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A Conserved epitope on the surface of carlaviruses Wieczorek, Andrew A. 1992

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A CONSERVED EPITOPE ON THE SURFACE OF CARLAVIRUSESbyAndrew Anthony WieczorekB.Sc., Simon Fraser University, 1981A THESIS SUBMITTED IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDepartment of Plant ScienceWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1992© Andrew Anthony WieczorekIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)  Department of  KAM SC-i CA.The University of British ColumbiaVancouver, CanadaDate  Japtuarli 1195 DE-6 (2/88)AbstractThe seed certification program in British Columbia relies heavily on doubleantibody sandwich-enzyme-linked immunosorbent assays (DAS-ELISA) fordetection of plant pathogens. Each individual sample requires a separate test foreach pathogen. The aim of this research was to simplify the procedure, whichwould result in considerable savings in both cost and labour. A method tofacilitate the testing by allowing several individual samples to be evaluated in asingle trial was developed by finding a unique antibody that could be used for thedetection of several viruses. A monoclonal antibody was raised to the coatprotein of potato virus M which also reacted to other members of the carlavirusgroup. Viruses morphologically similar to carlaviruses but belonging to thepotyvirus and potexvirus groups were also challenged with the PVM antibody, butfailed to react, despite evidence of conserved amino acid sequences in the coatproteins of carlaviruses and potexvirus. A variety of host species infected withviruses from all three groups were also tested to eliminate the possibility of areaction by the antibody to a plant componant. Partial characterization of theepitope to which the antibody reacts was also accomplished. It is postulated thatthe epitope is conformational in nature, since the antibody fails to detect coatprotein subunits in Western blots, and does not react to virus degraded by longterm storage either in purified form or in tissue. Since the epitope readily reactsin DAS-ELISA, it was concluded that the antigenic site must be located on thesurface of the virus particle. This is the first demonstration of an epitope thatiishows broad specificity within a virus group which is located on the surface of thevirus rather than buried in the viral core. The utilization of this epitope fordiagnostic purposes is discussed.iiiTable of ContentsAbstract^ iiList of Tables vList of Figures^ viList of Abbreviations viiiAcknowledgements^ ixINTRODUCTION 1MATERIALS AND METHODS^ 8Propagation of PVM^ 8Purification of PVM 8Immunization 9ELISA procedures 9Generation of hybridomas^ 11Cloning of the PVM coat protein gene^ 12Sequencing of the PVM coat protein gene 14Digestion of PVM coat protein with proteolytic enzymes^15Western blot analysis of coat protein 15Northern blot analysis^ 16Electron microscopy 17RESULTS^ 19Monoclonal antibodies to PVM^ 19Confirmation of the presence of a common epitope^ 19Reaction of monoclonal antibody 1C8 with various antigens^24Cloning and sequencing the PVM coat protein gene 32DISCUSSION^ 43Existence of common epitopes^ 43Generation of monoclonal antibodies 45Partial characterization of the epitope^ 49Cloning of the PVMcoat protein gene 54Current and future use of the 1C8 group specific antibody^60Appendix A^ 62REFERENCES 65ivList of TablespageResults of initial testing of monoclonal antibody culturesupernatants against a variety of carla- andpotexviruses.ELISA readings of reactions of monoclonal antibodies tothe homologous virus showing the specificity with whichmonoclonal antibodies raised against potato virus X(PVX), potato virus S (PVS), and potato virus M (PVM)react with the homologous virus.Detection of viruses trapped by polyclonal antiserashowing the presence of common epitopes.Detection of individual viruses with monoclonalantibodies after trapping with a mixture of rabbitpolyclonal antisera.TABLE 1.TABLE 2.TABLE 3.TABLE 4.20212325TABLE 5. Effect of host plant on reaction of monoclonal 1C8 tocarlaviruses.^ 26TABLE 6. Summary of fragments of the coat protein of potato virusM predicted from the complete cleavage by proteolyticenzymes.^ 29vList of FigurespageFig. 1^Effect of digestion with proteolytic enzymes on the sizeof the PVM coat protein subunits.^ 28Fig. 2^Western blot of PVM subunits cleaved by variousproteolytic enzymes and probed with rabbit anti-PVMantiserum.^ 30Fig. 3^Electron micrographs of negatively stained PVMparticles digested with various proteolytic enzymes.^31Fig. 4^Labelling of viruses using 10 nm colloidal goldconjugated to Protein A.^ 33Fig. 5^Electrophoretic analysis of PVM RNA under bothdenaturing and non-denaturing conditions.^ 34Fig. 6^Map of pT7/T3-18 plasmid and orientation of markersaround the insert.^ 36Fig. 7^Northern blot analysis of total RNA from infected andhealthy leaves from Solanum tuberosum.^ 37Fig. 8^Restriction map of potato virus M 3' end inserted intothe Sma I site of plasmid pT7/T3-18. 38Fig. 9^Western blot of total proteins from Escherichia coltcarrying plasmids with varying size deletions of the 5'end of p7.^ 40Fig. 10 Comparison of the amino acid sequence translated froma partial nucleotide sequence located in the 3' region ofthe p7 insert with the published sequence (67) of theentire coat protein open reading frame. 41Fig. 11 Comparison of a partial nucleotide sequence (shown asDNA) from the 3' end of the insert in plasmid p7 withthe coat protein coding region of the published sequenceof PVM (67). 42vipageFig. 12 Diagram of the two expected patterns of Western blotsthat would result from translation of a series of timedBal 31 exonuclease digestions of the 5' end of the PVMinsert in pT7/T3 from two possible orientations of thecoat protein gene and its surrounding sequences inpT7/T3 with respect to their orientation of the Lac Z'gene. 58viiList of AbbreviationsA405^ absorbance at 405 nmas amino acidBbSV^blueberry scorch virusCLV carnation latent virusDAS-ELISA^double antibody sandwich enzyme-linked immuno-sorbent assayDMSO^dimethyl sulphoxidDNA deoxyribonucleic acidE. coli^Eschericia coliEDTA ethylenediaminetetraacetic acidELISA^enzyme-linked immuno-sorbent assayFCS fetal calf serumgm^gramhr hourKb kilobaseKbp^kilobase pairsKDa kilodaltonsLB^Luria-BertainiMAb monoclonal antibodyMES^2(N-morpholino)ethanesulphonic acid14 microgramill^microlitremin minuteml millilitreng^nanogramNMV narcissus mosaic virusnt nucleotidePBS^phosphate buffered salinePVM potato virus MPVS^potato virus SPVX potato virus XRNA^ribonucleic acidSDS sodium dodecyl sylphateSSC^salt-sodium citrateSVX Silene pratensis potexvirusTAE^tris-acetate-ethylenediaminetetraacetic acidviiiAcknowledgementsI wish to thank Dr. David Theilman for his contribution to the portion of theproject that dealt with the cloning and sequencing of the PVM coat protein.Without his valuable advice, his liberal allowance in the use of both his own andhis technician's time, and his generous provision of space in his laboratory theproject would not have been accomplished. I wish to also thank Sandy Stewertfor her patience in showing me the cloning techniques, and Dr. Fran Leggett forthe electron micrographs. I would also like to acknowledge my committeemembers, Dr. R. Stace-Smith, Dr. R. Copeman, Dr. J. Tremaine, Dr. R. A. J.Warren, and especially Dr. D. Rochon for her critical reading of the thesis.Finally, I wish to recognize Dr. Marvin Weintraub without whose strong supportthis thesis would not have been attempted.ixINTRODUCTIONIn the detection of plant pathogens, numerous techniques, including serology,molecular biology (43) and bioassays have been utilized to identify the causalorganisms responsible for a variety of diseases. Each technique has itsadvantages and disadvantages. The method currently used to the greatest extentin routine diagnosis of viral pathogens is a group of serological methodscollectively known as ELISA protocols (8,19,43). Briefly, in these protocols,antigens are attached to a solid phase support (6), then detected with antibodiesthat are specific for the antigen. Detection is accomplished by a colorimetricconversion of substrate (7). This is accomplished by conjugating the originalantibody directly to enzymes (36), or by using commercially prepared broadspectrum conjugates. The conversion of the substrate provides a largeamplification of signal, allowing extreme sensitivity in the detection of the antigen(7).ELISA protocols rely on the specificity of antisera to detect and identifypathogens. The advantages of the method are that it is relatively easy to performby workers not having a high degree of technical training; can be done with aminimum of specialized equipment and facilities; can be used to test manydifferent samples in a single day; and is relatively inexpensive on a per samplebasis. Additionally, the sensitivity is such that only a small portion of a plant isnecessary for testing. A single leaf is often sufficient to give unequivocal resultswell before symptoms can be detected and the disease allowed to spread.1A disadvantage of ELISA is that there may be difficulty in raising antiserawith the desired characteristics. After the purification of antigens, contaminantsare often present that will induce antibodies that react non-specifically to eitherhealthy plant components or with benign relatives of the pathogen. These falsepositive tests have been eliminated to a large extent by using monoclonalantibodies instead of traditional polyclonal antisera (19,43).Though monoclonal antibodies (MAbs) are extremely specific to particularpathogens, this specificity may be a disadvantage (19). Monoclonal antibodiesraised against a particular strain of the virus may only react with that strain andfail to detect a related variant that causes the same disease in a differentgeographic region (47). There is also concern that subsequent mutation eventsmay result in the loss of the antigenic site (66). These problems can be overcomeby the careful selection of epitopes (26). This is done by screening a panel ofantibodies derived from a fusion experiment against all available strains of aparticular virus. If the antibody can detect all the variants in these tests, theantibody is probably against a highly conserved region of the virus (40), and theloss of this site by mutation would probably make the virus unable to replicate orcause it to assume a form incapable of causing disease.Classification of viruses into groups is to a large extent based on their physicalproperties, although when viruses have a similar or identical morphology, muchmore reliance is placed on chemical and biological factors. It could therefore beargued that there must be a specific physical determinant common to a group of2viruses that makes them related. If this determinant is expressed on the coatprotein of a virus, antibodies should be able to detect it. Confirmation oftaxonomic classification is often done by assessing the serological relatedness ofthe various members of the group (19,28,26). Polyclonal antisera raised toassociated members will usually cross-react to various degrees in reciprocalexperiments with other members of the group (27) . By evaluating the degree ofreaction in these experiments, one can speculate on the relatedness of the variousviruses and their evolutionary origins.The difficulty in these assessments arises in the heterologous nature of theantisera used. Polyclonal antisera afford the best chance of detecting commonepitopes since in theory antibodies to all possible epitopes are raised duringimmunization (62). Since polyclonal antisera contain various populations ofantibodies occurring in varying amounts (1), and since each antiserum has aunique composition, confusion can occur in assessing relatedness, which isparticularly problematic when assigning new members to existing groups. Oftenreciprocal testing with antisera do not give confirming results. Tests withantisera from alternate sources may