Archives of Virology
Archives of Virology 57, 339--348 (1978)
© by Springer-Verlag 1978
Structural Similarities of Hofl Cholera Virus With Toflaviruses By P.-J. E~ZMAI~~ and F. W~ILA~I) Federal Research Institute for Animal Virus Diseases, Tfibingen, Federal l~epublic of Germany With 5 Figures Accepted January 23, 1978
Summary Hog cholera virus grown in PK-15 cells was purified by eentrifugation through a sucrose cushion followed b y sucrose gradient centrifugation. Analysis of virus labeled externally with [3H]sodiuln borohydride on polyacrylamide gel electrophoresis revealed two glycoproteins, gp 55 and gp 46. A third structural polypeptide, p36, seems not to be glycosylated. The gp46 was also found in tile virus-free supernatant of infected cells. I t could be demonstrated by radioimmune precipitation of virus labeled with [asS]methionine that all three polypeptides are specific for hog cholera virions. Electron microscopically hog cholera virus appeared as a spherical particle with a diameter of 42=L8 rim. The virus particles frequently displayed a fringe of projections with a length of about 6 - - 8 rim. The similarities of hog cholera virus with Alphaviruses and Flaviviruses are discussed.
Introduetion Togaviruses are the simplest of the lipid-containing animal viruses that mature b y budding through the plasma membranes of infected cells. Virions contain single-stranded RNA of about 4 x 106 daltons and have icosahedral nucleocapsids surrounded b y a lipoprotein envelope including one or more glycopeptides (for reference see 6). Based on the susceptibility to lipid solvents (4) and on electron microscopic studies (9) hog cholera virus was considered earlier to be a member of the Togavirus family. I n an earlier report additional evidence was presented for the classification of the virus as a Togavirus (5). I t was shown t h a t hog cholera virus is composed of at least three potypeptides: p55, gp46, and p36. This polypeptide pattern of the virion is similar ot t h a t of Alphaviruses within the family Togaviridae which are composed of two glycoproteins in the viral membrane and of one core protein within the range of 50,000 and 30,000 daltons, respectively (for
0304-8608/78/0057/0339/$ 02.00
340
P..J. ENZ~IAN~ and F. WEtLAnD:
a r e v i e w see 15). A f u r t h e r gtycoprotein f o u n d in Semliki F o r e s t virus has a molecular weight of a b o u t 10,000 d a l t o n s (8). F u r t h e r s u p p o r t for t h e classification of hog cholera virus has been o b t a i n e d f r o m studies on t h e v i r a l I~NA w h i c h h a v e been show~a to h a v e a molecular weight of a b o u t 4× 106 d a l t o n s a n d a s e d i m e n t a t i o n coefficient of a b o u t 40 S (5). Because of t h e difficulties in discriminating b e t w e e n cellular a n d v i r a l polypeptides, t h e p r o t e i n composition of hog cholera virus has been r e i n v e s t i g a t e d a n d confirmed. These d a t a t o g e t h e r w i t h electron microscopic observations are p r e s e n t e d here.
Materials and Methods Cells and Virus PK-15 cells were grown in Earle's salt solution containing twice the amount of Eagle's vitamins, 5.0g/liter of lactalbumin hydrolysate, antibiotics (100 I U / m t penicillin and 50 ~g/ml streptomycin) and 10 per cent fetal calf serum. The hog cholera virus strain A L D 970a (12), was obtained from Dr. ti:orn, Tfibingen. The virus had been isolated from the serum of a viraemic pig infected with the ALD strain of hog cholera virus. Material from the third passage of this virus in PK-15 cells was used throughout this study. The virus was propagated in PK-15 cells during 72 hours in the presence of the medium described above except that 5 per cent fetal calf serum was used.
Puri]ieation o] Virus Hog cholera virus was concentrated by sedimentation through a 5 ml cushion of 20 per cent (w/w) sucrose in TEN-buffer (16) for two hours at 27,000 r p m in a SW27 rotor (Spinco, Beckman). F u r t h e r purification was achieved in a linear 20 to 50 per cent (w/w) sucrose gradient in TEN-buffer. MateriM in the density region of 1.15 to 1.16 g/ml was collected. For electron microscopic studies specimens of formaldehydefixed virus were prepared by adding the fixative (10 per cent formaldehyde in boratebuffer p H 9.0) to the clarified culture fluid harvested after about 48 hours p.i. to give a final concentration of 0.5 per cent. The virus was stored for about 24 hours at 4 ° C. The virus was then concentrated and purified as described above.
Labeling o] Virus Proteins 24 hours after infection of PK-15 cells with hog cholera virus the medium was replaced by fresh medium which consisted of ~ mixture of VM 3 A ( 18 ) and Eagle (10 : 1), 1 ~g/ml uctinomycin D (Serva, Heidelberg), and 10 y.Ci/ml p S ] m e t h i o n i n e (Buchler, Braunschweig). The labeling period was for 12--16 hours. The culture fluid was then harvested and cleared by centrifugation at 2200 × g for 30 minutes.
