Isolation and characterization of barley stripe mosaic virus protein

36, 87-93

VIROLOGY

Isolation

and

(1968)

Characterization

of Barley

D. J. GUMPP Departrnenf

of Rotan;/

and Microbiology, Accepted

Stripe

Mosaic

Virus

Protein’

R. I. HAMILTON3

AND

Monfana February

State

University,

Bozernan,

Montana

17, 1968

Degradation of barley stripe mosaic virus (BSMV) by calcium chloride yielded a nucleic acid-free protein component suitable for characterization. Immunodiffusion patterns of BSMV-protein showed several antigens: a slowly diffusing major antigen showing partial identity with BSMV, and two faster diffusing minor antigens identical to those formed on the degradation of BSMV or BSMV-protein with Leonil SA, an anionic detergent. These latter two minor antigens were identical to nonsedimentable virus-related antigens present in the crude sap of BSMV-infected barley. BSMV-protein above pH 6.3-6.4 has an electrophoretic mobility two-thirds that of BSMV. At pH 6.0 electrophoretic mobilities of the protein were indistinguishable from those of the intact virus. This increase in electrophoretic mobility was correlated with a polymerization of the protein into viruslike rods as observed by electron microscopy.

barley in t’he presence of 0.5 and 1.0 % Leonil SA (sodium dibutylnaphthalenesulfonate), an anionic detergent. This same front precipitation band was also observed by immunodiffusion of purified BSNV in gel containing the detergent (Hamilton and Ball, 1966). An increase in prominence of t#he front precipitat’ion band upon immunodiffusion was paralleled by a decrease in the prominence of the rear precipitation band (Hamilton, 1964). According to Hamilton and Ball (1966), BSRV was degraded by Leonil SA into faster diffusing virus-related ant’igens. Degradation of BSMV by dialysis against molar calcium chloride yielded an nntigenic protein fract’ion (Hamilton, 1965). This provided an opportunity to study some aspects of the structure and propert,ies of BSRIV by a partial characterization of the virus protein. A preliminary report of t,his work has been published (Gumpf and Hamilton, 1966).

INTRODUCTION

Much of the information pertaining to barley stripe mosaic virus (BSMV) is concerned with the serology of the virus and its related antigens. Immunodiffusion of purified BSRIV resulted in t,he formation of a precipitation band composed of two precipitation lines at t’he border of the ant’igen depot’ (Ball, 1961). ,4 front precipit,ation band of nonsedimentable antigens, analogous to Xprotein found in sap of ThlV-infected leaves, was observed in the immunodiffusion of sap from BSMV-infected barley (Hamilton and Ball, 1966). Hamilton (1964) reported the presence of a prominent front precipitation line upon immunodiff usion of sap from BSn IV-infected 1 Montana Agricultural Experiment Station Journal Series, Paper No. 834. This investigation was supported by Grant GM 11192-03 from the National lnstitute of General Medical Sciences, National Institutes of Health, Public Health Service. 2 Present address: Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska. 3 Present address: Department of Plant Pathology, Macdonald College of McGill University, Hte. Anne de Bellevue, Quebec, Canada.

MATERIALS

ANl>

METHODS

Preparation of az+us. A ?\lontnna isolate of BShlV derived from a single local lesion on Chenopoclium amaranticolor I,. aft,er isolation from Hordeuw awlgare I,. var. Vant,age? 87