also yield greatly differing results.Monoclonal antibodies have been used in an attempt to eliminate some of theconfusion resulting from the use of polyclonal antisera. The problem with thisapproach is that the specificity of the antibody ensures that it detects only singlediscrete antigenic sites, and thousands of antigenic sites can occur on the virus.Finding an antigenic site common to many virus strains can be likened to finding3a needle in a haystack. A single site may induce antibodies to either the linear orconformational form of that site (62), and this further decreases the possibility offinding a monoclonal antibody to an epitope shared by all members of the group.Despite these drawbacks, many attempts have been made to raise such anantibody. Evidence, both serological, and, as more viruses are cloned andsequenced, molecular, is corroborating the existence of conserved regions inviruses that can potentially be used to raise antisera (21).A variety of strategies can be used to increase the possibility of findingantibodies directed against these conserved regions. The simplest approach is toscreen vast numbers of monoclonal antibodies against a panel of "related" virusesand hope that through random chance the desired MAb can be found. By firstexposing the animal to one virus during immunization, then boosting with asecond virus, it may be possible to enrich for antibodies to common epitopes. Ifsufficient sequence information is available, fusion proteins or synthetic peptidesmay be used (55, 56, 20) as immunogens. These strategies either singly or incombination may eventually result in obtaining an antibody capable of detectingan epitope common to a virus group.An antibody of the type described could potentially be extremely useful. Inaddition to its benefits in taxonomic considerations, it would be very valuable incommercial testing operations particularly in situations where quarantinerestrictions exist. During the routine testing of thousands of plants, individualsare often tested for a number of viruses, and each of these requires a separate4test. Often, tests are for several related viruses. If an antibody exists to acommon epitope, numerous viruses could be detected in a single test. Onlyplants reacting positively to a common epitope would be subjected to a second testto determine the specific virus involved. Therefore a substantial savings in bothmaterial and labor would result.Potato virus M (PVM) was chosen as a model system to attempt to find such anantibody for several reasons. First, although PVM is not considered an importanteconomic problem, it can cause up to 15% yield loss. It is distributed world wide,and can show a range of symptoms that make it difficult to distinguish by visualinspection (17). Secondly, it is a member of the carlavirus group which has alarge number of both confirmed and potential members that can infect virtuallyall plant species (27). Third, considerable cross reactivity has been found to existbetween members of the group, yet no serological cross reactivity occurs betweencarlaviruses and any other virus group (5). Fourth, cloning and sequencing of arelated carlavirus, potato virus S (PVS) had been successfully done in an adjacentlaboratory (37), and both monoclonal and polyclonal antibodies were available toboth PVM and PVS. Finally, the laboratory in which the work was done has aclose association with the Food Production and Inspection Branch of AgricultureCanada, which provides quality control for the seed potato industry in thePemberton valley in British Columbia and in other regions of Canada. Thisassociation allows access to both healthy and infected potato samples from avariety of locations, and provides an opportunity for the side by side evaluations5of any new antibodies with existing polyclonal antibody technology. TheInspection Branch would also be in a position to implement any beneficialantibodies found in this study into its testing program.Common epitopes have been described in both structural and nonstructuralviral proteins (53, 21). In the case of nonstructural proteins, most are definedfrom the deduced amino acid sequences of open reading frames, and relativelylittle is known about either their structure or their function. Although someassignments have been made on the basis of comparison to other proteins ofknown function, many of the proteins have no known purpose, and are not evenconfirmed as being expressed during replication.In the case of coat proteins, an extensive analysis of potential and confirmedepitopes has been performed on members of the potyvirus group. By generating aseries of sequential artificial peptides, and comparing the amino acid sequence ofthese peptides among the various members of the potyvirus group, Shukla et al,(53) found a number of potential common epitopes. The presence of these epitopeswas confirmed by the reactions of both polyclonal antisera and monoclonalantibodies to these peptides. This supplied evidence for group specific epitopes inthe potyvirus group, and localized them to an internal conserved region buried inthe coat protein.Potato virus M is similar morphologically to potyviruses in that it is a long,flexuous rod (65). This resemblance may indicate a similar arrangement ofepitopes on the coat protein, and a group specific antigen could also exist among6carlaviruses as it does among potyviruses. Indeed, sequence analysis of the 3'terminal region of the RNA of five carlaviruses has revealed a conservednucleotide sequence (60). Though this region is not located in the coat proteingene, it may set a precedent for the existance of other conserved sequences.The existence of group specific epitopes in a related system, the homologyexisting at the 3' end in PVM, and the classical serological studies of the crossreactivity between the various polyclonals among the carlavirus group (27) led tothe belief that a group specific epitope could be found. The objectives of thisthesis were the following: 1) to determine if a conserved epitope existed that wasgroup specific for carlaviruses by raising a panel of monoclonal antibodies toregions of PVM, 2) to attempt to map the location of the epitope to a region of thecoat protein by generating a series of cDNA clones containing sequential deletionsin the coat protein gene of PVM, then expressing and sequencing these deletionsand 3) to assess the usefulness of any group specific antibodies obtained in plantquarantine programs.7MATERIALS AND METHODSPropagation of PVM. The source of PVM was infected Solanum tuberosumL. tubers, obtained from field samples collected by Dr. Richard Stace-Smith. Oncethe tubers were planted and sprouted, bulk propagation of tissue for subsequentpurification was done by taking cuttings from stems containing single leaves,dipping their ends in a commercial preparation of growth hormones and placingthem in soil. The planted cuttings were placed in a mist chamber forapproximately 1 week to root, then transplanted as necessary into larger pots.Purification of PVM. Tissue from the above ground portion of infectedplants was harvested and approximately 110-120 gm was homogenized using aWaring blender in a 500 ml volume of 0.1 M sodium borate buffer pH 8.0containing 0.01 M EDTA and 1% 0-mercaptoethanol. The homogenate wassqueezed through cheese cloth to remove plant debris, Triton X-100 was added toa final concentration of 2%, and the mixture stirred for 2 hr at room temperature.After stirring, particulates were removed by low speed centrifugation (9,000 rpmfor 20 min in a refrigerated Sorval RC-5B centrifuge using a GSA rotor). Thesupernatant was recovered and centrifuged at 35,000 rpm for 60-90 min in aBeckman 50.2 Ti rotor at 4 C. The pellet was resuspended in approximately 10mls of the borate buffer with a glass homogenizer . Additional debris wasremoved by centrifugation for 10 min at 10,000 rpm in a Beckman 30 rotor. CsC18was added to 40% (w/v) to the cleared supernatant and the solution centrifugedfor 16 hours at 50,000 rpm in a Beckman 70 Ti rotor at room temperature. Anopalescent band midway up the gradient was removed and dialyzed into boratebuffer. Concentration of the virus was determined by using the BioRad proteinassay (3) using a bovine serum albumin standard.Immunization. Balb/c mice were immunized with 20-50 ug of PVM dialyzedinto Dulbecco's phosphate buffered saline (PBS: 138 mM NaC1, 8.1 mM Na 2HPO4-71120, 1.2 mM KH2PO4 , and 2.7 mM KC1). The virus was emulsified with anequal volume of Fruend's incomplete adjuvant and injected subcutaneously. After1 mo, a second injection with an equal amount of virus in PBS without adjuvantwas administered intraperitoneally. The titre of the serum obtained from tailbleeds was assayed by ELISA. When necessary, additional injections wereadministered at 2 wk intervals. The mice were sacrificed 3-5 days post-injection,their spleens aseptically removed, and released 13-lymphocytes fused to FOX-NYmyeloma cells (Hyclone Laboratories). SDS-treated PVM was prepared by addingSDS to a final concentration of 2% to 1 ml of purified virus at 2-5 mg/ml, thenincubating with gentle stirring for 1 hr at room temperature. The virus wasdialyzed rigorously in 3-5 changes of PBS to rid the sample of all excess SDS.ELISA procedures. Clark and Adams (7) ELISA techniques were modified.ELISA plates (Linbro,Flow laboratories) were coated with either crude antisera at9a dilution of 1:500 in PBS or with gamma globulin fractions purified on a proteinA column (Pharmacia) (22,46), adjusted to a final concentration of 1 mg/ml anddiluted 1:1000 in PBS. The plates were generally left overnight at roomtemperature at this step. Blocking was done for 30 min at 37 C with 0.2% skimmilk powder in PBS or in 2% fetal calf serum in PBS. Antigen was added. Forplant sap, leaves were homogenized in 0.1 M Tris-HC1 pH 8.0 in a ratio of 1 gm oftissue to 10 ml of buffer. Incubation was for 1 hr at 37 C. Purified viruses werealso diluted in Tris buffer and generally adjusted so that a large excess(approximately 100 ng/well) was present to insure saturation. Monoclonalantibody culture fluids or the appropriate antibody at a previously determineddilution (in blocking buffer) was added next . Incubation was for 1 hr at 37 C.Goat anti mouse IgG and IgM (H and L) conjugated to alkaline phosphatase(BioCan Laboratories) diluted 1:3000 in blocking buffer was subsequently addedfor 1 hr at 37 C. Finally, substrate (para-nitrophenol phosphate at 1 mg/ml in10% diethanolamine pH 9.6, Sigma Laboratories) was added and the platesincubated at room temperature until sufficient color developed (usually 15 min to2 hr). All washing between steps was done with tap water. All volumes addedwere 100 ul except at the blocking step where 200 ul was added to ensure allpossible sites were saturated.For SDS denatured subunits 1 mg/ml of virus subunits was diluted 1:500 inPBS then added directly to ELISA plates. All subsequent steps and times werethe same10Viruses and polyclonal antisera used for ELISA protocols were kindly providedby Dr. Robert Martin, Dr. Richard Stace-Smith and Dr. Richard Hamilton.Viruses were supplied either as purified samples or as infected tissue.Generation of hybridomas. Briefly, six standard petri plates of FOX-NYmyelomas (Hyclone Laboratories) grown in Dulbecco's modified Eagles media(Gibco) containing 10% supplemented calf serum (HyClone) to approximately 70%congruency were fused with the spleen from an immunized mouse using 50%polyethylene glycol molecular weight 1500 with 10% dimethylsulphoxide (BDHChemicals) in Dulbecco's modified Eagles media. The resulting hybridomas wereplated into selective media (Dulbecco's modified Eagles media containing 4 X 10'M aminopterin, 7.5 X 10 -3M adenine, 1.6 X 10 -5M thymidine and 1 X 10 -4Mhypoxanthine and 20% calf serum). Seven days post fusion the media waswithdrawn and fresh selective media without the aminopterin was added. Tendays post fusion culture supernatants were screened for antibody productionusing ELISA. Detailed instructions can be found