viral polymerase (MacKenzie et al, 1989). With the exception of the 3' terminal U K ORF, which appears specific to members of the carlavirus group, all of these non-structural proteins are homologous with similar sized ORFs from PVX (Huisman et al, 1988) and other members of the potexvirus group. To date, no evidence exists to suggest a biological role for any of these small molecular weight non-structural proteins, and indeed, their accumulation in virus infected cells and tissue has yet to be demonstrated. The deduced amino acid sequence of the PVS 25K protein shares homology with similar sized non-structural protein ORFs from a number of potexviruses including PVX (Huisman et al, 1988) and WC1MV (Forster et al, 1988) as well as the other two members of the carlavirus group for which partial nucleotide sequence information is available, P V M (Rupasov et al, 1989) 90 and lily symptomless virus (LSV; Memelink et al, 1990). A characteristic of these 25K non-structural proteins is the presence of a conserved N terminal putative nucleotide triphosphate binding motif, G-GKSS/T. This and similar NTP binding domains have been observed in a number of known ATP- and GTP-binding proteins (Zimmern, 1987) and as well have been identified in the amino acid sequences of over 100 virally encoded proteins from members of diverse virus groups including dsDNA, ssDNA and ssRNA viruses (Gorbalenya & Koonin, 1989). The presence of such nucleotide binding domains within viral encoded proteins has led to the speculation that such proteins may be involved in duplex unwinding during D N A and R N A replication, transcription, recombination and repair, and perhaps also mRNA translation. In an effort to further characterize two of these gene products, the viral polymerase and the 25K non-structural protein, this chapter describes the preparation of specific monoclonal antibodies (MAbs) to chimeric fusion proteins expressed in E. coli. In recent years a number of vectors have been reported which simplify the purification of foreign proteins expressed in E. coli. Examples of these include vectors in which polypeptides are fused with bacterial 6-galactosidase, which can be subsequently purified by substrate immunoaffinity chromatography (Ullmann, 1984), or trpE fusion proteins (Stanley & Luzio, 1984), which, while mainly insoluble, can be purified from the insoluble fraction of lysed bacteria (Marston, 1986). Also, vectors which direct the 91 synthesis of polypeptides as fusions with staphylococcal protein A , whose products can be purified by affinity chromatography on IgG-Sepharose, have been constructed (Lowenadler et al, 1986). The use of such a vector for the preparation of MAbs to a chimeric fusion protein including the gene for the 3A non-structural protein of cucumber mosaic virus and their use in the in vivo localization of this viral encoded protein has been previously described (MacKenzie & Tremaine, 1988). Smith and Johnson, (1988), have recently described use of a novel expression vector in which foreign proteins are expressed as gene fusions with the enzyme glutathione S-transferase. This vector has the advantages that expressed fusion proteins are generally quite soluble and can be purified in a single step by affinity chromatography on immobilized glutathione, followed by elution under mild conditions using excess reduced glutathione. Also, the additional problem that protein A fusion proteins have, in binding to IgG and thus complicating immunological analyses, is circumvented. This chapter describes the preparation of monoclonal antibody reagents specific either for the PVS 25K non-structural protein or the viral replicase. In addition, and as a first step in the further characterization of the function of the 25K protein, transgenic tobacco plants (var Xanthi-nc) which constitutively express this protein have been produced. 92 3.2 MATERIALS AND METHODS 3.2.1 Expression of a Portion of the PVS 25K Protein in pGEX-2T. The preparation and isolation of recombinant clone pVS91 (see Fig. 3), which contains 2294 nucleotides corresponding to the 3' terminus of PVS has been previously described (Chapter 1). A subclone, pVS121, was generated from pVS91 by EcoRI digestion and re-ligation. Plasmid D N A from pVS121 was double digested with BamHI and EcoRI and a gel-purified fragment corresponding to 18 kDa from the C terminus of the 25K ORF was ligated into BamHI-EcoRI digested pGEX-2T (obtained courtesy of Dr. D.B. Smith, Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Victoria 3050, Australia) vector and used to transform DH5a cells to ampicillin resistance (Fig. 23). Subsequent clones were analyzed for expression of the correct sized fusion protein SDS-PAGE of total E. coli cell lysates from IPTG induced cultures. 