Comparison of Tymovirus Capsid Proteins in SDS-Polyacrylamide-Porosity Gradient Gels after Partial...

Phytopath. Z., 100, 347.-355 (1981)© 1981 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 / InterCode: PHYZA3

Institut fur Viruskrankheiten der Pflanzen und fur Biochemie,Biologische Bundesanstalt,

D-3300 Braunschweig, Bundesrepublik Deutschland

Comparison of Tymovirus Capsid Proteinsin SDS-Polyacrylamide-Porosity Gradient Gelsafter Partial Cleavage with Different Proteases

By

R. KOENIG, H . FRANCKSEN and H. STEGEMANN

With 2 figures

Received July 21, 1980

Capsid proteins of tymoviruses have heen compared hy means of sero-logy of intact viruses (KOENIG 1976, KOENIG et al. 1979) and by computeranalyses of the amino acid composition (PAUL et al. 1980). Both methods havetheir limitations. Serology can compare only surface regions of proteins. Dis-similarities occurring in the hidden regions inside the native proteins areregistered only if they influence the surface structure. Amino acid analysis,on the other hand, cannot take any sequential and structural features intoaccount. In the present study we have chedied for tymovirus capsid proteinsthe usefulness of a new method (CLEVELAND et al. 1976, STEGEMANN 1979),i.e. the comparison of SDS-PAGE patterns of protein fragments (poly-pep-tides) which are ohtained after partial digestion with different proteases inthe presence of SDS. We found that the separation of poly-peptides can hegreatly improved hy the use of SDS-PoroPAGE.

Materials and Methods

Material

The following tymoviruses were used whidi were purified by the bentonite methoddescribed by DUNN and HITCHBORN (1965) for turnip yellow mosaic virus (TYMV): several

Abbreviations: AA = Acrylamide; PAGE = Poiyacrylamide gel electrophoresis;PoroPAGE = Porosity gradient-PAGE; SDS = Sodium dodecyl sulfate.

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348 KOENIG, FRANCKSEN and STEGEMANN

strains (KOENIG et al. 1979) of Andean potato latent virus (APLV), eggplant mosaic virus(EMV), okra mosaic virus (OkMV), clitorea yellow vein virus (CYVV), TYMV, belladonnamottle virus (BMV), dulcamara mottle virus (DMV), Ononis yellow mosaic virus (OYMV)and Scrophularia mottle virus (ScrMV).

Papain (order-No. A-4762) was obtained from Sigma (Sigma Mundien, D-8021 Tauf-kirdien), Thermolysin (No. 161585) from Boehringer, D-6800 Mannheim, Staphylococcusaureus V8-protease (No. 36-900) from Miles Laboratories, Lyoner Strafie 32, D-6000 Frank-furt. SDS was purdiased from Serva, D-6900 Heidelberg.

Methods

Electrophoresis

Electrophoresis was done in slabs (3 X 226 X 180 mm) in the Mono-Phor or the Panta-Phor apparatus (Labor-Muller, D-3510 Hann. Munden). SDS-PAGE was performed accordingto the method of KOENIG et al. (1970) in 7.5 % gels made from Cyanogum® in 0.025 M sodiumphosphate buffer pH 7.1 containing 0.1 % SDS.

SDS-PoroPAGE was done according to the method of MARGOLIS and KENRICK (1968)in 10 to 25 % PAA gels in 0.125 M Tris/borate buffer pH 8.9 containing 0.1 % SDS. Themonomer was composed of acrylamide (AA) and N,N'-methylene-bisacrylamide (5 % based onsolid AA) which had been recrystallized from diloroform and acetone, respectively. Therunning time was 17 hours at 270 to 290 volts. Experimental details are described in the in-structions for the apparatus by STEGEMANN (1980), whidi will be sent on request.

Preparation of viral capsid protein subunits (protomers)

Virus preparations with roughly 2 to 10/(g virus per ml were first dialyzed against0.125 M Tris/HCl buffer pH 7.6 containing 0.1 % mercaptoethanol. Then, 1 to 2 ml virussuspension were heated in standard test tubes for 17 minutes in a boiling waterbath with anequal volume of a solution of 1 % SDS and 1 % mercaptoethanol in 0.125 M Tris/HCl bufferpH 6.8 to yield the protein subunits. The samples were then stored at —20 °C.