in the following references: (10,24, 31, 45, 64).Clones testing positive for desired antibody production were subjected tolimiting dilution until only one colony per well was visually confirmed. Thesewere retested by ELISA and positives recloned until all wells containing singlecolonies tested positive. These clones were transferred to 1 ml wells, thenexpanded to 10 ml wells. Hybridomas were frozen in Dulbecco's modified Eagles11media containing 20% Calf serum and 10% DMSO, then maintained below -133 Cfor long term storage.Ascites production was initiated by injecting Balb/c mice intraperitoneally withPristane (Sigma) 7-10 days before injecting hybridomas (4) . Each mouse wasthen injected intraperitoneally with one Petri plate (approx 1 X 10' cells) ofhybridoma cells centrifuged at 800 X G for 10 min and resuspended in 1 ml ofPBS. Ascites fluid was drained from the abdomen of mice 7-10 days afterinjection of cells using an 18 gauge needle.Cloning of the PVM coat protein gene. All following procedures wereperformed essentially as described in Maniatis et al. (39).PVM virion RNA waspurified from alkaline SDS treated particles by phenol/chloroform extractions andsubsequent ethanol precipitation . RNA purity and concentration was confirmedby the ratio of absorbance at 260 vs 280 nm, and the integrity was evaluatedusing non-denaturing 1% agarose-TAE gels. Formaldehyde denaturing gels wereused to determine their size (39).First-strand cDNA was synthesized using Molony murine leukemia virusreverse transcriptase (BRL) and oligo-d(12-18)T as primer (9). Conditions usedfor first strand synthesis were similar to those recommended by BRL using 6 jigsof purified PVM RNA and 2.5 ggs oligo-d(12-18)T. Second-strand DNA wassynthesized using Escherichia coli DNA polymerase I in the presence of RNase H(25). Synthetic double-stranded DNA was treated with phenol/chloroform, ethanol12precipitated and treated with T4 DNA polymerase to blunt the ends. DNA wassize fractionated by electrophoresis through a 1% agarose gel buffered with 0.04M Tris-acetate and .002 M EDTA pH 8.0 (TAE) and the slowest migrating bands(ca. 6 to 9 Kb) extracted using the Gene Clean (Bio 101) procedure, as describedin the accompanying manual. The purified DNA was then ligated into plasmidpT7/T3-18 (Pharmacia) prepared by cutting with Sma I.A portion of the ligation mixture from above was used to transform competentDH5a E. coli cells (BRL) and plated onto 1.5 % Luria-Bertaini (LB) agar platescontaining 50 lig /ml ampicillin with a top coat of 50 Ill of the chromogenicsubstrate 5-bromo-4-chloro-3-indoly1-13-D-galactopyranoside at 50 mg /ml inN,N'dimethylformamide (48).Selection of colonies containing plasmids with PVM coat protein inserts wereobtained in the following manner. Colonies as described above were duplicatedonto nitrocellulose membranes, lysed by hanging the membranes in achromatography tank saturated with chloroform vapour, and washed for 2 hr withPBS containing 0.5% skim milk powder, 50 gg /ml lysozyme and 1 gg /ml DNase.Filters were then probed with crude rabbit anti PVM polyclonal antiserum diluted1:500 in PBS containing 0.2% skim milk powder for 1 hr at room temperature.After rinsing in PBS ( three times for 10 minute each) goat anti rabbit antiserumconjugated to alkaline phosphatase (36) was diluted 1:1000 in 0.2% skim milkpowder in PBS and incubated for an additional hr. After a final rinsing, with 0.1M Tris-HC1 pH 8.0 the filters were developed with a precipitating substrate ( 0.3313mg/ml napthol-AS-MX phosphate and 3 mg/ml fast red TR salt (Sigma) in 0.1 MTris-HC1).Duplicate colonies reacting positively in the above test were transferred to 2ml of LB media containing 50 ug of ampicillin and grown overnight at 37 C.Plasmid DNA was extracted according to the method of Holmes and Quigly (23)and analyzed by electrophoresis following restriction enzyme digestion. Thelongest clone expressing a protein that reacted with the antibodies (designated p7)was used for all subsequent work.Sequencing of the PVM coat protein gene. Sequential overlappingdeletions for preparing templates for subsequent nucleotide sequencingprocedures were made using appropriate restriction enzymes and Bal 31 nuclease(34). To generate deletions in the 3' to 5' direction (with respect to the multiplecloning site), p7 was cut with Eco RI then treated with Bal 31 for varying lengthsof time. The Bal 31 was removed from the DNA by phenol/chloroform extractionfollowed by ethanol precipitation. The ends of the Bal 31 treated DNA wereblunted using T4 DNA polymerase (39), and then digested with Hind III. ThisDNA was then separated on a 1% TAE-agarose gel and the appropriate sizefragments extracted with GeneClean as described above and cloned directionallyinto pT3/T7-18 which had been previously digested with Hind III and Sma I.Deletions in the 5' to 3' direction were generated by digesting p7 with Pst I,treating with Bal 31, blunting as described above, then digesting with Eco RI.14After separating the fragments on an agarose gel, they were cloned into EcoR Iand Sma I cut pT7/T3-18 (Fig. 8).Sequencing was carried out according to Sanger (51) using a modified T7 DNApolymerase, Sequenase TM, (United States Biochemical Corp) (59, 58, 61).Analysis of results were done on an IBM personal computer using either theGeneMaster program (Biorad) or PCGene version 6.6 (Intelligenetics).Digestion of PVM coat protein with proteolytic enzymes. Five mg ofpurified PVM were digested overnight in a total volume of 1 ml at 4 C. Trypsinand endoproteinase Glu-C (V8) were added to a concentration of 10 ug / ml in 0.1M borate buffer pH 8.0. Pepsin was added to the same concentration afterdialyzing the virus into 0.5 M Na-acetate buffer pH 3.4.Western blot analysis of coat protein subunits. Protein wereelectrophoresed through varying percentage polyacrylamide gels according to themethod of Weber and Osborn (63) using the discontinuous buffer system ofLaemmli (32). Following separation, the gels were stained with 0.25% Coomassiebrilliant blue R250 in 10% acetic acid and 50% methanol. Proteins weretransferred to Immobilon membranes (Millipore) by electroblotting (100 V at 0.25amps in a BioRad miniblotter apparatus for 60 min in a buffer containing 25 mMTris, 192 mM glycine and 20% methanol at pH 8.3). After transfer the15membranes were probed usig rabbit anti-PVM polyclonal antiserum as describedabove (see section Cloning of PVM coat protein gene).Northern blot analysis. Total RNA from infected and healthy plants wasextracted by freezing tissue in liquid nitrogen, then suspending in 8M guanidineHC1, 20 mM MES, 20 mM EDTA and 50 mM mercaptoethanol, pH 7.0. Themixture was extracted with phenol/chloroform then 0.7 volumes of ethanol and 0.2volumes of acetic acid were added to selectively precipitate the RNA from DNAand contaminating proteins which remain in the supernatant (15, 35). The RNAswere separated on formaldehyde denaturing gels, then transferred tonitrocellulose by diffusion as described by Maniatis et a/.(39). Probes were madeby the nick repair method (18,41). DNA (0.1 ug) was treated with lowconcentrations of DNase to nick the strands, then repaired with DNA polymeraseI using a32P dATP (spec. act.= 3,000 Ci/mmole).Membranes were washed in prehybridization buffer (6X SSC, 10X Denhart'ssolution: .2%(w/v) bovine serum albumin, 0.2% (w/v) Ficoll (Pharmacia) and 0.2%(w/v) polyvinyl pyrollidone, 0.5% SDS and 50 ug / ml salmon sperm DNA) for 1 hrat 42 C. Prehybridization buffer was removed, then previously prepared cDNAprobes added in hybridization buffer (6X SSC, 0.5% SDS, 50 ug / ml salmon spermDNA and 50% formamide). The samples incubated overnight at 42 C. Blots werewashed three times for 10 min in 2 X SSC with 0.1% SDS at room temperature,then additionally for three times for 10 min in 0.1X SSC with 0.1% SDS at 65 C.16Membranes were exposed to Kodak X-Omat K diagnostic film overnight at roomtemperature.Electron microscopy. All immuno-electron microscopy samples wereprepared by Dr. Fran Legett, and reproduced with the permission of Dr. MarvinWeintraub. Purified virus or leaf tissue ground in 0.1 M Tris-HC1 buffer, pH 8.0,was adhered to 400 mesh nickel or copper grids previously coated with a film ofparlodion, then strengthened with a carbon coating. Blocking was performed byfloating the grids on 20 Ill droplets of 0.1 M Tris buffer pH 8.0 containing 1%bovine serum albumin (BSA) for 30 min, then transferring to 20 ul droplets ofantisera at an appropriate dilution in Tris. Washing was done by transferring thegrids sequentially to four 50 ill droplets of Tris for 5 min each, then the floatingthe grids on 20 ill droplets of suitably diluted Protein A or Protein G stabilized 10nm gold particles. After washing thoroughly with water, the viruse particles werestained with 4% uranyl acetate or 2% phosphotungstic acid.Gold particles (10 nm size) were prepared by adding 1 ml of 1% HAuC1 4 to 80ml of .45 11 filtered water, then heating the mixture to 60 C. Four ml of 1%sodium citrate and .070 ml of 1% tannic acid were diluted to 20 ml and added tothe gold solution to form the colloid.Protein A or Protein G was conjugated to the spheres by mixing theappropriate amount of protein with the gold in a total of 9.6 mis of the colloid.The amount of protein to be added was determined empirically by mixing a small17volume of gold and protein in a tube, then adding 5 M NaCl. The concentrationof protein to fully stabilize the gold is the lowest amount necessary to turn thesolution from blue to pink. After gently stirring for 15 min at room temperature,0.4 ml of 1% polyethylene glycol MW 20,000 was added to stabilize the mixture.The particles were concentrated by centrifugation in a Beckman SW-41 rotor for30 min at 20,000 rpm, then resuspending the pellet in either PBS or Tris buffer.18RESULTSMonoclonal antibodies to PVM. Initial attempts to raise monoclonalantibodies against PVM by injecting intact purified virus particles proved to beunsuccessful. When the fusions were screened, no clones showing a positivereaction to PVM were found. When the particles were treated with 1% SDS, andthen injected into mice, numerous positive clones were generated. Eighty cloneswere tested against a panel of viruses. One monoclonal antibody was found thatreacted in a group specific manner, detecting all the carlaviruses that were testedat this time, yet failing to react to any of the potex viruses (Table 1). Thismonoclonal antibody was labelled 1C8, and used for subsequent experiments.Confirmation of the presence of a common epitope among carlaviruses.Monoclonal antibodies against PVM and potato virus S (PVS) (relatedcarlaviruses) and potato virus X (PVX) (a potexvirus unrelated to thesecarlaviruses), were used to confirm the identity of the viruses. Polyclonal antiserawere used to confirm the presence of a common epitope among the carlavirusmembers. ELISA plates were coated with unfractionated polyclonal antisera toeach of the viruses tested. Monoclonal antibodies specific to each virus were thenused to determine whether the polyclonal antisera trapped only the homologousvirus to which it was raised, or whether it was able to trap other viruses.The specificity of the monoclonal antibodies to the virus that they were raisedagainst is shown in Table 2. Homologous polyclonal rabbit antiserum was used to19TABLE 1. Results of initial testing of monoclonal antibody culture supernatantsagainst a variety of carla- and potexvirusesProcedure^ Virus Number ofgroup cultures withA405 >10•Healthy potato tissue trapped with rabbit anti-PVM 1^0Infected potato tissue trapped with rabbit anti-PVM carlavirus 43Direct coated SDS treated PVM^ carlavirus 59Direct coated PVS2^ carlavirus^1Direct coated BbSV3^carlavirus^1Direct coated CLV4 carlavirus^1Direct coated PVX5^potex^0Direct coated NMV6 potex 0Direct coated SVX-4D97^potex^01 potato virus M2 potato virus S' blueberry scorch virus4 carnation latent virus5 potato virus X6 narcissus mosaic virus' potexvirus from Silene pratensis20TABLE 2. ELISA readings