93 Replicase 25K 7K PVS121 42K Amp Bam HI, Eco Rl Bam HI, Eco Rl Expression of chimeric fusion protein composed of 19 kD from C terminal region of PVS 25K ORF in frame with glutathione S-transferase gene. Affinity purification on glutathione - Agarose. Immunization, cell fusion and Isolation of MAb's to PVS 25K protein. Fig. 23. Schematic representation of protocol used for the expression of a portion of the PVS 25K ORF in pGEX-2T as an in-frame fusion protein. Plasmid pVS121, which contained an insert corresponding to approximately 18 kDa from the C-terminus of the 25K protein, was digested with SamHI and EcoRI. The resulting restriction fragment was gel purified and ligated into BamHI-EcoRI digested pGEX-2T and used to transform DH5a cells to ampicillin resistance. 94 3.2.2 Expression of a Portion of the PVS Viral Polymerase in pGEX-1. A subclone, pVS472, was generated from pVS49 (see Fig. 3) by ligation of an approximately 1.5 kbp EcoKl-Sall fragment into EcdRI-Sall digested Bluescript vector. Clone pVS472 was subsequently digested with BamHl and Sail and the gel purified restriction fragment blunt ended by treatment with T4 D N A polymerase and ligated into Smal/ calf intestinal phosphatase treated pGEX-1 vector. Following transformation, ampicillin resistant DH5a colonies were analyzed for expression of a correctly sized fusion protein, incorporating approximately 40.1 kDa from the C-terminus of the PVS polymerase, as described above. 3.2.3 SDS-PAGE Analysis of Chimeric Fusion Proteins. Whole cell extracts from un-induced and IPTG (isopropyl-fi-D-thiogalactopyranoside) induced cultures were analyzed by SDS-PAGE to confirm the expected sizes of expressed fusion proteins. Briefly, 2 ml cultures (1/10 dilution of overnight cultures) were incubated for 2 hrs at 37 °C, then IPTG was added to 1 m M and the cultures were maintained at 37 °C for an additional 4 hrs. Cells (0.5 ml) were harvested by centrifugation (4000 g x 10 min) and resuspended with 50 | i l of SDS-PAGE sample cocktail and 5 pi volumes were electrophoresed using the buffer system of Laemmli (1970). Following electrophoresis, gels were stained for protein using Coomassie Blue. 95 3.2.4 Purification ofpGEX Based Fusion Proteins. Glutathione S-transferase linked fusion proteins were purified from bacterial cell lysates by affinity chromatography on glutathione conjugated agarose (Smith & Johnson, 1988). Overnight cultures of E. coli were diluted 10 fold into fresh Luria broth (2 x 500 ml) and cultured for 2 hrs at 37 °C. Fusion protein synthesis was induced by addition of IPTG (0.5 m M final concentration) and the cultures were maintained at 37 °C for an additional 4 hrs. Cells were harvested by centrifugation (5000 x g for 15 min), resuspended with 45 ml of TST (10 m M Tris-HCl pH7.4, 0.15 M NaCl, 0.05% (v/v) Tween-20) containing 20 ug/ml PMSF (phenylmethylsulfonyl fluoride) as a protease inhibitor and disrupted by sonication. Triton X-100 was added to 1% (v/v) final concentration and the homogenate was then centrifuged at 13,000 x g for 15 min. Approximately 4 ml of a 50% (v/v) suspension glutathione-agarose (Sigma) beads was added to the supernatant and incubated for 1 hr with gentle agitation. The affinity beads were washed extensively with PBS by centrifugation and specifically bound material was eluted with excess free glutathione (5 mg/ml) in 50 m M Tris-HCl pH8.0. The eluant was dialysed against several changes of 10 m M sodium phosphate pH7.4 and lyophilized. 3.2.5 Immunization and Cell Fusion. Female B A L B / c mice were successively immunized at 10 day intervals by intraperitoneal injection with 100 ug quantities of affinity-purified fusion 96 proteins over a period of 6 weeks. Four days after the final immunization, spleen cells were harvested and fused with FXNY strain of mouse myeloma cells in the presence of 50% (w/v) polyethylene glycol 4000 (BDH). Hybridoma cells were cultured in Dulbecco's modified minimum essential medium containing 100 uM hypoxanthine, 0.4 uM aminopterin, 16 uM thymidine, 20% (v/v) fetal calf serum (FCS), and 5 x 10s syngeneic feeder thymocytes per ml. Specific antibody-secreting clones were identified by ELISA using purified fusion protein or glutathione S-transferase, purified from E. coli transformed with unmodified pGEX vector, as the immobilized antigen. These cultures were recloned by limiting dilution and stored frozen in liquid nitrogen in medium containing 20% (v/v) FCS and 10% (v/v) DMSO. For the production of large quantities of specific MAb, hybridoma cells were propagated as ascites tumours in B A L B / c mice previously injected with 0.5 ml of pristane (Sigma). 