Enzymatic splitting of viral capsid protein subunits

For proteolysis aliquots of the samples pretreated as mentioned above were incubatedfor 150 minutes at 37 °C with varying amounts of either papain, thermolysin or V8-protease(Table 1).

Proteolysis was stopped by boiling the samples for 5 minutes. Finally, 25 % (V/V) ofa solution containing 50 % sucrose and 0.01 % amido black was added.

Ten to 30,MI of the preparations were applied in SDS-PAGE, twice the amount wasused in SDS-PoroPAGE. If needed, equal volumes of individual preparations of the sameprotein treated with different amounts of enzyme were mixed to include all individual poly-peptide intermediates (Fig. 2) some of whidi may be seen only at a certain concentration ofan enzyme (Fig.l).

Table 1Concentrations of enzymes used with aliquots of viral capsid protein subunits

for partial proteolysis

Final concentration (/<g/ml)Enzyme *̂ I . I I j

a*) b c d

PapainThermolysinS. aureus V8-protease

0.0150.03.0

0.0500.59.0

0.1501.5

30

—5

10015

300

*) Code letters a to e as used in Fig. 2A, B, C.

Comparison of Tymovirus Capsid Proteins 349

Results and Discussion

Cleavage of capsid proteins

The buffer system 0.125 M Tris/HCl used by CLEVELAND et al. (1976)for proteolysis of test proteins (bovine serum albumin, alkaline phosphataseand tubulin) proved to be useful also for viral capsid protein subunits. Boratebuffers are not suitable due to complexing with trace elements. Since theoriginal virus preparations were stored in 0.05 M sodium citrate pH 7.0, whichinhibited thermolysin and caused protein subunits to form oligomers duringstorage, a dialysis step prior to enzymatic splitting was necessary. The pH ofthe dialysis buffer was adjusted to 7.6 to include the optimal range fordigestion also for thermolysin (pH 7—9). This enzyme was introduced by usfor the comparison of different potato proteins (STEGEMANN 1979). Theslightly higher pH as compared to that of CLEVELAND'S mixture did notnoticeably influence the splitting by papain and the S. aureus V8-protease.The addition of 0.1 % mercaptoethanol to the dialysis buffer prevented theoligomerization of capsid protein subunits without inhibiting proteolyticreactions. The reactivity of papain was activated.

The digestion rates for all enzyme/virus combinations were first dieckedby SDS-PACE as in Figure 1. Since SDS-PoroPAGE allowed a much better

Fig. 1. Poly-peptides of APVL-Col-2 capsid proteins after cleavage with Staphylococcus aureusV8-protease separated in SDS-PAGE. Equal amounts of virus protein were digested withincreasing enzyme concentrations. Final concentration of enzyme were 0, 1, 2, 4, 8, 16, 32, 64and 128 |ttg/ml sample (slot 1—9, left to right). Molecular weight marker proteins: Phos-phorylase b (97000), serum albumin (67 000), alcohol dehydrogenase (37 000), diymotrysinogen

A (25 700), cytochrome c (14 300) (slot 10)


2-A Protease S. aureus V8

virus Strain

group

Strain Mix

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

ScrMV

OYMV

DMV

rBMV <

ICYW

OkMV

EMV <

I

APLV <

Enzyme

1

^*

t

Al

pHud)

Hu-^" < Hu

[Ay

CCC <

1

Col- ^Caj

-

Col-3

Col-2

Caj

Bo-15

Ec-1

Col

i;

11

IIII

i1

111

11111111

. 1I

j l1

_ 1

1

1

111

11IIsu

1 il t li« I If

I t !It1

IIi l1II

1 A1 l|IIIInnII

ab

ab

e

d

d

d

e

e

c

de

de

d

ab

ab

b

b

ab

ab

+ In Fig. 2-C TYMV

++ Two different preparations of APLV-Hu

Fig. 2. A, B, C: Separacion in SDS-PoroPAGE of poly-peptides obtained from tymovirus capsidproteins after partial cleavage with S. aureus V8-protease, thermolysin and papain. For detailsof cleavage procedures and electrophoresis see Methods. Code letters in column "Mix" cor-