of reactions of monoclonal antibodies to thehomologous virus showing the specificity with which monoclonal antibodies raisedagainst potato virus X (PVX), potato virus S (PVS), or potato virus M (PVM) werereact with the homologous virusMonoclonal Immunized^ELISA readings (A 405 .)Antibody^AgainstPVX1a4^b5PVS2a^bPVM3a^b3A2-G7 PVX 1.215 0.003 0.001 0.013 0.003 0.0063B10-E4 PVX 1.781 0.005 0.011 0.007 0.008 0.005106-G10 PVS 0.017 0.009 2.691 0.001 0.022 0.009106-B7 PVS 0.02^0.011 2.73^0.001 0.011 0.0062H2-E7 PVS 0.007 0.004 2.324 0.032 0.029 0.0264D12-D4 PVM 0.045 0.008 0.032 0.039 0.916 0.0211D10-B9 PVM 0.026 0.008 0.002 0.008 2.24^0.0123B4-B3 PVM 0.034 0.013 0.014 0.027 2.208 0.0231 trapped with rabbit anti-PVX polyclonal antiserum2 trapped with rabbit anti-PVS polyclonal antiserum3 trapped with rabbit anti-PVM polyclonal antiserum4 purified virus at 0.5 mg/ml diluted 1:1000 in 2% fetal calf serum-PBS5 buffer control: no virus added to wells21trap each virus, then monoclonal antibodies were used to detect the presence ofthe virus. Each monoclonal antibody detected only its homologous virus, withoutreacting with any of the others trapped (Table 2). Monoclonals raised againstPVX detected PVX only. Similarly, monoclonal antibodies raised against PVMreacted only with PVM and those against PVS reacted solely with PVS. Thisspecificity allowed the confirmation of identity of the viruses used for subsequenttesting of the group specific monoclonal, and showed that there was nocontamination between the viruses which could account for false group results.The monoclonal antibodies could detect the individual viruses in a mixture ofviruses, and if a mixed infection occurred during the testing of 1C8, the viralspecific monoclonal antibodies could ascertain this (Table 3). The resultspresented in both Table 3 and Table 2 proved conclusively that the viral stockswere pure and uncontaminated by other viruses that could normally occur in anatural infection. Additionally, Table 3 showed that the polyclonal antisera toPVM cross reacts with other carlaviruses: rabbit anti-PVM trapped both PVM andPVS, confirming the presence of at least one common epitope. The PVS polyclonalantiserum, which would normally be expected to react in a reciprocal manner byalso trapping PVM failed to do so. These results could have two possibleexplanations. The anti-PVM polyclonal may have been raised against a viralpreparation of PVM contaminated with PVS particles, whereas the anti-PVSantisera was against a non-PVM contaminated source. The other possibility isthat the anti-PVM antisera has a larger componant of individual antibodies that22TABLE 3. Detection of viruses trapped by polyclonal antisera showing thepresence of common epitopes. Rabbit polyclonal antisera were used to trap a equalmixture s of 3 viruses, potato virus X (PVX), potato virus S (PVS), and potatovirus M (PVM) ), then monoclonal antibodies were used to detect which viruseswere trappedMonoclonal Immunizedantibody^againstELISA readings (A 405 .) when trapped withpolyclonal antiserum against:PVX PVS PVM3A2-G7 PVX 1.586 .000 .0063B10-E4 PVX 1.834 .020 .009106-G10 PVS .239 2.839 2.774106-B7 PVS .253 2.698 2.6782H2-E7 PVS .102 2.359 2.3634D12-D4 PVM .039 .072 .9621D10-B9 PVM .020 .089 2.2213B4-B3 PVM .089 .223 2.2701 each virus was at a final concentration of 500 ng/ml23are against conserved epitopes as compared to the anti-PVS antisera, which hasantibodies predominantly raised against epitopes unique to PVS.As a final confirmation of the ability to separately distinguish the individualviruses and as a test to confirm whether numerous viruses could be trapped in asingle well by a mixture of antisera, the experiment listed in Table 4 wasconducted. The results showed conclusively that even with crude antisera (notpurified) it was possible to trap a variety of viruses, even if a common epitope didnot exist.Reaction of monoclonal antibody 1C8 with various antigens. To eliminatethe possibility of anti-PVM monoclonal antibody 1C8 reacting to a host componentinduced upon infection with virus, and to determine if the antibody would detectcarlaviruses in hosts other than potato, several different host species infectedwith a variety of carlaviruses were tested. At the same time two members of thepotyvirus group, were also tested. Potyviruses are flexuous rods morphologicallysimilar to but longer than those in the carlavirus group. All host species infectedwith a carlavirus showed a strong positive result, except for pea streak viruswhich reacted extremely weakly with the antibody (Table 5). Subsequent hostrange tests cast doubt on the identity of the pea streak virus, which did notproduce the expected symptomatology in the range of hosts tested. Host plantsinfected with potyviruses, though showing strong symptoms, reacted with theantibody to the same levels as healthy controls in the carlavirus group.24TABLE 4. Detection of individual viruses with monoclonal antibodies aftertrapping with a mixture of rabbit polyclonal antisera. Polyclonal antisera raisedagainst potato virus X (PVX), potato virus S (PVS), and potato virus M (PVM)were mixed 1 together and used to trap purified virus mixturesMonoclonal Immunizedantibody^againstELISA readings (A405 )for virus mixturesPVS/PVX PVM/PVX PVS/PVM3A2-G7 PVX 0.566 0.655 0.00731310-E4 PVX 0.951 0.915 0.004106-G10 PVS 2.630 0.028 2.856106-B7 PVS 2.661 0.003 2.7392H2-E7 PVS 2.557 0.039 2.3964D12-D4 PVM 0.028 0.565 0.5001D10-B9 PVM 0.012 1.326 1.2363B4-B3 PVM 0.044 1.273 1.2221 final dilution of each individual antiserum was 1:150025TABLE 5. Effect of host plant on reaction of monoclonal 1C8 to carlaviruses 1Virus^Group Host^Absorbance at 405 nmInfected HealthyPea Streak^carlavirus Medigo sativa 0.108 0.067Potato virus M^carlavirus Solanum tuberosum 3.046 0.036Carnation Latent^carlavirus Chenopodium quinoa 2.835 0.078Potato virus S carlavirus Chenopodium quinoa 0.704 0.020Turnip mosaic^potyvirus Brassica pekinensis 0.020 0.021Zuchinni yellow mosaic potyvirus Cucurbita pepo 0.011 0.0171 Carlaviruses from various hosts were trapped with their homologous polyclonalantisera. Potyviruses were also trapped with their homologous antiserum toextend the specificity of the antibody and provide additional host species26Several procedures were performed in attempts to determine the nature of theepitope to which 1C8 was raised. Initially, ELISA was used to test whether theepitope was conformational or linear. Directly coated, purified PVM was testedusing homologous rabbit polyclonal serum and virus from infected plants. Noreaction occurred with directly coated virus, whereas both purified virus andvirus from infected plants did react (results not shown). To test the possibilitythat the purified virus failed to coat the plates when added directly to the wells ofan ELISA plate, a western blo was performed in order to visualize the virus.When a 12% polyacrylamide gel was loaded with intact purified virus and withvirus digested with the proteolytic enzymes trypsin, pepsin and V8 protease (Fig.1), a substantial reduction in the size of the viral coat protein subunit wasobserved. When compared with the predicted size fragments that would resultfrom complete cleavage (Table 6), it was clear that only partial digestion occurred.When the size of the proteins was determined, duplicate gels were transferredto Immobilon (Millipore) membranes. One blot was probed with 1C8 MAb andthe other with polyclonal rabbit antiserum. No reaction occurred in any of thelanes developed with the MAb (not shown). Rabbit polyclonal antibody reactedwith the untreated denatured virus, but not with any of the protease treatedsamples (Fig. 2). Electron micrographs of protease treated PVM (Fig. 3) showedthat a rod-like structure was still maintained by the virus, even though subunitsize was reduced by the treatment. These data suggest that polyclonal antiserum27ao1.3Cl)ESPco i  1co03^k Ei Seno. iti: I VbO coi--^a- > 2 zo-a( 97,400-.1( 66,200-4 42,69934,000 )0-^29,000^Imo^27,000 WC^swim26,000Imo-4( 31,000-41E 21,500Fig. 1. Effect of digestion with proteolytic enzymes on the size of PVM coatprotein subunits. Ten micrograms of purified PVM was digested withTrypsin, Pepsin or V8 protease. After treatment, the entire sample wasdenatured and electrophoresed through a 12% polyacrylamide gel (32).Bands were visualized by staining with 0.25% Coomassie brillient blue R-250in 10% acetic acid and 50% methanol. The size in Daltons of the PVMsubunits is shown on the left, and the size of the molecular weight markers isto the right.28TABLE 6. Summary of fragments of the coat protein of potato virus M lpredictedfrom the complete cleavage by proteolytic enzymesEnzyme^No. cut^No. of fragments No. single aa 2^sites released (> 1 aa)^releasedTrypsin^35^29^7Pepsin 17 18 0V8^42^40 31 based on published sequence of the coat protein gene using the IntelligeneticsPCGene prediction program2 amino acids2934,000* 29,000* 27,000* 26,000EmSIE^osa+E c c .§to Z —,,,b 0 - a .^a.I- 0^cozo — 11. >Fig. 2. Western blot of PVM subunits cleaved by various proteolytic enzymesand probed with rabbit anti-PVM antiserum. Digestion and electrophoresiswas done as described in Fig. 1. The proteins were electrophoreticallytransfered to Immobilon (Millipore) membranes, then probed with rabbitanti-PVM polyclonal antiserum. The bands were visualized using aprecipitating substrate (Fast Red TR salt). Numbers to the right show thesize in Daltons of the treated and untreated subunits. Expected sizes andposition of digested subunits is marked by *.30PVM control: buffer only PVM treated with pepsin MINPVM treated with trypsin 11■1 PVM treated with V8 proteaseFig. 3. Electron micrographs of negatively stained PVM particles digested withvarious proteolytic enzymes. Samples were adhered to 400 mesh carbon coatedgrids, then stained with 4% (w/v) aq. uranyl acetate. Bar indicates 100 nm.31raised against PVM reacts only to epitopes located on the surface of the viralparticle.As confirmation of the ELISA data, immuno-electron microscopy was used tospecifically label purified virus preparations with 1C8 (Fig. 4). Purified PVM,PVS and and an undescribed carlavirus from honeysuckle (Lenocira X brownii cv.Dropmore Scarlet) were adhered to coated grids and probed with antisera toPVM, PVS and monoclonal 1C8. Colloidal gold coupled to protein A or protein Gwas used to detect the presence of the antibody. Each virus was tested withpolyclonal antisera to provide positive controls. PVM virus reacted well withhomologous rabbit polyclonal antiserum, weakly with the monoclonal, and not atall with rabbit anti PVS. PVS and the honeysuckle carlavirus reacted with allthree antisera. The poorest labelling and reaction with the monoclonal antibodyoccurred with PVM. Though some slight labelling was seen, both other virusesshowed more bound label with the monoclonal antibody. In these other cases,there is no doubt that a specific interaction was seen. This provides visualevidence that the antibody reacts with virus particles, and not with some othercomponent, and that the antibody is not limited to PVM but does react with othercarlaviruses.Cloning and sequencing the PVM coat protein gene. RNA was extractedfrom purified PVM virus. When analyzed on a denaturing gel, the purifiedsample resolved as a single band of approximately 8.1 kb (Fig. 5). This RNApreparation was reverse transcribed into cDNA using an oligo- d(12-18)T primer,32Virus: PVM ...