3.2.6 ELISA. Hybridoma culture fluids were differentially screened against both the relevant specific fusion protein and glutathione S-transferase alone, or an unrelated glutathione S-transferase linked fusion protein. Linbro microtitre plate wells (Flow Laboratories) were coated overnight at 4 °C with 100 u,l of each specific antigen (pVS631 fusion protein, pVS707 fusion protein or glutathione S-transferase) at a concentration of 5 jxg/ml in 50 m M sodium carbonate buffer pH9.6. The wells were then rinsed, treated with blocking 97 buffer (10 m M Tris-Cl pH7.4, 0.15 M NaCl, 1% (w/v) bovine serum albumin, 0.05% (v/v) Tween-20, 0.1% (w/v) NaNg) for 60 min, and incubated with 100 u,l of hybridoma culture supernatant for 1 hr. After rinsing, individual wells were incubated with 100 ul of goat anti-mouse immunoglobulin-conjugated alkaline phosphatase (Bio/Can) at 200 ng/ml in blocking buffer for 90 min. Approximately 60-90 min after the addition of p-nitrophenyl phosphate substrate (0.5 mg/ml in 10% (v/v) diethanolamine pH9.8) the absorbance of each well at 405 nm was measured with a Titertek Multiskan M C (Flow Laboratories) plate reader interfaced with an IBM P C / A T microcomputer. 3.2.7 Transgenic Expression of the PVS 25K Protein in Transformed Tobacco. The gene coding for the PVS 25K non-structural protein was introduced into tobacco (var Xanthi-nc) via Agrobacterium mediated transformation. A n EcdRI fragment from plasmid pVS59, which contains an insert corresponding to approximately 2.5 kbp from the 3' terminus of PVS (see Fig. 3), was ligated into EcdRI, CIP treated, pCDX-1. This restriction fragment contained the entire coding sequence for the 25K protein, as well as 64 amino acids from the N -terminal region of the 12K non-structural protein. Following transformation into E. coli MM294, plasmid D N A from spectinomycin resistance colonies was screened by restriction enzyme digestion analysis for the presence of the appropriate insert, in the correct orientation. The T-region plasmid D N A from one of these clones, pVS427, was then introduced, by homologous 98 recombination, into the pTiB6S3SE disarmed helper Ti plasmid carried by A. tumefaciens strain GV3111SE, using a triparental mating procedure. One of the resulting Agrobacterium colonies which displayed spectinomycin, kanamycin and chloramphenicol resistance was then used for the transformation and regeneration of tobacco leaf disc explants as previously described. The ability of kanamycin resistant transgenic Xanthi to produce PVS 25K protein was assessed by Western immunoblotting using a specific monoclonal antibody. Samples of leaf tissue (100 mg) were homogenized with 100 ul of SDS-PAGE sample buffer (4% (w/v) SDS, 125 m M Tris-HCl pH6.8,10% (v/v) 2-mercaptoethanol, 0.04% (w/v) bromophenol blue, 20% (v/v) glycerol) and incubated at 95°C for 5 min. Following centrifugation at 13,000 x g for 5 min in an Eppendorf microcentrifuge, 15 ul aliquots were loaded onto a 12% polyacrylamide gel and, after electrophoresis using the buffer system of Laemmli (1970), separated proteins were blotted onto Immobilon membrane (Millipore) in 25 m M Tris, 192 m M glycine, 20% (v/v) methanol pH8.3 at 100V (0.25 A) for 60 min at 4 °C. The transfer blots were treated with blocking buffer (10 m M Tris-HCl pH7.4, 0.15 M NaCl, 1% (w/v) BSA, 0.05% (v/v) Tween-20, 0.1% (w/v) NaNg) for 60 min and incubated with M A b 2E3 hybridoma culture supernatant (1 /50 in blocking buffer) for 60-90 min. Following rinsing with PBS plus 0.1% (v/v) Tween-20, blots were incubated with [125I]-labelled goat anti-mouse Ig (1.5 x 106 cpm/ug; 1.0 x 106 cpm/ml in blocking buffer) for 60-90 min followed by 99 exhaustive rinsing with PBS-Tween and overnight autoradiography (-70 °C). 100 3.3 RESULTS 3.3.1 Preparation and Purification of pVS631 andpVS707 Fusion Proteins. Plasmids pVS631 and pVS707 contained in-frame gene fusions coding for a chimeric protein consisting of glutathione S-transferase linked to either an 18 kDa, or 40 kDa, portion from the C-terminal regions of the 25K non-structural protein or PVS viral polymerase, respectively. Induction of the tac (hybrid trp-lac) promoter with IPTG in cells transformed with these vectors resulted in the synthesis of fusion proteins of Mr 44K or Mr 65K for clones pVS631 and pVS707 respectively, of which about 26 kDa was contributed by the glutathione S-transferase gene product (Fig. 24). In each case the respective fusion proteins accumulated intracellularly, were soluble, and could be purified to near homogeneity by a single step affinity chromatography on immobilized glutathione. Typical yields of purified fusion protein ranged from 5-10 mg per litre of cells. 