A study was performed to determine some properties of the potato virus M (PVM) in crude sap

and in pure preparations. In crude sap the virus was shown to have a dilution end point value of

10- 4, longevity in vitro particles sedimented as one peak with a sedimentation coefficient value of

162 s. The buoyant density in cesium chloride was 1.304 gm/cm ~ Electrophoresis in polyacrylamide

gels showed that the virus possessed only one protein subunit with a molecular weight of 39,300

daltons

Ahmad, Ismail

Stace-Smith, R

Wright, N.S

Universiti Putra Malaysia Institutional Repository

Pertanika 8(1), 73 -77 (1985)Some properties of Potato Virus M (PVM)in Crude Sap and in Pure PreparationsISMAIL AHMAD, R. STACE-SMITH* and N.S. WRIGHT*Department of Microbiology,Faculty of Life Sciences,Universiti Kebangsaan Malaysia,Bangi, Selangor, Malaysia.Key words: Potato Virus M; crude sap; pure preparations.ABSTRAKSatu kajian telah dilakukan untuk menentukan beberapa ciri virus M (PVM) kentang dalamsap kasar dan 'dalam sediaan tulen. Dalam sap kasar virus tersebut telah dz'tunjukkan mempunyainilai, titik pencairan terakhir sebanyak 10 -4, pemandinan in vitro selama 3 han, dan titikpenyanaktifan terma setinggi 65 - 70°C. Zarah-zarah virus dalam sediaan tulen mendap sebagai satupuncak mempunyai pekal£ pemendapan bernilai 162 s. Ketumpatan apungan dalam sesium kloridaadalah 1.304 g / cm ~ Elektroforesis dalam gel poliakrilamid menunjukkan virus tersebut mengan-dungi hanyasatu subunit protein yang mempunyai berat molekul setinggi 39, 300 dalton.ABSTRACTA study was performed to determine some properties of the potato virus M (PVM) in crude sapand in pure preparations. In crude sap the virus was shown to have a dilutz'on end point value of10- 4, longevity in vitro particles sedimented as one peak with a sedimentation coefficient value of162 s. The buoyant density in cesium chloride was 1.304 gm/cm ~ Electrophoresis in polyacrylamidegels showed that the virus possessed only one protein subunit with a molecular weight of 39,300daltons.INTRODUCTIONA virus identified as potato virus M (PVM)was isolated in British Columbia, Canada, andstudies on host range (Ahmad et al., 1978) andpurification (Ahmad, 1984) have been carriedout. Some properties of the virus in crude sapand in pure preparations, albeit using differentisolates, have been reported by several workersnotably Bagnall et al. (1956), Hiruki (1973) andmore recently Tavantzis (1982) and Ahmad(1984) (Table 1). However, the virus remains asone of the least characterised carlaviruses.Hence, this study aims to report some' propertiesof the virus in crude sap and in pure prepara-tions.MATERIALS AND METHODSVirusesThe PVM isolate used in this study wassimilarly maintained and purified as previouslyreported (Ahmad et al., 1978; Ahmad, 1984).Purified U strains of tobacco mosaic virusI .(TMV) for the centrifugation experiment waskindly provided by Dr. J.A. Dodds. **C~nada Agriculture Research Station, Vancouver, Canada.*(Former post-doctoral fellow, Canada Agriculture Research Station, Vancouver, Canada).ISMAIL AHMAD, R. STACE-SMOTH AND N.S. WRIGHTTABLE ISome properties of potato virus M (PVM) in crude sap and in pure preparationPropertiesParticle length (nm)Particle width (nm)Longevity in vitro (days)Thermal inactivation point (C)Dilution end pointAmax(nm)Amin(nm)A /A.max mmSedimentation coefficientBuoyant density (g/cm1Protein subunit molecular weight (daltons)Estimated RNA content in virion (%)ISelected references only.Reported values 1645 (g)2, 650 (c), 651 (a), 660 (f)13 (a, g)2 (b,c,d,e), 3 (j)60 - 65 (h), 65 -70 (b,c,e,j), 68 -71 (i)10 -3 (e), 10 -4(d,h,j)260 (a,g)245 (a,g)1.21 (a,g), 1.23 (a) .162 S (j)31.304 (j) 339,300 (j) 35 - 6 (g), 6 (j)2Sources of data: (a) Ahmad, 1984; (b) Bagnall et at., 1956; (c) Bagnall and Larson, 1957; (d) Hiruki, 1973; (e) Hario et at.,1969; (f) Rozendaal and van Slogteren, 1957; (g) Tavantzis, 1982; (h) Tu and Hiruki, 1970; (i) Wetter and Brandes, 1956;(j) present study.