Extrinsic Labeling o/Glycoproteins With Sodium Barohydride Galactose oxidase-tritiated borohydride labeling of hog cholera virus was performed by modification of the techniques of GA~MBEnG and HA~:OMOR~ (7). Hog cholera virus was purified as described and was then pelleted through 10 per cent glycerol in TEN-buffer. Following gentle resuspension in 0.2 ml TEN-buffer, the virus was incubated for 10 minutes at 37 ° C with 500 U neuraminidase (Behring). The virus was then repelleted as described above, resuspended in 0.2 ml TEN-buffer and treated for 15 minutes at 37°C with 500 U galactose oxidase (Sigma). Again the virus was repelleted and incubated for 30 minutes at 20°C with 1 mCi of tritiated sodium borohydride (Buchler, Braunschweig) dissolved in 10 ~I of 0.01 ~ ]~TaOH. The labeled virus was precipitated with 5 per cent trichloroacetic acid. F u r t h e r t r e a t m e n t was as described for P A G E analysis.
Structure of I-Iog Cholera Virus
341
Radioimmune Precipitation The method of radioimmune precipitation used was similar to that described by I~LE et al. (11). Hog cholera virus (0.1 ml) labeled with [ssS]methionine and purified b y density gradient centrifugation was mixed with an equal volume of TEN-buffer containing I per cent Triton X-100. The virus was disrupted at 20 ° C for 30 minutes and then diluted twofold with TEN-buffer. The extract was incubated with an equal volume of hog cholera antibody (pig gammaglobulin fraction from an immune serum, kindly provided b y Dr. Kern, Tfibingen) at 4 ° C overnight. To the immune complexes 0.1 ml of antiglobulin (Miles, antipig IgG) was added. The precipitates were allowed to form at 4 ° C for two hours, collected by centrifugation at 2200 × g for 30 minutes, az~d washed two times with TEN-buffer. After the last wash the precipitates were resuspended in 0.5 ml of TE:N-buffer containing 2 per cent SDS and t per cent 2-mercapteethanol, and the solutions were heated for 5 minutes at 95 ~ C.
Polyaerylamide Gel Eleetrophoresis (PAGE) Viral proteins were precipitated by 5 per cent trichloroacetic acid. The pellet was washed three times with ethanol and then dissolved in 0.3 ml of TEl~l-buffer containing 2 per cent SDS and 1 per cent 2-mercaptoethanol by heating for 5 minutes at about 95 ° C. Electrophoresis of viral proteins in polyacrylamide gels using a continuous system (0.1 y! phosphate buffer) was carried out as described earlier (16). Optimal separation of viral proteins in PAGE was achieved by using a SDS-mixture (MCB--Pierce) described earlier (14). Electrophoresis was performed in cyclindrieal gels (5 × 60 ram) and vertical slab gels (130 × 110 ram). Molecular weights were estimated b y the procedure of SHAPI~aO et al. (19). ]~adioactive labeled proteins were mixed with unlabeled marker proteins of defined molecular weights (bovine serl~m albumin, rabbit immune globulin G, ovalbumin, and chymotrypsinogen) and coelectrophoresed with each radioactive sample to determine the relative electrophoretic mobilities. After destaining, cylindrical gels were fractionated and counted as described earlier (5). Slab gels were dried and autoradiographed on Agfa-Gevaert Osray T 4 films. Exposure time varied from 3 to 9 days.
Electron Microscopic Preparation Negative staining of the particles was done with an 1 per cent unbuffered solution of uranyt acetate or with phosphotungstic acid (2 per cent) at p H 6.8.
Results P A G E o/ Hog Cholera Virus Structural Proteins Purified hog cholera virus, labeled with [ssS]methionine was disrupted a n d the s t r u c t u r a l proteins were f r a c t i o n a t e d b y electrophoresis in polyacrylamide gels. I n Figure 1 a polypeptide p a t t e r n is shown which is u s u a l l y o b t a i n e d after SDSt r e a t m e n t of viral proteins followed b y electrophoretic s e p a r a t i o n i n P A G E using a c o n t i n u o u s buffer-system. Three m a i n proteins with molecu]ar weights of 54,000--56,000; 45,000--47,000; a n d 35,000--37,000 were f o u n d in all experim e n t s performed. However a n o t h e r polypeptide with a molecular weight of a b o u t 65,000 d a l t o n s also m i g h t be of viral origin a l t h o u g h it was n o t consistently e n c o u n t e r e d in the polypeptide p a t t e r n . I t was t h o u g h t t h a t this p r o t e i n m i g h t be a n u n c l e a r e d precursor of one of the s t r u c t u r a l proteins which is only sometimes i n c o r p o r a t e d into the virion. Several other proteins with molecular weights in the range of a b o u t 30,000--35,000 a n d 18,000--25,000 were f o u n d sometimes. I t appeared t h a t these polypeptides were d e g r a d a t i o n products which arose from proteolytie cleavage because t h e y occurred m a i n l y i n older preparations.