1 2 3 4 3 1 . 0 -2 1 . 5 -Fig. 24. Coomassie Blue staining patterns of E. coli cell extracts and purified fusion proteins separated by SDS-PAGE. Samples were total cell extracts from E. coli containing the pVS631 expression vector following (lane 1) or prior (lane 2) to induction with 0.1 mM IPTG, purified pVS631 fusion protein following glutathione-agarose affinity chromatography (lane 3), total cell extracts from E. coli containing plasmid pVS707 (lane 4) following IPTG induction, and affinity purified pVS707 fusion protein (lane 5). Mr standards x 10"3 are indicated. 101 3.3.2 Cell Fusion and MAb Production. The fusion of immune splenocytes from two B A L B / c mice, immunized with either pVS631 or pVS707 fusion protein, with mouse myeloma cells yielded two hybridoma cell lines that secreted antibody specific for the PVS 25K protein and 3 cell lines which secreted antibody specific for the viral polymerase. A greater number of MAbs were isolated which were specific for the glutathione S-transferase portion of the respective fusion proteins, as indicated by their cross-reaction between each of these different fusion proteins in ELISA. Fig. 25. Binding of MAb's 2E3 and 3E2 to fusion proteins pVS631 and pVS707 by ELISA. Microtitre plate wells were coated with either affinity purified pVS631 fusion protein, containing a portion of the PVS 25K protein, or pVS707 fusion protein which contained an approximately 40 kDa portion of the C-terminal region of the viral polymerase. Antigen coated wells were then incubated with either MAb 2E3 ( • ) , specific for the 25K protein, or MAb 3E2 (A), followed by alkaline phosphatase conjugated goat anti-mouse Ig. 10 100 1000 Reciprocal Dilution of Culture Ruid ELISA titration data for two particular MAbs showing specificity for either the 25K non-structural protein or a portion of the viral replicase are presented in Fig. 25. M A b 2E3 bound specifically to affinity purified fusion 102 protein pVS631 (Fig. 25 top panel) and did not bind at all to the pVS707 chimeric protein. In contrast MAb 3E2 bound specifically to that portion of the viral replicase expressed in fusion protein pVS707 (Fig. 25 bottom panel) and did not bind to pVS631 fusion protein. 3.3.3 Expression of the PVS 25K protein in virus infected and transgenic tobacco plants. The in vivo expression of the 25K non-structural protein in either PVS infected tissue or in transgenic Xanthi tobacco engineered to express this protein was determined by Western immunoblotting of leaf tissue extracts (Fig. 26). Significant quantities of 25K protein could be detected in SDS extracted tissue samples from PVS infected Chenopodium quinoa (Fig. 26, lane a) and no reactivity was observed with similar samples from non-infected control tissue (Fig. 26, lane d). Similar immunoblots of tissue samples from plants infected with either PVX, WCIMV (potexviruses), or another carlavirus, PVM, a b e d 25k-Fig. 26. Western immunoblot analysis of PVS 25K expression in either PVS infected tissue, or in tissue from transgenic Xanthi tobacco plants engineered to express this protein. Total SDS soluble protein extracts from PVS infected Chenopodium quinoa (lane 1), two different transgenic plant lines (lanes 2,3) or uninfected, non-transformed Xanthi tobacco (lane 4) were separated by SDS-PAGE and immunoblots probed with MAb 2E3 followed by ( 1 2 5 l)-labelled goat anti-mouse Ig. The mobility of the 25K protein is indicated. 103 showed no labelling with M A b 2E3 (data not shown). The labelling of PVS 25K protein in tissue extracts obtained from two transgenic Xanthi plants is also shown in Fig. 26, lanes b-c. 104 3.4 DISCUSSION The construction of cloned chimeric fusion proteins for the preparation of specific monoclonal antibody reagents has proven to be a valuable technique for studying the expression and localization of non-structural viral encoded proteins. In addition, the ability to generate transgenic plants expressing such virally encoded non-structural proteins, either individually or in various combinations, promises to be a powerful tool for studying and elucidating their function. In this study, portions of the PVS genome encoding two non-structural proteins, the C terminal 41 kDa region of the viral polymerase and an approximately 18 kDa portion from the C terminus of the 25K ORF, were inserted into the protein expression vector pGEX. Chimeric fusion proteins expressed in E. coli were then purified and used to produce specific monoclonal antibodies. Antibody 2E3 was shown by solid phase ELISA to be specific for the 25K protein while M A b 3E2 bound specifically to that portion of the viral polymerase