~ot previously reported.Physical Properties in vitroThe dilution end point (DEP), longevity invt'tro (LIV), and thermal inactivation point(TIP) were the properties of virus in crude sapbeing determined. Crude sap used for determingthese properties was extracted from infectedpotato (Solanum tuberosum L. designatedBanana selection) or tomato (Lycopersiconesculentum Mill. cv. Rutgers) plants. Extractionwas achieved by macerating' leaves of eitherplant in a mortar and then filtering it throughmuslin cloth.The determination of DEP was performedwith crude sap which has been diluted in 0.005M borate buffer, pH 7.8, to 10 -I, 10 -2, 10 -3,10 -4, 10 -5, or 10 -6 of ,the original concentra-tion. The determination of TIP was carried outas follows: Two milliliters of crude sap wereplaced in thin-walled serological test-tubes (75X 10 mm) which were then incubated for 10minutes in a water bath set at 30 C, 40 C, 50 C,60 C, 65 C, 70 C, 75 Cor 80 C. After 10 minutesthe sap was immediately cooled by plunging thetest-tubes into a beaker of ice for 30 seconds. Inthe determination of LIV, crude sap was in-cubated at room temperature for I, 2, 3, 4, or 5days.The infectivity of the virus following eachtreatment was assayed onto the primary leavesof 10-12-day-old Red Kedney been plants(Phaseolus wlgaris L. cv. Red Kidney) (Hiruki,1970). One half-leaf was inoculated with un-treated freshly extracted sap while one-half leafof the opposite primary leaf was inoculated withbuffer only. The variously treated sap was theninoculated randomly onto the remaining half-leaves. The formation oflocallesions was observ-ed 6 -12 days after inoculation.74 PERTAN1KA VOL. 8 NO.1, 1985· PROPERTIES OF POTATO VIRUS IN CRUDE SAP AND IN PURE PREPARATIONSProperties in Purified PreparationsCentrifugation experiments for the deter-mination of sedimentation coefficient andbuoyant density of the virus was carried out in aSpinco Medel E analytical ultracentrifuge.Purified virus at concentrations ranging from0.25 to 0.8 mg per ml in 0.005 M borate buffer,pH 7.8, was centrifuged at 21,740 rpm and 20 C.The sedimentation coefficient was then estimat-ed graphically (Markham, 1960) using picturesof Schlieren patterns taken at four-minute inter-vals.The bouyant density was estimated by theequilibrium method in cesium chloride(Chervenka, 1973). Test samples containing30 - 50 p..g of PVM and 436.9 mg of cesiumchloride per ml of 0.005 M borate buffer, pH7.8, were centrifuged for 12 hours at 44,770 rpmand 25 C. The actual densities of the cesiumchloride solutions were determined from theirrefractive indices which were read using a refrac-tometer. In some rotor cells, 100 ug of tobaccomosaic virus (TMV) U I strain were added toPVM samples from comparison.Polyacrylamide Gel ElectrophoresisPolyacrylamide gel electrophoresis for thedetermination of the subunit protein molecularweight was performed with gel concentrations of4% - 9%. Virus particles were dissociated in 0.1M phosphate buffer, pH 7.2, containing 4 Murea, 1% sodium dodecyl sulphase (SDS) and1% mercaptoethanol (Dunker and Rueckert,1969). Five proteins of known molecular weightswere used as standards. Bovine serum albumin(62,000); ovalbumin (43,000); alcohol dehydro-genase (29,.000); carbonic anhydrase (29,000);myoglobin (17,200). Electrophoresis was per-formed at a constant voltage of 50 V for 3 - 4hours using 0.1 M phosphate-O.l % SDS solutionat pH 7.2 as the running buffer (Weber andOsborne, 1969). The molecular weight of PVMprotein subunit was estimated graphically fromthe relative mobilities of standard and testproteins (Shapiro