■^Virus: PVS^ Virus: Lonicera carlavirusAntibody: rabbit anti PVM^Antibody: rabbit anti PVM^Antibody: rabbit anti PVMVirus: PVM^ Virus: PVS^ Virus: Lonicera carlavirusAntibody: monoclonal 1C8^Antibody: monoclonal 1C8^Antibody: monclonal 1C8Fig. 4. Labelling of viruses using 10 nm colloidal gold conjugated to Protein A.Purified virus or leaf dips of several viruses were probed with the antibodiesindicated, then visualized with Protein A conjugated to 10 nm colloidal gold.Samples were post-stained with 4% (w/v) aq. uranyl acetate. Bar indicates 100 nm.33A.ugs of PVM RNA2.0 1.0 .511:5cazcczcca.I 23,130 by9,419 by6,557 by"M, 4,371 byid 2,322 byill^2,028 by-4^564 by 4.40 Kb )II"1.35 Kb ►.24 Kb Po9.49 Kb 11.7.46 Kb 11.'Fig. 5. Electrophoretic analysis of PVM RNA under both denaturing andnon-denaturing conditions. (A.) Three different concentrations of PVMRNA (2.0, 1.0 and 0.5ug) were electrophoresed through a non-denaturing1% agarose gel bufferred with TAE. Numbers on the right indicate thesize of HinD III digested lambda DNA in base pairs. (B.) 1 ug of PVMRNA was electrophoresed through a 1% agarose gel containing thedenaturant formaldehyde. RNA size standards (BRL) are shown in Kb onthe left.34since RNA from other carlaviruses had previously been shown to contain apoly(A) tail. The resulting cDNA was blunted with T4 DNA polymerase, clonedinto the Sma I site of plasmid pT7/T3-18 (Fig. 6), and then transformed intocompetent DH5a strain of Escherichia coli . After duplicate plates of resultingcolonies were made, the original colonies were transferred to nitrocellulosemembranes, lysed, and screened for production of coat protein with rabbit anti-PVM antiserum.Colonies shown to be positive for coat protein production were then picked fromoriginal plates and grown overnight in 2 ml of selective media. Plasmid DNA wasextracted and analyzed. The longest clone (ca. 5 Kb), designated p7, was thenshown to be derived from PVM viral RNA due to its specific hybridization to totalRNA extracted from infected potato tissue in Northern blots (Fig. 7). To furtherconfirm that the coat protein region was indeed contained in this area, bacteriallysates containing various sized plasmids were electrophoresed throughpolyacrylamide gels, transferred to Immobilon and probed with polyclonalantibodies to coat protein. The results (not shown) confirmed that the plasmidcontained the entire coat protein gene, based on a size comparison with a purifiedvirus sample loaded in a concurrent lane.A restriction enzyme cleavage map of the cDNA of clone p7 was constructed(Fig. 8). Common six base cutting enzymes were used in double digests to cutthe p7 DNA both singly and in combination. The fragments were run out on a 1%TAE agarose gel and their sizes determined. The fragments were then assembled35PVM insert^GGGM13 primerT3 promoter pUC 18 MCS T7 promoterpBR322 onFig. 6. Map of pT7/T3-18 plasmid and orientation of markers around the insert.Lac Z' means B-galactosidase gene region, fl on means origin of replication for flphage, pBR322 means origin of replication form plasmid pBR322, MCS meansmultiple cloning site, T3, T7 promoter are RNA polymerase promoter sites and m13primer is a sequence derived from M13. Amp resistance means ampicillinresistance. CCC and GGG are the flanking nucleotides immediatly adjacent to thePVM derived cDNA insert.36>..013a)4.0 'S03 4)0 "-= c B.-4 23,130 by-4 9,419 by-41( 6,557 by-4 4,371 by..01 2,322 by.44 2,028 by564 byEC44■4a) 2PVMSub-GenomicRNAA.Non-denaturing^ 1% Agarose with1% Agarose gel FormaldehydeFig. 7. Northern blot analysis of total RNA extracted from infectedand healthy leaves from Solanum tuberosum. (A.) 1.0 ug of totalRNA electrophoresed on a non-denaturing 1% agarose gel. (B.)Same RNA samples as in A. electrophoresed through 1% agarose gelusing formaldehyde as a denaturant, then transferred by capillaryaction to nitrocellulose and probed with a DNA probe prepared bynick repair of p7. A probe derived from lambda DNA was used as acontrol in the far right lane.37Fig. 8. Restriction enzyme cleavage map of clone p7. Common restriction siteswithin the virus are shown as well as the sites remaining in the multiple cloningregion of the plasmid after insertion. Location of the sequence homologous to the 3'end of the published sequence of the coat protein gene (67) is indicated by the solidbox. Length of the cloned region in Kb, and the relative location of commonrestriction enzyme sites with respect to the start of the cloned PVM region isindicated by the numbers below the hatched region.38in an orderly manner on the basis of their sizes to generate a map of thearrangement of restriction sites.Once the arrangement of the restriction sites was known, the information wasused to generate a series of deletions that would allow sequencing of the region.Bal 31 exonuclease, which removes base pairs bidirectionally was added to thepreparation of cleaved plasmid DNA, then aliquots were removed at various timeintervals and the reactions stopped to prevent further deletion. The result was aseries of fragments of varying lengths, classes of which differed by approximately200 base pairs. Deletions were done from both sides in the presumed region ofthe coat protein.Once construction of the deletions was completed, E. coli extracts containingplasmids from the 5' end of the cloned region (with respect to its orientation inthe multiple cloning site of the original p7 plasmid) were loaded on a 12%acrylamide gel and total protein was transferred to Immobilon membranes.These were probed with polyclonal rabbit antiserum raised against PVM coatprotein. All deletions produced the same size protein, which was slightly higherin electrophoretic mobility than the coat protein from purified virus (Fig. 9).Several months after the start of sequencing, the entire 3' region waspublished by another group (49). Comparison of existing sequence informationwith that of the published sequence showed sufficient similarity (Fig. 10 and Fig.11) that sequencing was terminated at this point, and the published sequenceused for discussion of further aspects of this thesis.39Deletion numberB.^42 24 28^PVM coatProteinFig. 9. Western blot of total proteins from Escherichia coli carrying plasmids withvarying sized deletions of the 5' end of p7. (A.) Restriction digest maps of 3deletions used for the analysis showing the remaining restriction enzymecleavage sites. Box indicates cDNA derived from PVM RNA. Solid line indicatesplasmid sequence. Numbers to the right indicate the arbitrary designation of theplasmid. Numbers below show the sizes in Kb of the insert. (B.) Western blot oftotal protein from E. coli was performed as described in Fig. 1. The blot wasprobed with rabbit anti-PVM polyclonal antiserum, and visualized withprecipitating substrate. Numbers above each lane correspond to the deletionsshown in A.^ 40Published - MGDSTKKAETAKDEGTSQERREARPLPTAADFEGKDTSENTDGRAADADG -50Published - EMSLERRLDSLREFLRERRGAIRVTNPGLETGRPRLQLAENMRPDPTNPY -100Published - NRPSIEALSRIKPIAISNNMATSEDMMRIYVNLEGLGVPTEHVQQVVIQA -150Published - VLFCKDASSSVFLDPRGSFEWPRGAITADAVLAVLKKDAETLRRVCRLYA -200111111^111111111111111P7 - DAVLAVMKKDAETLRRVCRLYA -22Published - PVTWNHMLTHNAPPADWAAMGFQYEDRFAAFDCFDYVENTAAVQPLEGLI -25011111111111^1111111111111111111111^111111111111111P7 PVTWNHMLTHNSPPADWAAMGFQYEDRFAAFDCFIYVENTAAVQPLEGLI -72Published - RRPTPREKVAHNTHKDIAVRGANRNQVFSSLNAEVTGGMNGPELTRDYVK -3001111111111111111^1^11^111111111^111111111111111^1P7 RRPTPREKVAHNTHKDMALRGRNRNQVFSSLSAEVTGGMNGPELTRDFGK -122Published - SNRK -304II^1P7 - SNNK -126Identity : 116^(^92.1%)Number of gaps inserted in Published: 0Number of gaps inserted in p7: 0Fig. 10. Comparison of the amino acid sequence translated from a partialnucleotide sequence located in the 3' region of the p7 insert with the publishedsequence (67) of the entire coat protein open reading frame. Alignment andtranslation were done using the Intelligenetics PCGene program Numbers to theright indicate the number of the amino acid in the sequence, with the first aminoacid being the extreme amino-terminal residue in the series.41Published - TGCGAAGGGTGTGTAGGCTGTATGCCCCGGTGACATGGAATCATATGCTG -7850III^11111111^11111^II^11111^111111111111^IIP7 ^GGTTTGTAGGCTATATGCACCCGTGACCTGGAATCATATGTTG -90Published - ACGCACAACGCGCCTCCGGCCGATTGGGCTGCCATGGGGTTTCAGTATGA -790011^111111^1^11^11111^11111111^11111111^11^11^11^11P 7 - ACTCACAACTCTCCCCCGGCTGATTGGGCAGCCATGGGTTTCCAATACGA -140Published - GGATCGCTTCGCTGCTTTCGACTGCTTTGATTACGTTGAGAATACTGCTG -7950III^11^11^11111^11111111111^11111^11111^11^11^1P 7 - GGACCGGTTTGCTGCATTCGACTGCTTCATCTACGTCGAGAACACAGCGG -190Published - CAGTCCAACCCCTAGAGGGATTGATCAGGCGACCTACCCCAAGGGAAAAG -80001^11111^11^11^11^11111111^III^1^11^11^11^11^11^IIIP7 CCGTCCAGCCACTGGAAGGATTGATAAGGAGGCCCACTCCGAGAGAGAAG -240Published - GTAGCTCACAATACGCACAAAGACATCGCAGTGCGTGGAGCAAATCGCAA -8050II^11111111111^11111^II^II^II^1111^11^II^11111P7 GTTGCTCACAATACCCACAAGGATATGGCTTTGCGCGGTCGTAACCGCAA -290Published - TCAGGTGTTCAGCTCTCTCAATGCCGAGGTCACTGGTGGTATGAATGGTC -8100111111111111111^11^1^111111111^11^11111^11111111^1P7 TCAGGTGTTCAGCTCCCTTAGTGCCGAGGTGACCGGTGGCATGAATGGCC -340Published - CGGAGCTCACTAGAGATTATGTAAAGTCTAATAGAAAATGAAGGACGTAA -81501^11^1111111111111^1P 7 CCGAACTCACTAGAGATTT CG^ 361Identity : 300^(^78.9%)Number of gaps inserted in Pubished: 0Number of gaps inserted in p7: 14Fig. 11. Comparison of a partial nucleotide sequence (shown as DNA) from the 3'end of the insert in plasmid p7 with the coat protein coding region of thepublished sequence of PVM (67). Alignment was done using the IntelligeneticsPCGene program. Numbers to the right of the published sequence indicate theposition of the nucleotide in the sequence, with position one being the firstnucleotide in the PVM genome. Numbers to the right of the derived sequenceindicate the position of bases in a contiguous series of a partial sequence of the p7plasmid. The first position is arbitrarily assigned to the first nucleotide in theseries.42DISCUSSIONExistence of common epitopes among carlaviruses. Serological crossreactivity between members of the carlavirus group are, on the whole, quiteextensive (27). Though some members do not show any tendency to cross react,as indicated by lilac mottle virus or muskmelon vein necrotic virus, others, suchas carnation latent virus, the type member of the group show extensive crossreactivity with other carlaviruses. This indicates that there are common epitopespresent, and possibilities exist for the development of group specific antibodies,which could conceivably be universal for carlavirus group recognition. The factthat some of the members of the group fail to cross react is probably due to thecomposition of the polyclonal antisera used to test for the reaction. This issupported by the data in Table 2, where it is shown that rabbit anti-PVMpolyclonal can trap PVS, proving that a shared epitope exists, yet rabbit anti-PVSfails to react in a reciprocle test. Since polyclonal antisera is made up of aheterologous population of antibodies, sometimes epitopes can be masked orpredominant epitopes may exist that only allow minor epitopes to be detected bysmall populations of antibodies in the serum which would not give sufficientsignal to be differentiated from background. The possibility of not finding sharedepitopes is also increased by the fact that most antisera are raised with thepurpose of specifically identifying the virus in question; if extensive cross43reactions occur, the antisera is usually considered to be of poor quality anddiscarded in favour of one that is more selective.The advantage of group specific antibodies is shown by a comparison of resultsof Tables 2 and 3. Although incubated under identical conditions, optical densityvalues for viruses trapped with a mixture of polyclonal antisera in a single wellwere lower overall compared to those trapped with a single antiserum. In adouble antibody sandwich (DAS)-ELISA, assuming conditions of saturation at allsteps (14,57), the limiting factor would be how much virus can be trapped on theplate, which in turn is determined by the amount of trapping antiserum that canadhere to the individual well (6). Assuming that no other factors interfere, mixedantisera will bind in a stoichiometric manner: if three antisera at equalconcentrations are loaded on the well, each will bind to approximately one third ofthe possible sites. Each antiserum will then only be able to trap one third of thevirus particles as compared to coating the plate with the same concentration of asingle antiserum, ultimately resulting in a decrease in optical density whensubstrate is added. As