2- B Thermolysin

Mix

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

1W

11

t

i

i

aI1) J

11

11 fw '

7

1

fl

US111Ifi

it

IUlM

Cf

bde

bde

bde

abe

bee

abe

be

bee

bee

bee

bede

ed

bee

bee

bee

abee

bee

18 II ttf bee

2-C

13

1i 1

11•1111

11II

u111

Papain

)

n "II\IIH0i•

II1 1

1111(1

OHnmmin

'Mix

' b e

! abe

iabe

ae

be

be

be

abe

be

be

abe

be

be

be

be

be

abc

abe

respond to those in Table 1 and indicate the enzyme concentrations in the single digestionpreparations which have been mixed for a better representation of intermediate digestionproducts. Only the lower parts of the gels are shown representing the molecular weight rangefrom about 10 to 37 kilo daltons in Fig. 2A and from 10 to 25 kilo daltons in Fig. 2B and 2C,respectively. The range from ca. 13 to 18 kilo daltons (indicated by the arrows) is mostrelevant. The last slot in Fig. 2A shows V8-protease in the highest concentration used fordigestion; thermolysin and papain in the concentration used were not detectable with the

Coomassie Blue stain. Running direction was from left to right


differentiation of the splitting products, only patterns obtained with thismethod are discussed in the following section. Optimal comparisons of allsplitting products in a single gel were achieved with mixtures of digestsobtained with different amounts of each enzyme. These mixtures containedin a more even distribution poly-peptides of up to 4 different digestion steps.

The most characteristic splitting products were separated in the mole-cular weight range between 13 and 18 kilo-daltons. When peptide patternsobtained with different viruses or different preparations of the same virus(Figs. 2 A, B, C, lines 10 and 11, two preparations of APLV-Hu) are com-pared, it has to be kept in mind that the peptides in this molecular weightrange are only in a transient state and that these intermediate degradationproducts can be digested further. Thus, not the relative strengths of individualbands but only the migration rates are important for the interpretation of thepatterns. Even the lack of certain bands or the occurrence of additional bandsmay not always signalize a difference between two proteins as it is seen inFigure 2 A with two preparations of APLV-Hu.

The cleavage of uncoiled proteins in the presence of SDS may not strictlyfollow the rules of splitting characteristics with native proteins. Nevertheless,the resulting patterns of poly-peptides in SDS-PoroPAGE are depending uponthe enzyme used. Therefore it is assumed that papain still splits peptide bondsinvolving basic amino acids, leucine or glycine. Thermolysin supposedlycleaves the peptides on the amino side of leucine and phenylalanine, V8-protease on the carboxyl side of glutamic and aspartic acid.

Surprising and not understood yet was the resistance of the capsid proteinsubunits of some viruses to digestion, especially with V8-protease. Already ata concentration of 3/ig/ml, this enzyme caused a considerable degree of clea-vage with strains of APLV. With OkMV, DMV or EMV-t, on the otherhand, concentrations 100 times as high were necessary to bring about at leastsome cleavage of the protein subunits. The amount of enzyme necessary ispossibly typical for each virus.

Similarities of peptide patterns in relation to the closenessof serological relationships (Figs. 2 A, B, C)

The APLV isolates Hu and Ay cause rather different symptoms on testplants. Serologically, however, they cannot be distinguished (FRIBOURG et al.1977, KOENIG et al. 1979). The patterns of the poly-peptides confirm thesimilarity of their capsid proteins (Fig. 2, lines 10 to 12): after cleavage withpapain and thermolysin they appear identical and after cleavage with V8-protease nearly so, with the possible occurrence of one extra-band withAPLV-Ay. Likewise, BMV-1 and BMV-3 cannot be distinguished serologic-ally, although they differ in other properties (PAUL 1969, KOENIG 1969).Again the similarity of their capsid proteins is confirmed by the similarity ofpoly-peptide patterns (Fig. 2 A, B, lines 4 and 5). APLV-Col and Ec-1 appear


identical with most antisera tested (KOENIG et al. 1979), their poly-peptidepatterns are also very similar (Fig. 2, lines 17 and 18).