et al., 1969).RESULTSPhysical Properties in vitroLocal lesions were observed on Red Kidneybean half-leaves which had been inoculated withsap diluted up to 10 -4. Thus, the DEP value ofPVM in potato or tomato sap was 10 -~ In theexperiment to obtain the LIV value, virus incrude sap was infective after 1, 2, or3 days ofincubation at room temperature prior to ino·cu-ltion onto Red Kidney bean half-leaves. Virus insap incubated for 4 days perior to inoculationdid not show any infectivity indicating that theLIV value was 3 days. In another experiment,virus in sap heated for 10 min at 65 C or lowerremained infective but virus in sap heated at 70C was not infective. Thus, TIP of PVMfromtomato or potato sap was 65 - 70 C.Properties in Punfied PreparationsStudies with purified preparations showedthat the virus sedimented as one peak duringanalytical ultracentrifugation. The individualvalues of sedimentation coefficient obtainedfrom six determinations ranged from 159 S to166 S with a mean value of 162 S. The Schlierenpattern obtained from equilibrium banding ofvirus in cesium chloride showed that PVM par-ticles formed a band closer to the centre of therotor cell than did TMV particles (Fig. 1). Therange of values obtained from four individualestimations was 1.301-1.309 g/cmB with amean value of 1.304. g/cm~. Tobacco mosaicvirus which was run concurrently with PVM hada buoyant density of 1.324 g/cm ~.Polyacrylamide Gel ElectrophoresisDissociated virus moved through the poly-acrylamide gel as one band. This indicated thatPVM contained only one protein subunit. Themolecular weight of the protein subunit differedslightly when different gel concentrations wereused. The values estimated were 38,900,39,100,39,00,39,600, 39,700 and 39,800 daltons for 4,5, 6, 7, 8 and 9% polyacrylamide gels, respec-tively. The mean value for PVM protein subunitmodelucar weight was 39,300 daltons (Fig. 2).PERTANIKA VOL. 8 NO. I, 1985 75ISMAIL AHMAD, R. STACE·SMOTH AND.N.S. WRIGHl:DISCUSSIONTMV-PVM\The present study showed that the physicalproperties z'n vitro of PVM, such a DEP value of10 -4and a TIP value of 65 -70 C were similar tosome previously reported values (Table 1).However, LIV value of 3days found in this studyis slightly higher than the value of 2 days report-ed by Bagnal et ai. (1956) and Hiruki (1973).This difference is probably not important sinceslight variations in physical properties z'n vitro ofplant viruses is a common phenomenon especial-ly when experiments were performed using di£1e-rent isolates (Francki, 1980).Fig. 1. Schlieren pattern ofPVM and TM V bandsfollowing isopycnic centnfugatio1t in cesium chlon·de.6 BSAThis is the first report on sedimentationproperties of PVM. The values of 162 sand1 .304 g/~m B for the sedimentation coefficientand the buoyant density, respectively, reportedin this paper were typical properties of viruses ofthe carlavirus group (Matthews, 1981). Thesevalues indicated that PVM has an RNA contentof about 6% (Gibbs and Harrison, 1976; Sehgalet ai., 1976). The closeness of buoyant densityestimation was confirmed by the value of 1.324g/cm B obtained for the bouyant densities ofTMV U I strain are 1.325 g/cm B (Siegal andHudson, 1959) and 1.324 g/cm B (Sehgal et ai.,1970).52.0.6 0.8 1.0REL. MOBILITYMGAlthough the molecular weight of the PVMprotein subunit appeared to increase with highergel concentrations, the differences were not sig-nificant (P > 0.05). The average value of 39,000daltons was higher when compared with those ofother carlaviruses for which a range from 31,000- 36;100 daltons has been recorded (Wetter andMilne, 1981). In our tests, the PVM proteinsubunit consistently moved slower than alcoholdehydrogenase which has a molec1Jlar weight of37,000 daltons (Fig. 3). This confirms the con-clusion that the molecular weight of PVMprotein subunits of other carlaviruses.REFERENCESFig. 