ss

GUMPF

AND

was used t,hroughout this investigat,ion. The virus was previously shown to sediment in the ult’racentrifuge at t,he same rate as the type strain of BSMV (AC 69, obtained from the American Type Culture Collection) and to react wit#h antiserum to t’he same strain. BSMV was cult,ured in barley (Horcleulrz vulgare L. var. Blackhulless), in a greenhouse maintained at about 20”. Young plants at the three leaf stage were inoculated by rubbing the leaves with steam-sterilized cheeseclot’h pads soaked with an extract from BSMV-infected leaves containing Celit,e4 as an abrasive. Infected leaves of these plants were harvested 2 weeks after inoculation. The leaves were homogenized in a food chopper and were stored at - 10” when not’ immediat’ely used. The virus was isolated and purified as described by Hamilton and Ball (1966). Isolation of protein. BSMV-protein free of nucleic acid was obtained by a modificat’ion of Yamazaki and Kaesberg’s (1963) method. Purified virus, suspended in 0.02 n/r tris- (hydroxymethyl)aminomethane (Tris)0.0064 171 citrate, pH 6.5, was dialyzed against 1 M CaClz buffered at pH 6.3 with 0.02 M Tris-0.0064 111 citrate, at 4” for 12 hours. After dialysis the precipitated virus nucleic acid was removed by centrifugation at 20,000 g for 30 minutes. The supernatant fluid, which contained almost exclusively protein, was dialyzed against several changes of 0.02 Al Tris-0.0024 M HCl buffer at pH S.5 for 24 hours to remove the calcium and chloride ions. During the early stages of dialysis the protein precipitated, but went back into solution with continued dialysis. The ions were removed, then the prot’ein solution was centrifuged for 1.5 hours at 144,SS0 g to remove any undegraded virus. The supernatant fluid was used as the purified protein preparation. Concentrations of protein were determined spectrophotometrically with a Beckman Model DU spectrophotometer with a l-cm light path. An absorbance of 1.7 at 280 rnp was equivalent to 1 mg/ml of BSMV-protein. Protein concentrations were det’ermined using the Folin phenol reagent 4 Diatomaceous Mnnville Company.

silica

manufactured

by Johns-

HAMILTON

(Lowry et al., 1951), biuret (Gornall et cd., 1949), and dry weight determinations. Production of antisera. Purified virus and protein preparations diluted with 0.01 M pot’assium phosphate&.14 M NaCl, pH 7.0, to a concentration of 1 mg/ml of virus5 and protein, respectively, and emulsified w&h an equal volume of Freund’s complete adjuvant, were injected into rabbits. Three intramuscular injections of 0.5 mg, 0.5 ml per hind leg, were given at 7-day intervals. Serum was collected on the second and third week after the final injection and stored at -10”. Serum titers mere determined by the microprecipitin method (van Slogteren, 1954) using both BSMV and BSMV-protein at 0.5 mg/ml as the constant antigen concentration. Tit’ers of BSMV antiserum and BSMV-protein antiserum were 1:512 and 1: 128, respectively. Immunodiffusion patterns of the BSMV antiserum gave better resolution against both BSMV and BSMV-protein than did the BSMV-protein antiserum. Amino acid analysis. BSMV-protein samples were hydrolyzed with about 100 times their weight of constant-boiling HCl at 110” in a vacuum for 24 hours. To remove the HCl, the samples were lyophilized and then redissolved in small amounts of distilled water and lyophilized again. This process was repeated Ohree times. Two samples of the hydrolyzates weighing 0.55 mg and 0.67 mg, respectively, were dissolved in 1 ml of distilled water and analyzed on a Technicon automatic amino acid analyzer. Electron microscopy. Purified virus and protein suspensions at concentrations of 1 mg/ml were dialyzed at room temperature against 0.1 M potassium phosphate buffers at pH 6.0, 7.0, and 8.0 for 2 hours. Following dialysis the protein preparations were made 1% with respect to sodium azide. All preparations were then combined with equal volumes of 2 % phosphotungstic acid, adjust,ed to the appropriate pH with 2 N XaOH, and incubat,ed for 2-5 minutes with 5 Virus concentration was determined by relating absorbancy at 260 rnp of a range of BSMT’ concentrations obtained gravimetrically. An absorption of 2.8 at 260 rnp in a l-cm light path was equivalent to a concentration of 1 mg/ml.