APLV Bo-15 and Caj belong to the same strain group (Col-Caj group) asCol and Ec-1, however, they represent different sub-groups and the majorityof our antisera produced weak spurs when the former two isolates were testedside by side with the latter two in agar gel double diffusion tests (KOENIGet al. 1979). After cleavage with V8-protease, the four isolates appear rathersimilar, however, after cleavage with thermolysin and also papain the dif-ferences are clearly evident (Fig. 2, lines 15 to 18). The APLV isolates Col-2and Col-3 whidi serologically and on the basis of their poly-peptide patterns(Fig. 2, lines 13 and 14) appear very similar belong to the CCC-group ofAPLV strains (KOENIG et al. 1979). These isolates usually form strong spurswhen tested side by side with members of the Col-Caj group, such as Col, Ec-1,Bo-15, Caj. After cleavage with thermolysin and papain the isolates of theCCC-group can clearly be distinguished from those of the Col-Caj group(Fig. 2, lines 15 to 18), whereas after cleavage with V8-protease the similari-ties are more pronounced. APLV-Hu and Ay (Fig. 2, lines 10 to 12) belong toa third APLV strain group (Hu-group) and very strong spurs are alwaysobserved when isolates of this group are tested side by side with isolates ofthe other two strain groups (CCC and Col-Caj) (KOENIG et al. 1979). Poly-peptide patterns obtained after cleavage with all three enzymes confirm thesedifferences.

Because of close serological relationships the APLV isolates have beenconsidered to be strains of EMV (CIBBS and HARRISON 1973). KOENIG et al.(1979) found that the Hu-group is more closely related to EMV-t and EMV-ALthan to the original APLV-isolate Col (GIBBS et al. 1966). The greater simi-larity of EMV-t to Hu rather than to Col becomes also evident after cleavagewith V8-protease (Fig. 2A, lines 8, 10, 11 and 18); greater similarity of EMV-Al to Hu rather than to Col is seen after cleavage with papain (Fig. 2 C,lines 9, 10, 11, 18).

BMV and DMV have an average serological differentiation index in reci-procal tests (RT-SDI) of 2, i.e. they are still relatively closely related (KOENIG1976). Their poly-peptide patterns obtained after cleavage with V8-protease(Fig. 2 A, lines 3 to 5) and thermolysin still show many similarities. OYMVand ScrMV also have a RT-SDI of 2, their poly-peptide patterns appearsimilar after cleavage with thermolysin, but much less so after cleavage withV8-protease or papain (Fig. 2, lines 1 and 2). Similarities in the patterns areeven seen with OkMV and C Y W after cleavage with V8-protease (Fig. 2 A,lines 6 and 7). These two viruses have a RT-SDI of 5.

From the data of a computer analysis of the amino acid compositionsPAXJL et al. (1980) concluded that a relatively close relationship may existbetween EMV-t and OkMV which is not apparent in serological tests (KOENIG1976). The poly-peptide patterns of these two viruses (Fig. 2, lines 7 and 8)are rather different after cleavage with thermolysin and papain, there are,however, some similarities after cleavage with V8-protease which makes ittempting to speculate on "hidden relationships" between the two viruses.

Phytopath. Z., Bd. 100, Heft 4 23


However, it must not be overlooked that similarities which are seen in peptidepatterns only after cleavage with one enzyme might well be chance similaritiesproduced by equally sized poly-peptides of different composition. Experi-ments with SDS-degraded EMV-t and OkMV and antiserum to the latter gaveno evidence for "hidden relationships" which could become evident after SDS-treatment. Thus, with tymoviruses, poly-peptide analyses in SDS-polyacryl-amide gels seem to be useful mainly for the typing of strain groups and thecomparison of closely related viruses.

Summary

Capsid proteins from several tymoviruses were partially proteolyzed withStaphylococcus aureus V8-protease, thermolysin or papain, respectively. Thepoly-peptide patterns obtained after poiyacrylamide porosity gradient gelelectrophoresis in the presence of sodium dodecyl sulfate were usually ratherdissimilar with serologically distantly related viruses. However, with closelyrelated viruses, especially with strains of Andean potato latent virus, a goodagreement was observed between serological typing and similarities in peptidepatterns.