2. Electrophoresis of PVM protein subunitsthrough 7% polyacrylamide gel using bovine serumalbumin (BSA), ovalbumin (OA), alcoholdehydrogenase (AD), carbonic anlydrase (CA)and myoglobin (!tfc) as' protein standards.AHMAD, LB. (1984): Purification of potato virusM (PVM). Saz'ns Malaysz'a (in press).AHMAD, LB., WRIGHT, N.S. and STACESMITH,R. (1978): Potato virus M (PVM): Suscepti.76 PERTAN1KA VOL. 8 NO. I, 1985PROPERTIES OF POTATO VIRUS IN CRUDE SAP AND IN PURE PREPARATIONSbility and host range studies. Proc. Pl. Port.Con! Kuala Lumpur, 1978. pp. 93 -100.BAGNALL, R.h. and LARSON, R.H. (1957):Potato virus M (Abstr.) Phytopath. 47: 3.BAGNALL. R.H., LARSON, R.H. and WALKER,J.C. (1956): Potato virus M, S, and X inrelation to interveinal mosaic of the IrishCobbler variety. Res. Bull. Agric. Exp.Stain. Univ. Wzsh. No. 198.CHERVENKA, C.H. (1973): A Manual ofMethodsfor the Analytical Ultracentrtfuge. Back-man Instrument, Palo Alto, California.USA.DUNKER, A.K. and. RUECKERT, R. (1969):Observations on molecular weight deter-mination on polyacrylamide gel.]. Biol.Chem. 244: 5074 - 5080.FRANCKI, R.I.B. (1980): Limited value of thethermal inactivation point, longevity in vitroand dilution end point as criteria for thecharacterization, identification and classifi-cation of plant viruses. Intervirology. 13:91- 98.GIBBS, A. and HARRISON, B. (1976): Plant Viro-logy: The Prindples. Edward Arnold LTD,London. p. 121.HIRUKI, C. (1970): Red Kidney bean, a success-ful bioassay host for quantitative qualitativework with potato virus M. Phytopath. 60:739 -740.HIRUKI, C. (1980): Detection of potato virus Min tubers, sprouts, and leaves of potato byFrench bean test. Potato Res: 16: 202 - 212.HORIO, Y., YANO, I. and ESUMI, K. (1969):Potato virus M found in Japan'. Ann. Phyo-path. Soc. Japan. 35: 47 - 54.MARKHAM; R. (1960): A graphical method forrapid determination of sedimentation co-efficient. Biochem.]. 77: 516 - 515.MATTHEWS, R.E.F. (1982): Classification andnomenclature of viruses. Intervirology. 15:1-199.ROZENDAAL, R and V AN SLOGTEREN, D.H.M.(1957): A potato virus identified with potatovirus M and its relatIonship with potato virusS. Pro. 3rd. Con! Potato Virus Diseases.Lisse-W ageIiingen, pp. 20 - 37.SEHGAL, O.P., JONGHo, J., BHALL, R.B.,SOONG, M.M. and KRAUSE, F.F. (1970):Corellation between bouyant density andribonucleic acid content in viruses. Phyto-pathology. 60: 1778 -1783.SHAPIRO, A.L., VINULA, E. and MAIZEL, J.V.(1967): Molecular weight estimation of poly-peptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophs. Res.Comm. 28: 815 - 820.SIEGEL, A. and HUDSON, W. (1959): Equili-brium centrifugation of two strains oftobacco mosaic virus in density gradient.Biochim. Biophs. Acta. 34: 3514-3515.T AVANTZIS, S.M. (1982): Improved purificationof two potato carlaviruses. Phytopath. 73:190 -194.Tu,J.C. and HIRUKI, C. (1970): Ultrastructureof potato infected with potato virus M.Virology. 42: 238 - 242.WEBER,K. and OSBORNE, M. (1969): The relia-bility of molecular weight determinations bydodecyl sulfate-polyacrylamide gel electro-phoresis.]. Biol. Chem. 224: :M06 - 4412.WETTER, C. and BRANDES, J. (1956): Unter-suchungen uber das K.{rtoffel-S-Virus.Phytopath. Z. 26: 81- 92.WETTER, C. and MILNE, R.G. (1981): Carla-viruses. Pages 696 - 730 in: Handbook ofPlant Virus Infections and· ComparativeDiagnosis. E. Kurstak, ed. Elsevier/North-Holland Biomedical Press, Amsterdam.(Received 27 September, 1984)PERTANlKA VOL. 8 NO. I, 1985 77

Some properties of Potato Virus M (PVM) in Crude Sap and in Pure Preparations

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Some properties of Potato Virus M (PVM) in Crude Sap and in Pure Preparations

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