more and more polyclonal antisera are mixed, a limit willquickly be reached where the maximum amount of virus trapped will not bedistinguishable from background. Practically, this limits the number of virusesthat can be screened in a single test.If a common epitope were present on the viruses tested, this limitation wouldnot exist. The dominant limiting factor that would then become the concentrationof the virus present in the sample. A single common epitope would allow testing44for a much larger number of viruses in a single ELISA well than could be doneusing combined antisera, provided that the viruses being tested for contained thisepitope in a conserved region.Generation of monoclonal antibodies. Commonly, to generate monoclonalantibodies against a virus, the virus is purified to homogeneity, then injected atan appropriate concentration into a mouse. When this approach was attemptedwith PVM, no clones secreting antibodies were found on subsequent testing.Direct observation of the virus preparations under polarized light showed a greatamount of light scattering rather than the clear light path which would indicatewell dissolved virus. The virus preparation appeared very opaque. Initially, itwas thought that this was simply due to the instability of the virus, and that aninsoluble precipitate was being formed. After low speed centrifugation ( 15,000 XG, 10 minutes) the precipitate was not removed, and the solution remainedopaque.When the purified virus was examined using the electron microscope many ofthe particles were lined up and aggregated in an end to end mannner as well asalong the long axis. The particles probably formed large aggregates which stillremained in solution, and remained in this form when injected into the mouse.These aggregates may have been too large to allow recognition of the virus asnon-self in the mouse, and too big to stimulate any immune response. The resultwould be that no monoclonal antibodies would be generated, since no B-cellactivation would occur.45As a test of this theory, the virus was modified to prevent the aggregation. Ifthe aggregates were the result of specific attractions on the surface of the virus, itwas felt that modification of the virus either by denaturation or partial removal ofresidues on the surface of the coat protein may prevent this interaction. Bothchemical and enzymatic treatments were attempted.Trypsin cleaves proteins at carboxylic groups of amides of arginine and lysineat neutral pH. Endoproteinase Glu-C (Staphylococcus aureus V8) acts at morediscrete sites also under neutral or slightly basic pH. Pepsin cleaves at carboxylicacid groups of aromatic amino acids not adjacent to valine, alanine and glycine.The pH optimum for this enzyme is 2.0 . If both pepsin and trypsin cleaved thevirus into fragments too small to use, it was hoped that the more specific V8protease would provide a gentler cleavage that would result in peptides ofsufficient size to generate a representative panel of antibodies which hopefullywould contain group specific epitopes. Conversly, if a sufficiently high degree ofcleavage did not occur under neutral conditions, subjecting the virus to acidicconditions might provide conformational changes that could expose alternatecleavage sites. If complete cleavage of the virus failed to generate appropriatesized peptides, then partial cleavage with low enzyme concentrations and shortexposure times would be attempted.The untreated coat protein (Fig. 1 and Fig. 2) migrated as a 34K protein, whichcorresponds to the reported value . The treatments with trypsin, pepsin and V846protease resulted in subunit molecular weights of approximately 29K, 26K and27K, respectively.Electron microscopy of treated particles (Fig. 3 ) showed some minormorphological change in the particles, but the overall rod shape is stillmaintained. Shukla et al (54) obtained similar results with potyviruses whichwere similarly treated. Though the cleavages were performed with sufficientenzyme over sufficient time to result in complete cleavage of the coat protein,there was only partial cleavage in all cases. After the complete sequence of thecoat protein was determined (49, 67) a prediction of cleavage sites was madepossible (Table 6). This table confirms that only partial cleavage occurred, as thesize of the subunits obtained is substantially larger than those predicted undercomplete cleavage conditions. Predominantly only one fragment remained asopposed to the numerous fragments predicted. This could be due either to sterichindrance of the enzyme activity by the helical structure of the particles, or to theprotection of cleavage sites by the organization of the coat protein subunits insuch a way that these sites are bound to the RNA core of the particle (54).The virus particles were chemically modified by SDS, which dissociates thevirus into its subunit components. Since this was most easily accomplished, micewere injected before the digests were analyzed, and the first fusions wereperformed on this form of the virus. One of the fusions from this experimentyielded the results presented in Table 1. Initially, the fusion was screened bydirectly coating the SDS denatured particles on ELISA plates. Positive clones47were selected and expanded into 1 ml wells. After two days in 1 ml cultures, theclones had multiplied sufficiently to yield enough antibody to test against thepanel of viruses listed (Table 1). A single clone produced an antibody thatappeared to be group specific in this limited test; one antibody detected allcarlaviruses, but did not react against any of the potexviruses. Since thisapproach was successful, fusions with the enzymatically modified virus were notdone. Further effort was channelled into assessing the range and specificity ofthe antibody.As the antibody appeared to react against a broad range of infected samples,the possibility occurred that it may not be reacting specifically to the viruspresent but rather to a compound generated by the host plant in response toinfection. Since "stress related" compounds may be induced to any general stress(12), this possible explanation was tested. Two experiments were set up to verifythat the antibody was reacting with the viruses specifically. The first experimentwas to broaden the host range. If a particular host was producing a stresscompound to which the antibody was reacting, it would be unlikely that widelydiffering species would all produce the same compound in response to infection.The results are summarized in Table 5. When carlaviruses and potyviruses wereused to infect a diversity of species to "stress" these plants, the expected resultwould be that if the antibody were to a stress compound, only the original host,potato, would show a reaction. If, on the other hand, there was a general stresscompound produced universally by plants, hosts infected with potyviruses could48also be expected to produce the compound, and should produce a reaction. Allplants infected with potyviruses that were used showed disease symptoms. Theresults (Table 3) clearly indicate that all species infected with carlavirusesreacted. Plants stressed with potyviruses failed to show any reaction whatsoever,and absorbance readings were in the range of those with healthy controls.Another concern was that the virus may react universally with flexuous rods,and may be finding a conformational epitope necessary to form a rod structure.Carlaviruses are flexuous filaments 600-700 nm long by 12-13 nm wide.Potexviruses are structurally very similar except for a shorter length: they are470-580 nm in length and 11-13 nm wide. Potyviruses are at the other end of thespectrum in length: they are slightly longer being 680-900 nm long by 12 nmwide. There is no known serological cross reactions between the groups, but thecoat protein amino acid sequences of carlaviruses and potexviruses show somesimilarities (37,67).ELISA (Tablel and Table 5) clearly failed to detect any viruses from putativelyrelated groups. The antibody was tested against members of both the poty- andpotexvirus groups and showed no reaction. All evidence strongly shows that it isa common epitope which is specific to carlaviruses that is being detected.Partial characterization of the epitope to which 1C8 reacts. Westernanalysis of the proteolytically cleaved PVM subunits determined that there was acomplete elimination of antigenic sites when probed with homologous polyclonalantiserum (Fig. 2). Amino acid sequence studies (52) would suggest that the sites49eliminated by the enzyme are either at the 3' or 5' terminus of the coat protein.Work by Shukla et al (52) on potyviruses showed that when analysed by Westernblotting and amino acid sequencing, partial digestion of potyvirus particles bytrypsin specifically removed the N and C terminal portions of the protein, andthat this was sufficient to eliminate the antigenicity of the particles. Shuklasuggested that both the C terminal and N terminal portions of the protein areexposed on the outside of the viral subunit particle in the assembled virus. Otherrod-shaped viruses such as potexviruses (30) and tobamoviruses (2) have beenshown to have this configuration. They are also known to have protruding N andC terminal regions that seem to protect the core regions. In addition, thedegradation of filamentous viruses on storage is also thought to involve thisregion (42).As the nucleotide sequences of members of the carla, potex and potyvirusgroups became known, comparison of the sequence of the coat protein with otherfilamentous viruses showed that a region exists in potato virus M that has 55amino acids identical to that of potato virus X (50). Previous work done on thesequence of potato virus S (37) also found a significant region of amino acidsequence homology between PVS and several potexviruses. Since no significantserological relationship has been found between these two groups of virus, theauthors suggesed that this region is buried within the inside of the particle whenit is properly assemble into intact virions , and normally not accessible toantibodies raised against whole virus.50By applying this information to the results of the ELISA data and the Westernblots, as well as the sequence information recently obtained about the coat proteinof PVM, it is most probable that a similarity exists with carlaviruses, and that themonoclonal antibody is against an epitope located somewhere in the amino orcarboxy terminal regions.Antibodies react to either linear or conformation epitopes. The linear type ismade up of a specific sequence of amino acids and is not dependant on threedimensional spatial configuration. These epitopes are readily detected indenaturing systems, since they do not depend on the shape that the proteinassumes, but only on the combination and length of the amino acid string.Typically these epitopes are from 4-7 amino acids in length (16). Conformationalepitopes are subject to native folding of the protein and are made up of 15-22discontinuous residues, 5-6 of which contribute most of the binding energy (33).They are absolutely dependent on either the entire protein or the specific area inwhich they are located being in a natural configuration. The amount of nativeconformational retention necessary is totally dependent on the location of theepitope: since they are discontinuous and rely on folding to bring them inproximity to each other to react with the antibody, they can exhibit a varyingdegree of tolerence to non-native conformation. If all of the residues are locatedon one small region, they may allow some degree of unfolding before they aredestroyed.51The most sensitive conformational epitopes are those that depend on theinteraction of more than one protein component. In the case of filamentous rods,the helical structure (54) of the subunits imposes an additional restriction if theepitope happens to bridge the subunits, or be dependent on their alignment. Thishas been shown in the case of PVX, where even direct binding to ELISA plates orto nitrocellulose is