Zusammenfassung

Vergleichende Untersuchungen uber die Hullproteine von Tymovirenin SDS-Polyacrylamid-Porositatsgradienten-Gelen

nadi partieller Spaltung mit versdiiedenen Proteasen

Die Hullproteine von verschiedenen Tymoviren wurden mit Staphylo-coccus aureus V8-Protease, Thermolysin oder Papain partiell hydrolysiert. Beiserologisch entfernt verwandten Viren waren die nach Gel-Elektrophorese inPolyacrylamid-Porositats-Gradienten in Gegenwart von Natriumdodecyl-sulfat erhaltenen Peptidmuster im allgemeinen sehr untersdiiedlich. Bei sero-logisdi nahe verwandten Viren, besonders bei Stammen des Andean potatolatent virus, ergab sich dagegen eine gute Ubereinstimmung zwischen serologi-scher Typisierung und Ahnlidikeiten im Polypeptidmuster.

The skillful tedinical assistance of Miss Angelika SIEG and the financial support givenby the Deutsche Forsdiungsgemeinschaft are gratefully acknowledged.

Literature

CLEVELAND, D . W., S. G. FISCHER, M. W. KIRSCHNER, and U. K. LAEMMLI, 1977: Peptidemapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electro-phoresis. J. Biol. Chem. 252,1102—1106.

DUNN, D . B., and J. H. HITCHBORN, 1965: The use of bentonite in the purification of plantviruses. Virology 25,171—192.

FRIBOURG, C. E., R. A. C. JONES, and R. KOENIG, 1977: Host plant reactions, physicalproperties and serology of three isolates of Andean potato latent virus from Peru.Ann. Appl. Biol. 86, 373—380.


GIBBS, A. J., and B. D. 1-IARRISON, 1973: Eggplant mosaic virus. Plant Virus Description No.124. Commonw. Mycol. Inst., Ass. Appl. Biol. Hertsfordshire, Berks, England.

, E. HECHT-POINAR, and R. D. WOODS, 1966: Some properties of three related viruses:Andean potato latent, dulcamara mottle, and ononis yellow mosaic. J. Gen. Microbiol.44,117—193.

KOENIG, R., 1969: Behavior of belladonna mottle virus in serological tests in the presence oforganic mercury compounds. Virology 38, 140—144.

, H. STEGEMANN, H . FRANCKSEN, and H. L. PAUL, 1970: Protein subunits in the potatovirus X group. Determination of the molecular weights by polyacrylamide electro-phoresis. Biodiim. Biophys. Acta 207,184—189.

, 1976: A loop-structure in the serological classification system of tymoviruses. Virology72,1—5.

, C. E. FRIBOURG, and R. A. C. JONES, 1979: Symptomatological, serological and electro-phoretic diversity of isolates of Andean potato latent virus from different regions inthe Andes. Phytopathology 69, 748—752.

MARGOLIS, J., and K. G. KENRICK, 1968: Polyacrylamide gel electrophoresis in a continuousmolecular sieve gradient. Anal. Biodiem. 25, 347—365.

PAUL, H . L., 1969: Untersudiungen uber ein neues, isometrisches Virus aus Atropa belladonnaL. III. Die mengenmaCige Zusammensetzung des belladonna mottle virus aus Protein-huUen und Nukleoproteidteildien und deren Inhomogenitat. Phytopath. Z. 65,257—262.

, A. GIBBS, and B. WITTMANN-LIEBOLD, 1980: The relationships of certain tymovirusesassessed from the amino acid composition of their coat proteins. Intervirology (inpress).

STEGEMANN, H . , 1979: SDS-gel-electrophoresis in Polyacrylamide, merits and limits. In: P. G.RiGHETTi, C. J. VAN Oss, and J. W. VANDERHOFF (Eds.), Electrokinetic SeparationMethods, 313—336. Elsevier, North-Holland, Amsterdam.

, 1980: Gel-Elektrophorese und Fokussierung in Platten, S.I—29. Gebraudisanweisungfur Studenten und tedin. Assistenten vom Institut fur Biochemie, Messeweg 11, D-3300Braunsdiweig (1st ed. 1968).

Authors' address: Dr. R. KOENIG, H . FRANCKSEN, Prof. Dr. H. STEGEMANN, BiologisdieBundesanstalt, Messeweg 11, D-3300 Braunsdiweig.

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