BARLEY

STRIPE

MOSAIC

shaking at room temperature. The suspensions were then sprayed on grids coated with collodion film, examined and photographed in a Siemcns Elmiskop I electron microscope at a magnification of 20,000 diameters. Serological studies. Immunoelectrophoretic analyses of BSRlV and BS:\IV-protein were done using a glass photographic plate coated with a film of l’ormvar and bordered by a %-mm layer of masking tape as a support, for t,he 1% agurose bed. Electrical continuit,y between the agarose and buffer solut,ions was maintained with filter paper s:trips. Circular wells, 7 mm in diameter, \vere cut, in the agarose bed with a cork borer, and the wells were filled w&h t,he ant’igens. Buffers used were 0.01 pot’assium phosphate wit’h 0.02 AI potassium chloride, at, pH 6.0, 7.0, and S.O. The buffers used as the agarose solvent contained 0.02 % sodium azide. ,411 anCgens were dialyzed 2 hours prior to electrophoresis in the electrophoresis buffers at room t#emperature, and t,he pH 6.0 protein preparations n-ere made 1% wit#h respect t’o sodium azide. El&rophoresis was done at 4” with a current of 5 mamp per slide, giving a potent’ial of about’ 7 volts/cm for 4 hours. Aft,er electrophoresis had been concluded, a trench \v:ts cut in the agarose bed parallel t,o the direction of current, flow and filled with undiluted antiserum. Identification of the transported antigens was facilitated by setting up an immunodiffusion system in the agarose bed on a straight line with, and at various dist’ances from the origin. The wells were then charged with t,he same ant’igens as t,hose t#hat, were subjected t)o elect’rophoresis. The plates were incubated at 20” in a humid environment. Migration distances were determined by measuring the distance between the origin and the arc formed by t#he immunoprecipitin lines. Immunodiffusion analyses of BSMV, BSMV-protein and BSMV-related antigens was done by the double diffusion met#hod (Ouchterlony, 1958). &uantit,ies of the same 1% agarose gel used for immunoelect,rophoresis were used for t#he diffusion medium. Antigens were placed in wells arranged around a central well filled with urldiluted antiserum. The diffusion systems

VIRUS

s9

PROTEIN

were incubated ment .

at 20” in a humid

environ-

RESULTS

Ultraviolet Absoqdion

Spectra

BSRIV had an ulkaviolet absorption spectrum with a maximum bet,n-een 260 and 270 mp, a minimum betn-een 250 and 255 mp, a 260:280 rat,io of 1.0-1.2, and a max: min ratio of 1.02-1.20. B&\IF’-protein had an ultraviolet, absorpt’ion speckurn typical of protein free of nucleic acid with a maximum between 27s and 2SO rnp, a minimum at, 250 rnp, a 260:280 ratio of 0.47-0.57, and a max:min ratio of 2.1K1.43. Amino ,Icirl Snalysis The partial amino acid composit,ion of BSMV-protein is presented in Table 1. These results are an average of t,wo separate determinations. Xo analyses were performed to determine the concentrat’ions of trypt’ophan, or cyst,ine and cysteine, which are destroyed and oxidized, respectively, by acid hydrolysis. The values given for serine and t#hreomne were not exkapolated to zero hydrolysis time. Electwn d~ic~~oscopy Elect,ron micrographs of BShlV and BSJIV-protein are shown in Fig. 1. It was TABLE AMISO

1

ACID COMPOSITION OE’ BSMV-.PROTEIN pi) OF 130.97 pi OF TOT.IL RECOVERED AMIXO ACIDS

Amino

acid

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isolellcine Leucine Tyrosine Phenylalanine Ammonia Lysine Histidine Arginine

PS

Percent

18.11 5.M 5.10 li. 06 G.45 3.57 lO.G5 8.03 1.20 13.71 5.11 5.12 2.68 5.85 3.41 15.33

13.83 4.32 3.89 13.18 4.02 2.73 8.13 6.13 3.21 10.47 4.15 3.91 2.05 4.47 2.60 11.71

(IN

90

FIG. 1. BSMV and BSM\‘-protein at pH 7.0. (2h) Polymerized BSM\* ing “stacked-disk” appearance.