sufficient to destroy certain determinants (29).Two strategies can be used to map sequential epitopes. The first method is tomake a series of nested deletions in the coat protein cDNA such that the deletionsare translated and expressed. This results in protein fragments covering theentire region of interest in varying lengths, allowing the linear assignment ofepitopes once these fragments are reacted with the antibodies of interest. Thereason that this method was chosen was that the deletions have a dual purpose:once translated and expressed they can localize the region of the epitope, and theDNA clones from which they are derived can be used to obtain the sequence of thecoat protein, which, at the time of initiation of the project, was not known.A second method for mapping epitopes is to use a series of overlappingsynthetic peptides (53, 55). This method is dependent on the previous knowlegeof the amino acid sequence of the region of interest: this was unknown for PVM souse of this method was not possible.Tertiary structure epitopes can only be mapped by crystallographic methodswhich involve reacting the antibodies with the virus under conditions such thatcrystals of the complex can be grown and subjected to X-ray techniques.52Computer modelling is then used to solve the structure. These techniques arebeyond the capability of the laboratory at which the work was done. Crystals aredifficult to grow, the equipment is expensive, and only limited number of facilitiesexist in the world that have this capability and expertise.Monoclonal 1C8 was tested under a variety of conditions to attempt todetermine the epitope. There was some discrepancy in the results between theinitial screen and subsequent performance of the antibody. Tests of the antibodyin Western blots proved negative in all cases. Direct coating of virus to plates inELISA was also sufficient to remove 95% of the values obtained when comparedto virus preparations trapped with homologous antisera. On storage, both withpurified virus and in tissue, there was a degradation of reaction with time. Virusstored at -20°C in 50% glycerol gradually showed degradation similar to mostfilamentous particles, and storage of tissue at this temperature also resulted in aloss of reactivity to the antibody. Storage at room temperature or 4 C greatlyaccelerated the rate of loss of reactivity. This is consistent with the results foundby Kaftanova et al (44). Optimum sensitivity and maximum absorbance valueswere obtained with freshly ground tissue in double antibody sandwich ELISA.This reaction profile suggests that the epitope is very sensitive toconformational change. A discrepancy exists in this behaviour since the antibodywas derived from a denatured virus preparation. Initial testing for the formationof monoclonal antibodies was also with directly coated SDS denatured coatprotein. Since the SDS was dialyzed away before injection and testing to53minimize interference with subsequent handling, it is possible that a degree of re-folding of the virus occured both during the immunization process and in theELISA testing. This resumption of native conformation must be sufficient for theinitial formation of antibodies and subsequent detection of the antibody. No otherexplanation seems plausible.Since the epitope is conformational, only X-ray crystallography can be used toaccurately determine its location.Cloning of the PVMcoat protein gene. In order to determine the possiblebinding site of any antibodies generated, the RNA from purified virus was reversetranscribed into cDNA, then cloned in an E. coli plasmid. Though this part of thethesis was made redundant when the complete sequence of PVM was published(67), several interesting aspects became evident during the project. Initially,these were treated as experimental discrepancies, and, it was felt they would beresolved once the complete sequence of the region surrounding the coat proteinwas determined. After the sequence became known from published data, severalsubsequent papers did clarify some of the mysteries that had occurred.Since the project was built around analysis of the coat protein, a strategy wasderived which would assure that the cloned cDNA would contain the coat proteingene. To isolate this region in the minimum amount of time, and to ensure thatit could be used for epitope mapping (that it contained the region that specified itsantigenicity) the cDNA was cloned into a vector that contained a constitutivelyexpressed Lac Z' gene which would be fused to the coat protein region. The Lac Z'54gene would provide all necessary upstream transcriptional and translationalsignals for expression of the viral proteins. Polyclonal antiserum would then beused to probe for the expression of the protein.The limitations of this particular strategy were: 1) that only about one third ofthe clones containing the protein region would be detected (only those properly inframe with the Lac Z' gene product); 2) that the addition of the fusion portionmight interfere with the binding of the antibodies (38, 56); and 3) that since thereare probable proteins coded downstream (i.e. towards the 3' end) to the coatprotein, which can be deduced by comparison to sequences of other carlaviruses,termination signals may be encountered before expression of coat protein occurs.For these reasons the number clones that it would be possible to detect could beseverely limited, and it was feared that only a few, if any, would arise. Thisstrategy had been used previously in the successful cloning of PVS, but yieldswere only two to three colonies/plate (D. MacKenzie, personal communication).When the eight plates of bacteria were assayed, instead of the low numbersexpected, each contained between 30 to 100 positive colonies. Colonies wererandomly selected, expanded, and mapped with restriction enzymes. The longestclone whose product reacted positively with the antiserum was kept forsubsequent use (Fig. 8).A series of nested deletions were made from this clone to allow sequencing ofthe region (Fig. 9). As sequence information became available, it was analyzed bytranslating the regions, and looking for continuous reading frames. No55continuous coding was possible unless the strand was reversed andcomplemented. This indicated that the clone was in the opposite orientation tothat which was originally thought. The possibility that an error had been madewith the plasmid choice and that the 19 version of the plasmid with a reversedLac Z' with respect to the multiple cloning site had been mistakenly used waschecked by sequencing the plasmid in the overlap region between the Lac Z' geneand the multiple cloning site, then comparing it to the sequence obtained from themanufacturer. The sequence information confirmed that the correct plasmidpT7/T3-18 had been used. The conclusion was that the protein could not possiblybe transcribing from the Lac Z' gene.At the same time that these deletions were being sequenced, a Westernanalysis was done of these clones. Although the deletions were of varying sizes,they all produced the same size coat protein, which migrated slightly higher onan acrylamide gel and was therefore slightly larger than the coat protein of PVM(Fig. 10). If transcription was initiating from the Lac Z gene, and the coat proteingene was in the correct reading frame with respect to it, progressively smallersized proteins were expected to be produced as the deletions would be reducingthe protein size by deleting the carboxy terminus.At the time of the experiments, neither the sequence nor the location of thecoat protein gene was known. The size of the fragments used to determine thesize of the coat proteins in the Western blot (Fig. 10) were larger than the codingregion for the coat protein (now known to be from position 7227 to 8142 on the56genome) which is 915 base pairs in length. It is conceivable that an in framefusion with the Lac Z' gene at one of the earliest amino acids could still give theresults shown in this figure.Recently, a paper by Foster and Mills (13) described three potential ribosomalbinding sites in PVM as well as other carlaviruses based on the strong sequencehomology of these regions to the ribosomal binding sites of E.coli RNA polymerase13 and the A protein from the E.coli phage MS2. The first two binding sites areclose together and appear in front of the putative 25K protein open readingframe, which is located upstream of the coat protein (Fig. 12). The second bindingsite is located immediately in front of the coat protein gene.Based on the information on the location of the sites described in the paper,the sizes of the proteins that were translated from these sites were determinedusing the PCGENE program. If translation is initiated at the third binding site,a protein only seven residues longer than the coat protein would be made. Thesize of this protein would be 34,347 as compared to 33,645. This could account forthe slight shift in size seen on the Western blot (Fig. 10), if these proteins wereproduced. Translation initiation at the first or second site, proximal to the 25 Kopen reading frame would result in a protein of approximately 77 K but wouldnecessitate at least five stop codons to be read through before the coat proteinwould be translated. No start codons are encountered at either location, but thestart codon for the coat protein is only seven residues away. The first start codonnext to the 25 K ribosome site is 49 amino acids away.57Fig. 12. Diagram of the two expected patterns of Western blots that would resultfrom translation of a series of timed Bal 31 exonuclease digestions of the 5' end ofthe PVM insert in pT7/T3 from two possible orientations of the coat protein geneand its surrounding sequences in pT7/T3 with respect to their orientation of theLac Z' gene. (A.) The resulting pattern if the coat protein is in the oppositedirection to Lac Z' and using its own ribosomal binding site. (B.) The resultingpattern if the coat protein is in the same orientation as the Lac Z' gene and beingtranslated using the Lac Z' ribosome binding site.^ 58Though the size discrepancy is still not fully accounted for, the evidencestrongly points to initiation of ribosome binding at the third site. The first pieceof evidence is the much larger than expected number of positive clones that werefound when the library was screened. The second is the necessity for reversal ofthe sequence information in order to translate the protein. The third is theamount of homology between E.coli binding sites and the putative binding siteslocated by computer analysis. The fourth line of evidence is that when thelocation of the sequence of the 3' end of the coat protein is mapped back to theoriginal p7 clone, it falls in a region between 4.14 Kb and 4.7 Kb. Since p7 is only4.9 Kb in length, the orientation accounts for the results seen in the Westernanalysis. These results provide experimental evidence not only for the existenceof the site but for its utilization.The sequencing was discontinued for two main reasons. The first was thepublication of the complete sequence by Zavriev et al (67). Comparison of apertinent portion of the sequence information showed 92% homology at the aminoacid level and almost 80% homology at the nucleic acid level. There was no doubtthat it was the same virus although possibly a slightly different strain. Thesecond reason was that the epitope at this time was determined to beconformationally dependent and the antibody would only work if the virus was inits native form. Generation of sequential deletions would not determine the siteof antibody binding.59Current and future use of the 1C8 group specific antibody. In Canada,a program exists for quality control of seed potatoes. This is carried out by theAgriculture Canada, Food Production and Inspection Branch. The programconsists essentially of three parts: 1) virus inactivation by heat therapy of potatoplants; 2) production of nuclear stock for the elite seed program, which allows thepropagation of disease free plants through