contained within the glutathione S-transferase chimeric protein, pVS707. Conclusive evidence that the PVS 25K non-structural protein was in fact expressed at significant levels in vivo in virus infected tissue was obtained from Western immunoblots of total SDS soluble protein extracts which had been labelled with M A b 2E3. Similar blots from non-infected control tissue, or from plants infected with PVX, WC1MV or P V M , showed no labelling with 2E3 105 antibody indicating that this antibody was specific for the 25K protein and did not cross-react with the homologous protein from other viruses. While far from complete, the work described in this chapter lays a foundation for future studies concerning the biological roles of the non-structural gene products encoded by the PVS genome. The relatively high level of amino acid sequence conservation exhibited by the 25K proteins from members of both the carlavirus and potexvirus groups is probably indicative of its importance in the processes of either viral replication or transport. The use of transgenic plants constitutively expressing the 25K protein should prove to be a valuable tool for the future systematic characterization of this protein. Of interest also would be to determine whether one or more of the MAbs generated against the viral polymerase are effective in inhibiting viral template specific polymerase activity. Such antibodies could have significant potential in the development of alternate strategies for genetically engineered virus resistance (see Future Prospects). 106 F U T U R E PROSPECTS Although the work presented here contributes significantly to an understanding of the molecular biology of PVS and the strong evolutionary relationship which exists between members of the carlavirus and potexvirus groups, a complete appreciation of the roles for the various viral gene products is still lacking. Much work remains to be done with respect to defining the biological roles of the various small molecular weight non-structural proteins (UK, 7K, 12K and 25K) encoded by the PVS genome. By analogy with other simple R N A plant viruses, it is probable that one of these putative non-structural proteins is involved in the cell-to-cell spread of PVS during systemic infection, and the most likely candidates in this regard include the 12K and 25K proteins. The production of specific monoclonal antibody reagents to recombinant viral gene products expressed in E. coli, together with the ability to constitutively express these proteins in transgenic plants, either individually or in combination, promises to be a valuable approach for further characterizing their biological functions in vivo. Currently, experiments are underway to ascertain whether transgenic plants expressing the PVS 25K protein are able to complement the movement of the LS-1 strain of TMV, a temperature sensitive mutant whose inability to spread systemically at the restrictive temperature (33 °C) has been traced to a single amino acid substitution (pro —> ser) within the 30K non-structural protein (Ohno et al, 107 1983). While the prospect of introducing resistance to various viral pathogens into commercially important crop cultivars via A. tumefaciens mediated gene transfer technology has yielded promising results not only with PVS, but a growing list of other plant viruses, the current methodology also suffers from significant drawbacks. The most successful approach to date has been to mimic the phenomenon of classical cross-protection by expression of viral capsid protein(s) in transgenic plants. However, this form of coat protein mediated resistance tends to be quite specific for the homologous virus, or very closely related strains, and the engineering of resistance to a number of unrelated viruses would necessitate the expression of each of their individual coat protein genes in transgenic plants. While this would be feasible in the case of a small number of viruses, and has already been demonstrated for two unrelated viruses of potato, PVX and PVY (Lawson et ah, 1990), it would become exceedingly cumbersome as the number of viruses increased. A n alternative approach would be to exploit one function which is common to virtually all simple R N A viruses, genome replication. The polymerases from a large number of D N A and R N A viruses share a high degree of homology centred around the consensus sequence T/SG—T—NT(22 aa)GDD (Cornelissen & Van Vloten-Doting, 1988), and the possibility exists of generating monoclonal antibody probes which would cross-react with, and inhibit the activity of, a number of these polymerases from different viruses. 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