GUMPF

AND

in negative protein at pH

HAMILTON

contrast 6.0. (2~)

evident that at pH S.0 the purified BSMVprotein wax in small disk-shaped particles, while at pH 6.0 the protein had polymerized into either smoot)h viruslike rods or loose “stacked-disk” rod forms, both of indefinit,e lengths. The m+esenceof a darklv stained central zone extending the length of t,he virus and polymerized protein forms, is evidence for a hollow central core. Existence

with 2y0 phosphotlmgstic Polymerized BSMY-protein

acid. (2a) BSMV at pH 6.0, show-

of this central core in the polymerized protein would indicate an orderly arrangement of the prot#ein upon polymerization. Serological Conapayison of BXMV BSM V-protein

and

Inzmunocliffusion. BSAIV produced two precipitin lines in a major precipitin band close to the antigen depot, (Fig. 21. BSMV-

BA4RI,EY

STRIPE

MOStlIC

FIG. 2. Immlmodiffusion patterns of BSMT’ alld BS;11\7-proteill at pll 7.0. Well 1, BPIv~\~protein; well 8. plwified BSM\.. Well 3 was filled with lmdiluted BSM\. antiserum. a, BSRI\~-protein major alrtigen; b alld c, BSM\‘-protein minor antigell; tl, BSiM\. major alltigell.

protein produced a major precipitin band close to t’he antigen depot and two minor precipitin bands midway between the antigen and antiserum depots (Fig. 2). Hooking of t’he major BSAIV-protein precipitin band into the BSMV precipitin band indicates a partial identity of antigens displaying similar serological characteristics. The minor BSMV-protein antigens were not, fourld in purified BSMV preparations, but, were always present in the protein preparations used in these experiments. Incubation of BSMV or BSRIV-protein with 0.5 ‘55 Leonil SA at room temperature resuked in the formation of t’wo precipitin bands. The band formed closest to the antiserum well was composed of two precipitin lines. The t,wo precipitated antigens were identical to the minor BSMV-protein antigens and the antigens derived by Leonil SA degradation of BUN. The curvature of the major precipitin bands bordering the BSAIV-protein wells was a function of antigen concentration and pH. In these serological invest,igations the ant,igen concent,rations were the same for each system. The difference in curvature observed for the major BSMV-protein antigen was therefore a function of the pH. The degree of curvature of the precipitin bands t’oward the antigen depot was greater at pH 6.0 than at pH 7.0. The pH also affect,ed t,he diffusion rate of BSMV-protein. At pH 7.0 the protein diffused a measurable diskmce from t,he antigen depot, but at

VIRITS

PROTEIN

91

pH 6.0 it remained at the border of the antigen well. The observed increase in curvature and decrease in diffusion rnte at pH 6.0 resulted from increased antigen size caused bv polymerization of the prokin. BSnIV and “its related antigens displayed identical immunodiff usion patterns at pH 7.0 and S.0, except for less curvature of the major components of BS:\IV and BSMV-protein at, pH S.O. The serological comparison suggested the following classification of the antigens: the major BSRIV-prot,ein antigen is partially identical or similar to t#he major BSi\lV antigen found in purified BSi\IV preparations and in crude sap from BSRIV-infected plank; t’wo minor BSI\ IV-protein antigens, found as components of BSnIVprotein preparations derived by calcium chloride degradation of BS1\IV, are identical to minor nnt,igens found in crude sap from infected plants, or to minor antigens formed by 1,eonil SA degradat,ion of virus or virus protein. Immunodiff usion patterns using BSh IVprotein ant’iserum were identical to t,hose observed with BSJIV antiserum. BSMV antiserum was used solely because it allowed for bet’ter resolution of the antigen mixtures. Inzn~unoelect~op~~~esis. BSMV antiserum was used in the immunoelect~rophoretic analyses of BSMV and BSI11V-protein. BSJIV and t’he BSMV-protein antigens at pH 6.0, 7.0, and 8.0 migrated anodically when subject’ed to electrophoresis. Figures 3 and 4 show immunoelectrophorctic patterns and illustrat8e the method of antigen identification. BSlUV and BSMV-protein migrat)ed at the same rate at pH 6.0 (Fig. 3) ; at pH values above 6.3-6.4 the major B&\IV-prot’ein ant,igen migrated at a rate of about two-thirds that of B:\LSV (Fig. 4). The minor BSMV-protein ant’igens migrated identically, at a rate slightly less than that of the major BSJIV-protein antigen. Incubat’ion of BSnlV or BSRIVprot’ein with 0.5 % Leonil SA resulted in the formation of two antigens, identical serologically to the minor :ant)igens of BSMV-protein, which exhibited a high mobility rat’e independent of pH between 6.0 and 8.0.