sterile tissue culture methods; and 3)routine testing for any latent infections once the plants are placed in the field,Although the branch has integrated monoclonal antibodies into its procedures,a fear exists that due to the high degree of specificity of monoclonal antibodies,strains of the virus may arise that do not contain the epitope towards which theantibodies are raised, and these would be missed in the testing protocol. Thespecificity of 1C8 towards a broad group of viruses has resulted in its acceptanceand integration into the first two aspects of the program. Because it detects bothPVS and PVM in a single test it would also provide a significant improvement inquality without increasing the work load, since to date the program does notroutinely screen for the presence of PVMThe disadvantage of the antibody for field testing might result from theinstability of the epitope under long term storage conditions. Dr. Robert Martin,who attempted to use this antibody for testing for blueberry scorch virus in fieldsamples found that there was significant degradation of the carlavirus inblueberry tissue, which seemed to eliminate the epitope, making the antibodyuseless for his purposes, even though the antibody did react to purified virus60preparations stored in the freezer (personal communication). This antibody hasbeen used successfully in a field survey for elderberry carlavirus in Sambucuscanadensis (11) indicating that the ability of the antibody to detect carlaviruses instored tissue may depend on the stability of the individual virus or on the methodof harvesting and storage of the tissue. Trials of the antibody for latent virussurvey in the potato program will be initiated this winter.61Appendix A: Complete nucleotide sequence of potato virus M(67).Ptmpvmcg Length: 8535 October 25, 1992 19:38 Type: N Check: 4922 ..1 NNTAAACAAA CATACAATAT CTGGACTTAC AC TACAATAT AC TACCAGGA51 AATACTATAT TCGGTCTAAG TCAGCATGGC AGTCACATAC AGAACGCCAA101 TGGAAGATAT TGTTAATTGC TTCGAGCCAG CAACTCAGGC TGTGATAGCT151 AATAGCGCTG CTACACTGTA CAAGAACTTC GAGGAGCAAC AC TGCCAATA201 CTTCAATTAC TACCTTTCTC CCTTGGCGAA AAGGAAATTG AGCATGGCAG251 GCATATACTT GAGTCCGTAC TCGGCAGTCG TGCATTCGCA TCCGGTTTGT301 AAGACGCTGG AAAATTACAT AT TGTATAGT GTTTTACCTT CGTACATAAA351 TTCTAGCTTT TACTTTGTAG GTATTAAGGA GAGAAAACTG CAGCTGTTGA401 AATCAAAATG CAAAAATTTG GACAGTGTGC AGGTGGTGAA TAGATACGTG451 ACCAGTGCAG ACAGAATGAG GTACACAAAT GATTTCGTGC CATATGGCTC501 ATACGAGCAT GAATGCCTGG TGCACAAAGG AGTTGGTCTG GACAACGAAG551 CGCTCAGAGG AC TAGTAGGT CCACTAAGGC GTCACAAAGC AAAAAACCTA601 TTTTTCCATG AT GAGT TGCA TTACTGGAGT AGTAAGGTGC TTATTGATTT651 CTTAGATGTC ATGCGTCCAG ATAAGC TACT TGGTACTGTT GTGTACCCCC701 CAGAAT TACT AT TCAAGCCA ACACGTAGCT TGAATGAGTG GT GCTACAC T751 TAT GATATAG TGGGGGACAC ACTGATGTTT TTCCCTGATG GCGTGCAGAG801 CGAGGGC TAT CAGCAGCCAT TAAAGGGTGG TTACCTACTG GGGGCAAGGA851 GTTTGAAATT GCCGGACGGT ACAGTGTACA TGGTTGATGT GCTGTGCAGT901 AAATTTCCCC ACCATTTGAT TTCGATAACA AAAGGTGAAG CGGCAGCGCC951 GACGCATCGT GCGTTCGGCC CAT T TGAGGC GGTTGCATCG GAAGC T T T GA1001 AAGCGACCCT TAGT CC TGAT TACCCGTGTG CTTTCCCCGT TAGCTATGAG1051 GTGGTTAACA AGATTTACAG GTACTTACGT ACACTGAAAA AACCCGATGA1101 GCAGTCCGCC ATAGCAAAGC TAAGCCAAAT AAT T GC TGAG CCGTCCGGGA1151 GGGAAATTGA TT TCGTGGAG TGCTTCGCGC GGCTGGTGAT TCACAATTCT1201 AGCATGTGCG CCACAATCAT GCCAGAGCAA CTGAAAGAAT TCATGGGGAA1251 CTGGCTCGGA AAGATGCCTT CTGTGCTGGC ACGCCGCT TT AGTAGTGTTA1301 GAGCTGTGTG TGTGAACAAA TTCATCCGGG GT C TAAAACC GTACAGCTTC1351 ACCCTGCGCT TGAATGAGAT AACCTGGTGG AACATTTGGG AAAACAGT TA1401 CGCCTGGTTC TTTGATACAG ATGCTGAGGT CGACGTACCA GAAAAATTGG1451 ACTCGCTATT CATGGGAGAA GGTGCTGGCC TTGTTGCACA TATCACCTCT1501 AGGCCCTATG TAGGGACAGT CCCGTTAGCA GACCGGGAGT GGAATGCCCT1551 GT TGTGCATG GACTCGCAGA AGTTGTTGCA CGCAATGAGG CGCATGTTCA1601 TGAGAGGCGC TTGGGGGGCG CACATGTGCG TCATTTCCAG GGAAT TT T TG1651 CTCAAATATG TGGAGGCAAG GT TGAAATCA AGCTGTTTAA TTGCAAAGGC1701 CCGGAGAAGG GGTCAACACA AAGAGAAGCT TGAGGCATGG GAAGTCCTGG1751 GGTTGAAGAG CTCAGATGCA CTGTTTAGGG CCATGACGTA CC TGTGCAAC1801 GCGAGATTGG AGCCCATGTT CTCTGAGTCA GGCCTGAGAT TTTTCTTAAC1851 GCGCGGAAGG AATAAT C T GT ACGGCCTCAC CAATTATACA GAGGGAAAGC1901 GTGCAGTAAC TGGGGTGCAG AACCTATGGA GCAATGTGGT GCATGAGGTG1951 AGTACCAAGC GGCACAAAGG CATGATAAGG CTAGAGAAGG CCCGAGTTAC2001 AGAGCAGCCC AGAAGTGAGT TCGCAAGTTG CGTGTTAGAG CCCGAGGTAT2051 GGCGCGATGT GGAAGCTGCG CTCGATATCG AATTGGGCGA AGTTGCTTGT2101 GCTTGCAACG CACGATTCGT GCAAGGGGTG GTACTGAGCA ACCAGGCAGG2151 TCTTAATGTC CGTGAGCAAG TTGCAGGTGC TTCTGTGGGG CTGTACACGA2201 AAGATAGAAG CAACTTGAAG TGGGGTAACA GTGAGCTGCT TAGCAATGGT2251 TGGGGAAGGA GCTTGAGCGT CT GGATGGAG ATTAACTCCG TGAGCCAAAA2301 AT TTGATGTC GCCGTGCGTT TGAGTTACAG CAAGGAGACT CAAATGAATG2351 TGCTGCTGCC GAGCCTTGAT GGAATAGAAC GGGGCGCGGG CGCAACTGTG2401 GTTAATCTGC GGAAGTGTGG TGCATTCATC GTAAGGTGCG CTCGAGGGTG2451 GAGACTGGCG CTGGCGTGGA TGGACCACAT TTGTTTGGAG GTGATGGCCA2501 ACGTTGCATA CGGTCATGAA TGCTATATGA GGTCTTGGGG CACAATGGAT2551 GTTGTGGTCT TCCTGAAAAG GGCCACTGTT TCTGAGCAGG TAACTTTTGA2601 GAGTGCACAG GAGGTGGGCC CCATTGAGGG TAAGAGTGAT TCGGGGGCAC2651 CAGGAGTTGG AGTGAACCTC GACTTGGGTG GGGTCGTTGG CAGCGAGTAC2701 CCCGCCAATG GTGCTGAGCG ATATAAGCGG GTGTCTGGGC CCGGTGATGG2751 TTGCTGTTGC TGGCACAGTT TTGCATACCT AGTTGGCATG CACCACATGG2801 AGTTGAAGCG ATTGTGCACT TCTCATGTTT TTGAAAATGC CGCACTCAAT622851 GT TGAGCTGG AGCAGTGCAA GGCATCTGGC GCAT TCGT CA CTCATGCCGC2901 CATACTGGCA ACAGCTT T GA GACTCAGAGC TGAAATTAGA GTGCACAACG2951 CTGGCACAGG TAGAGTTCAT CGTTTTGCTC CCAAGCAGAA GAACATGGCA3001 CT TGATTTGT GGCTCGAGTC GGAGCAC TAT GAACCACAGG TACTTCGCAA3051 TGGTTGTGTA ATTGAATCCG TGGCACAAGC ACTGGGCACG CGGAATGCCG3101 ATATCCTGGC TGTTGTAGAA GAGCGGTGCT GTGAGGAGGT TGTTGAAAGC3151 GTGCAAGCTG GTCTTGGTCT AAATCTGCAT CATGTGGAGA TTGTGCTGCA3201 ATGTTTCGAC AT TGTAGGGC AT TGCAAC T T AGGGGATAAG GAAATCACGC3251 TTAATGCTGG TGGTAAGATG CCCTTCTGCT TCGATATCTC TGATGAACAC3301 ATGAGTTTTT GCGGACGGCG CAAAGACCCC ATCTGCAAGT TAGTAAGTGG3351 TGCATTACAC GGCAAAATGT TTGCCGAATC TGCGTTGCTA GATCTGGAGA3401 ACTGCGGCTT AAAAATAGAC TTCGAACCAA ATTGGAATCG CGCAGGAATG3451 CTCGCAGATA GCATGTAT CA AGGAGCCACA GGAGTTTTGG GTTCTGCACT3501 CTTCAATAAT AAGAGAAATA TGCGTGAGAA ATTTGTGCGT AATGTATCTT3551 TGAGCTTGCA TGCGATAGTG GGAACCTTTG GCTCTGGGAA GAGTACGCTG3601 TTCAAAAACC TACTGAAGTA TGGTGCAGGC AAATCGCTGG ATTTTGTGTC3651 ACCGAGGCGT GCGTTGGCCG AAGACTTCAA GCGTACAGTT GGGATGAACG3701 AGCGTGGCGG GAGAGCAAAA GCAGGGCAAG AGAACTGGAG AGTTACCACG3751 TTGGAGACAT TCTTAGCAAG AGTGGAATTT CTAACAGAGG GCCAGGTGGT3801 TATTTTGGAC GAGATGCAGC TGTATCCACC TGGGTACTTT GACCTAGTTG3851 TGAGTATGCT TAAAGTGGAT GTGAGACTTT TCCTCGTGGG CGATCCTGCA3901 CAAAGCGACT ACGACAGCGA GAAGGATAGA TTGGTGCTGG GAGCTATGGA3951 GGAGAACATG AGCGTCGTGC TTGGGGCACG CGAGTACAAT TACAAAGTGC4001 GGAGTCATCG GTTTTTGAAT TGCAATTTCA TAGGGAGACT TCCATGTGAA4051 ATAAATAAAG AT GAT T GCAC GAT TGATGAG CCTCACATTA TGCGCATGCA4101 CC T TGAAAAT CT TCTGGACG TGGCAGAAGA GTATAAATCT GTGGTGCTCG4151 TAAGCTCTTT TGATGAGAAA ATGGTAGTGT GCGCGCATCT CCCAGAGGCG4201 AAGGTGCTCA CT T TTGGAGA GAGCACTGGA TTAACATTCA TGCATGGCAC4251 AATTTACATC TCCGCGGTGT CAGAGAGGAC TAATGAGCGA AGATGGATAA4301 CGGCTCTCCG TCGATTTCGC TTCAATTTGT GTTTTGTGAA TTGCAGCGGG4351 ATGGATTATC AGCAATTGGC AGGGAGATAC AAAGGTCGAG TGCGGTCCAA4401 ATTCCTGTGC AAGAC TGC TA TTCCTGACGA TCTAAATAGC ATGCTGCCCG4451 GCCAAGCACT CTTTAAGAGT GAGTACCCGC GATTGATTGG TAAAGATGAG4501 GGTGTCAGAG AAGAGAAGCT TGCAGGCGAT CCATGGCTCA AAACAATGAT4551 TAATCTATAT CAAGCACCGG AGGTGGAAAT TGCAGAAGAG CCTGAGGTGG4601 TGATGCAGGA GGAATGGT TT CGCACACATT TGCCGCGTGA TGAGTTGGAG4651 AGCGTTAGAG CGCAATGGGT TCACAAGATA TTAGCCAAGG AGTACAGAGA4701 GGTGCGCATG GGAGATATGG TGTCAGAGCA ATTCACTCAC GATCACACCA4751 AACAACTGGG TGCGAAGCAA CTCACAAATG CAGCTGAGAG ATTCGAGACC4801 ATATACCCCA GGCATAGAGC TAGTGACACC GTCACTTTTC TAATGGCCGT4851 GAAGAAAAGA TTGAGCTTCT CTAACCCTGG GAAGGAAAAG GGAAACTTGT4901 TCCATGCAGC CAGC TAT GGT AAAGCATTGC TATCAGAATT CCTCAAGCGT4951 GTGCCGCTAA AGCCGAACCA CAATGTGCGG TTTATGGAGG AAGCACTGTG5001 GAACTTCGAA GAGAAGAAGC TGAGCAAAAG TGCTGCCACA ATTGAGAATC5051 ACTCTGGACG CT CATGCCGG GAT TGGCC TA CAGATGTGGC CCAGATTTTC5101 TCAAAAAGTC AGTTGTGCAC CAAATTCGAC AATAGGTTCA GGGTTGCTAA5151 AGCAGCGCAG AGCATCGTGT GTTTTCAACA TGCGGTCTTG TGTCGTTTTG5201 CGCCCTACAT GCGATACATT GAGATGAAAG TGCACGAGGT GCTGCCGAAG5251 AATTACTACA TCCACTCAGG AAAGGGTTTG GAGGAGCTGG ATGCGTGGGT5301 CAAGAAAGGG AAGTTTGACC GGATTTGCAC GGAGTCTGAT TATGAGGCAT5351 TCGATGCGTC ACAAGATGAA TTTATCATGG CTTTCGAGCT GGAATTGATG5401 AAGTACTTAA GGTTACCAAG TGATCTAATC GAGGATTACA AGTTCATCAA5451 GACTAGCCTA GGATCTAAAT TGGGCAATTT TGCTATAATG CGCTTCTCCG5501 GGGAGGCAAG CACTTTTCTG TTTAACACAC TGGCCAATAT GTTGTTCACC5551 TTTATGAGGT ACAACATACG GGGTGATGAA TTCATATGCT TTGCTGGGGA5601 TGATATGTGC GCGTCGCGAA GAT TGCAACC CACAAAGAAG TTTGCTCACT5651 TCCTAGACAA GC T TAAACTG AAAGCGAAGG TGCAATTCGT GCAATTCGTG5701 AATAAACCAA CTTTTTGCGG TTGGCACCTG TGTCCCGATG GTATATATAA5751 AAAGCCGCAA CTTGTGCTAG AGAGAATGTG CATCGCGAAA GAGATGAACA5801 ACCTGAGCAA TTGCATTGAT AATTACGCCA TTGAGGTGGC GTACGCATAT5851 AAGTTGGGGG AGAAGGCTGT GAATAGAATG GATGAGGAGG AAGTCGCGGC5901 GTTCTACAAC TGCGTGAGAA TCATAGTGCG AAACAAACAC CTCATTCGCT635951 CTGATGTGAA ACAAGTATTT GAAGTGCT TT AATTAGTGTA GCTTAGGTAT6001 TGCTATTGTA TTGAATATTT ATGGATGTGA TTGTAGATTT GTTGTATAAA6051 TACAAGTTTG AGCGTTTAAG TAATAAGT TA GTGTGCCCTA TAGTTGTTCA6101 CTGTGTGCCT GGGGCTGGCA AGAGTAGCTT AATTCGCGAG TTGTTAGAAT6151 TAGATAGTCG CT TCTGTGCA TACACAGCTG GTGTAGAGGA CCAACCAAGG6201 TTGAGCGGGA AT TGGATCAG GAAGTGGAGC GGGCAACAAC CGGAAGGCAA6251 AT TTGTGGT T CTGGACGAGT ACACTCTGTT GACTGAAGTG CCTCCGGTAT6301 TTGCATTGTT CGGTGACCCC ATACAATCGA ACACAAGCGC CGTTCAGCGT6351 GCTGACTTTG TGTGCTCAGT GAGTAGAAGA TTCGGCAGTG CCACGTGCGG6401 GCTGTTACGG GAGTTGGGCT GGAACGTTCG AAGTGAAAAG GCTGACCTGG6451 TGCAAGTATC TGATATATAC ACAAAAGACC CCCTGGGCAA AGTTGTGTTC6501 TCAGAGGAGG AAGTGGGTTG CTTGCTGAGA TCACACGGGG TGGAAGCATT6551 GAGCTTGCAG GAAATAACAG GCCAAACTTT CGAGGTGGTA ACGTTCGTGA6601 CTTCAGAGAA TTCTCCAGTG AT CAATCGAG CGGCTGCCTA TCAGTGCATG6651 ACAAGGCATC GAACGGCTCT GCACATCTTG TGTCCTGATG CCACTTACAC6701 CGCCGCCTGA CTTCACAAAG GTATACCTTT CTGCTGCACT CGGGGTGTCG6751 CTTGCTCTAG TTGTTTGGCT GC T TATAAGG AGTACACTAC CTGTGGTGGG6801 GGATAGAGAT CACAACTTGC CACACGGAGG TTGGTACAGG GACGGGACCA6851 AATCAGTGTT TTACAACAGC CCCGGCCGGC TCAACTCAAT AGAGGCTAGA6901 AAAGCTCCGC TACTTGGTCA ACCTTGGGCT ATCGTCGTCC TGCTAGTACT6951 GCTTATCTGG GCGAGTCACA AGCTAGGAAG GCCTAACTGT AGAGCCTGTG7001 CGGGCTCACA CACATGATAG TGTATGTACT TGTAGGACTG AGCGCCTTCT7051 GCATTGTGCT GTATTTGATC TCTCAGGGAC AGTCTGACTG CGTGGTGCTA7101 ATCACCGGCG AATCAGTGCG CGTGCAAGGG TGCCGAATTG ACGGTGAGTT7151 CGGAAGTGTG CTATCAAAAT TGAAGCCGTT TGGGTGTGGT TCCTTTAGGT7201 CATAAGGTGA ATCTGAAATA GTGAGTATGG GAGATTCAAC GAAGAAAGCT7251 GAAACTGCCA AAGATGAGGG CAC T TCGCAA GAAAGGAGAG AAGCGCGACC7301 ACTGCCGACT GCTGCTGACT TTGAGGGGAA GGACACATCG GAGAACACTG7351 ATGGGCGTGC TGCAGATGCT GAT GGAGAAA TGTCATTGGA GCGGAGGCTT7401 GACAGCCTCC GAGAAT T CC T GCGAGAGCGG AGGGGCGCAA TTCGAGTGAC7451 AAACCCAGGG TTAGAGACTG GCAGGCCAAG GTTGCAGCTA GCTGAAAATA7501 TGCGCCCTGA TCCCACGAAT CCGTACAACA GGCCGTCCAT AGAAGCTCTC7551 AGCCGGATCA AGCCAATCGC GAT C TCAATiC AATATGGCCA CATCTGAGGA7601 TATGATGCGC ATATAT GT GA ACCTGGAGGG GCTAGGGGTG CCGACTGAGC7651 ACGTGCAGCA GGTAGTGATT CAGGCTGTGC TATTTTGCAA GGACGCAAGC7701 AGCTCCGTAT TCCTGGATCC GCGAGGCTCG TTCGAGTGGC CAAGAGGTGC7751 TATAACTGCA GATGCCGTCT TGGCTGTGCT GAAGAAGGAT GCAGAAACAC7801 TGCGAAGGGT GTGTAGGCTG TATGCCCCGG TGACATGGAA TCATATGCTG7851 ACGCACAACG CGCCTCCGGC CGATTGGGCT GCCATGGGGT TTCAGTATGA7901 GGATCGCTTC GCTGCTTTCG ACTGCTTTGA TTACGTTGAG AATACTGCTG7951 CAGTCCAACC CC TAGAGGGA TTGATCAGGC GACCTACCCC AAGGGAAAAG8001 GTAGCTCACA ATACGCACAA AGACATCGCA GTGCGTGGAG CAAATCGCAA8051 TCAGGTGTTC AGCTCTCTCA ATGCCGAGGT CACTGGTGGT ATGAATGGTC8101 CGGAGCTCAC TAGAGAT TAT GTAAAGTCTA ATAGAAAATG AAGGACGTAA8151 CCAAGGTGGC TTTACTTATA GCGAGAGCTA TGTGTGCCTC TTCAGGTACC8201 TTTGTGTTTG AACTAGCTTT TAGTATTACT GAGTATACGG GTCGACCACT8251 TGGCGGTGGG AGATCCAAGT ACGCACGTCG TAGACGTGCT ATTAGTATAG8301 CTAGGTGTCA CAGGTGC TAT CGCCTCTGGC CCCCAACTGT GTTTACTACT8351 AGGTGTGATA ATAAACATTG TGTGCCTGGT ATCTCTTACA ATGTGCGCGT8401 GGCGCAAT TT AT TGATGAAG GAGTAACCGA GGTGATACCT TCAGTCATCA8451 ACAAGCGAGA GTAGCCAT TA AATCCTATTT AATATATAAC GTGTGCTACT8501 ATAAATAAAA CTTGGTTTTT AACTATTTTT AGCCA64REFERENCES1. 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