92

GUMPF

AND

FIG. 3. Immunoelert rophoresis patterns at pH 6.0, of BSRI\-, BHM\‘-protein, and antigens formed by the degradation of BSM\. or BSM\‘-protein with 0.57; Leonil SA. Well 1, purified BSMJ’; wells 2 and da, BSMTor BSM\--protein with 1.0% sodium azide. The trenches were filled with undiluted BSMV antiserum. a, BSMY-protein major antigen; b and c, BSMV-prot.ein minor antigens.

FIG. 4. Immunoeleetrophoresis patterns at pH 8.0 of BSM1-, BSM\.-prot,ein, and antigelis formed by t,he degradation of BSM\and BSMY-protein with 0.5:/; Leonil HA. Wells 1 and Ia, purified BSMV; wells 2 and da, BSMV-protein. The trenches were filled with uudiluted BSMV antiserum. a, BSMV-protein major antigen; 6 and c, BSMY-protein minor antigens. DISCUSSION

Only calcium chloride degradation of BSMV was used in this investigation be‘cause it was simple to perform. Ultraviolet absorption data of BSMV-protein isolated by this method indicated that it was free of contaminating nucleic acid and it proved satisfactory for the work. There was no indication that BSAIVprotein possessed a particularly unique

HAMILTON

amino acid composit’ion which would int’erfere with polymerizat.ion. BSMV-protein had a high aspartic and glutamic acid content which undoubtedly confers a negative charge to the protein at pH values 6.0, 7.0, and 8.0. This conclusion was supported by the high anodic mobility of BSMV and BSAIV-protein during electrophoresis. Electron micrographs of the protein at pH 6.0 showed two polymerized forms: a smooth rodlike form and a loosely polymerized “stacked-disk” form. Both types of polymerization have been reported for TMV-protein (Takahashi and Ishii, 1952, 1953; Franklin and Commoner, 1955). It should also be mentioned that at, a pH above 6.3-6.4, or n-hen polymerization had not occurred, BSAIV-protein was in the form of disks. This was also observed with nonpolymerized TJLV-protein (Takahashi and Ishii, 1952). Shortly after complet,ion of this work, the observations of Kiselev et al. (1966) were brought to the attention of the investigators. They likewise report’ed the repolymerization of BShIV-protein into disks, rodlike stacked disks, and helical rods. The antigenic components of BSAIV and BSA’ZV-protein reacted with both BSSIV antiserum and BSAIV-protein antiserum. The major BSXIV-protein antigen, present very close to the antigen depot, had reactions of partial identity with the major BSMV antigen present in purified BSSIV and in sap from BSYIV-infected plants. Reactions of parGal identity rather than reactions of complete identity behween BSMV and BSAIV-protein could result from an incomplete or imperfect polymerization of the protein. Minor antigens, whet~herin sap from infected plants, formed by calcium chloride degradation of BS\IV, or formed on degradation of BSMV or BSMV-protein with detergent, diffused rapidly and were precipitated in front precipitation bands about half way between the antigen and antiserum depot,s. These rapidly diffusing antigens appeared t,o be the same as the nonsedimentable ant’igens observed in extractas of BSMV-infected barley (Hamilt’on and Ball, 1966): which

BARLEY

STILTPF:

MOSAIC

in turn are analogous to the low molecular \veight, TAN X-protein (Takahashi and Ishii, 19.53). I3SAIVprotein, when in the nonpol\,mwized form had an elect)rophoret,ic mobility less t,han that of BSA\IV. This same electrophoretic behavior has been previously noted bet’\veerl TJLV and TAN-protein. Kramer :~nd Wittman (1958) reasoned that the prot,ein fragments obtained on degradat’ion of Tl\IV are not uniformly charged, the part forming the outside wall being more negatively charged than that forming the inside I\-all. On degradation both sides would be exposed t.o t,he medium which indicat,es t.h:tt, t.he less negat,ively charged inside ~~11 would effectively negate some of the outside ~~111 charges, thcreblv reducing electrophoretic mobility. When BSAIV-protein is polymerized, the mobilities of the virus and prot,ein are the same. The protein in thi:: stnt#e has the same surface pot’ential as the intact virus. From this it is evident that t,he virus nucleic acid plays 110 role in the determinat.ion of surface potent,ial. L\Iinor ant,igens, derived by calcium chloride degr:tdat,ion of BSMV, had lowr mobilit,ies t.hau either t.he virus or t,he major protein wtigen regardless of pH. Antigens formed by Leonil SA degradation of BSALV or BSAiV-protein were unique in having t’he highest mobility of an>- of t,he components. This mobility n-as the same at, all pH values. Putnam (19-2s) reported t’hat anionic detergents wmbine wit,h plant virus proteins clw,ving protein molecules and remaining :w a complex, J\-hich \\-ould then tend to increase anodic mobilit\-. This was t)he case \vith HSMV-protein. Leonil SA degraded both BSAW and BSMV-protein int,o two identical a11 tigeri complexes, serologically to the tlvo minor antigens found in sap from BSRIV-infected barley leaves and as componcnt,s of BSAIV-protein, but, with greatly increased clectrophorctic mobility. These minor antigens possessed the same electrophorctic mobilities, but their differing diffusion rat,es made them detectable. REFERENCES E. M. Idrlltificatioll Ph,vt.opathology

B.\LL,

(1961). “Serological Tests for the of Plant \.iruses.” American Society. Ithaca, New E-ark.

\.IRUS

PROTEIN

93

FR.\NIetermination of serum proteins by means of the Biuret reaction. J. Riol. Chem. lii, 751-7M. GUIVWH, I). J., and H.\.\IILTOS, R. I. (1966). Serological strtdies of barley stripe mosaic virus protein. Phytopalhology 36, 879 (Abstract). II.ZMILTON, R. I. (196l). Serodiagnosis of barley stripe mosaic facilitated by detergent,. Ph!ltopalhology 54, 1290-1291. H.~I\IILTOPIT, R. I. (1965). Isolation and properties of barley stripe mosaic virus protein. Phylapaihology 55, 1060 (Abstract). H.QWI.TON, R. I., aud Bar,~, E. M. (1966). Antigenie analysis of extracts from barley infected with barley stripe mosaic virus. Virology 30, 661-672. KISELEV, N. 8., B~al~~~,cou, I. G., K.\FTINOV:\, A. S., and Nov~rcov, 1.. K. (1966). A study of virus prot,ein repolytnerization and resynthesis of some rodlike viruses. Riokhimiya 31, 670-678. KRAMER, E., and WITTMAN, II. G. (1958). Electrophoretisohe ITntersuchungen der A-Prot.eine drier Tabakmosaikvirus Stiimme. X. :\-n//uforsch. 13b, 3&33. LWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement, with the Folio phenol reagent. J. &id. Chem. 193, 265-275. OCCHTERLONY, 0. (1958). I)iffusiori-irl-gels methods for immunological analysis. In “Progress in Allergy” (P. Kallos, ed.), T-01. 5, pp. l-78. Karger, Basel. PUTNAM, F. W. (1948). The interactions of proteins and synthetic detergents. B&an. Z’rotein Chem. 4, 79-122. TAKAH.~SHI, W. N., and ISHII, M. (1952). The formation of rod-shaped particles resembling tobacco mosaic virtzs by polymerization of a protein from mosaic-diseased tobacco leaves. Phytopathology 42, G9&691. TAKAH.%BHI, W. N., and ISHII, %I. (1953). A macro molecular protein associated wit’h tobacco mosaic virus infection: Its isolation and properties. Am. J. No(any 40, 85-90. v.1~ QLOGTEREN, 1). II RI. (1954). (E. Streugers et al., eds.), pp. 51-54. In “Proceedings of the Pecolld Conference on Potato \?rus I)iseases” \-eenman & Zonen, Wageniugen. ~.LM.U.LICI, II., and K.YESBERG, P. (1963). Isolation and characterization of a protein subunit of broad bean mottle virrls. d. Mot. Viol. 6,405373.