342
P.-J. E~z~A~ 55 46
and
F. WEILAND
:
36
~5 ¸
%
10 ¸
×
E
¢:L U
5
to
15
20
25
30
~
~5
50
5s
60
fractions
Fig'. 1. PAGE of hog" cholera virus proteins Hog cholera virus labeled with [3aS]methionine was disrupted with SDS, mixed with unlabeled marker proteins and eleetrophoresed in polyacrylamide gels
Viral Glycoprotein8 By labeling of purified hog cholera virus with [3H]sodium borohydride it was shown that the two polypeptides 54,000--56,000 and 45,000--47,000 contained carbohydride chains (Fig. 2A). These proteins were therefore designated gp55 and gp46. Ill addition to the gp55 and gp46 several larger and minor glycoproteins with molecular weights above 70,000 and below 30,000 were labeled also. It seems that these products originated either from the enzymatic system used for the labeling procedure or arose from proteolytie cleavage of larger proteins possibly by the proteolytic activities described for the enzyme galactose oxidase (20). Only the labeled glycopeptides gp55 and gp46 coincided with the peptides shown in Figure 1. Of the three main peaks gp55, g p ¢ 6 , and p36, gp46 was usually most prominent (Fig. 1). It was also shown that this main structural protein was present in the supernatant of infected cells. After pelleting the virus labeled with [35S]methionine the proteins of the supernatant were precipitated by ammonium sulfate (30 per cent, w/w). In PAGE it was shown that gp46 was the main labeled product in the supernatant (Fig. 2B). In the supernatant of uninfected cells, this glycopeptide was never found. Radioimmune Precipitation o/Hog Cholera Virus It seemed, further necessary to prove that the demonstrated viral polypeptides were specific for hog cholera virions, because it has been shown by LAIJDE (13) using ~ntracel]nlar virus harvested by disruption of the infected cells that hog cholera virions may be contaminated with cellular membranes after isopycnic gradient eentrifugation. Material from cells was found in the same density region as hog cholera virions. Therefore we tried to show by radioimmune precipitation
Structure of I-Iog Cholera Virus
343
A
4055
46
30,
% x
20
E
c~
®
.... ® 5
I0
15
20
25
30
35
~0
45
50
55
60
fractions
g
Is46
5
10
15
20
25
30
35
/,0
/.5
50
55
60
fractions
Fig. 2. PAGE of hog cholera virus glyeoproteins A. Virus labeled externally with [sI-I]sodium borohydride; t3. Supernatant of hog cholera virus infected PK-15 celts from which virions are removed by ultraeentrifugation. Molecular weights were determined by internal standardization in both runs as deseribed
which proteins were specific for hog cholera virions. Concentrated hog cholera virus labeled with [35S]methionine and purified by density gradient centrifugation was treated with immune serum from pigs and then precipitated by a second antibody from rabbit against pig IgG. The precipitate was washed and redissolved in TEN-buffer containing SDS and electrophoresed in a slab gel. In Figure 3 (right panel) an autoradiogram of a slab gel is shown. The resultant eleetrophoretie profile containing the main peaks gp55, gp46, and p36 was comparable with that shown before (Fig. 1). As a control the left panel shows an autoradiogram of hog cholera virM proteins not precipitated by immune sera. Centrifugation of hog eholera virus through a cushion of 20 per cent sucrose as it was usually done
344
P.-J. ENZlVIANN
and
F. WEILAND
:
seemed therefore necessary for effective purification. By this step material derived from cells with the same buoyant density as the virus will be eliminated as a result of its lower sedimentation coefficient.
Fig. 3. PAGE of radioimmune precipitated hog cholera virus Autoradiograms of virus-specific proteins separal~ed by SDS-polyaerylamide slab gel eleetrophoresis. The derails of slab gel eleetrophoresis and autoradiography are described in Materials and Methods. Left panel: hog cholera virus labeled with [35S]methionine and purified by density gradient centrifugation. Right panel: The same virus as in the left panel, but precipitated by immune sofa
Electron Microscopic Studies Electron microscopy of purified hog cholera virus usually revealed the structures as seen in Figure 4. When virus concentrated and purified as described was used a population of roughly spherical particles varying in size was found. The mean diameter of the virions was determined to be 4~2~L8nm. In several preparations particles were found to contain surface projections but most of the virions always seemed to be disintegrated. After fixation of infectious cell culture fluid with formaldehyde followed by concentration and purification of the virus, the particles frequently displayed a fringe of projections with a length of about 6 to 8 nm (Fig. 5).
Structure of Hog Cholera Virus
345
Fig. 4. Electron micrographs of hog cholera virus particles The virus preparation was purified as described and stained with uranyl acetate (bar = 50 nm)
Discussion
I n order to determine the glycoproteins of hog cholera virus the galaetose oxidase-tritiated borohydride method of external labeling was applied. By this treatment terminal galactosyl and N-aeetylgalactosaminyl residues are oxidized to the corresponding aldehydes b y the enzyme galactose oxidase (1). These aldehydes are then reduced with tritiated borohydride b y the method of GA~I~I~I~G and HAKO~ORI (7). Because sialic acids are often linked to subterminal galactosyl residues, the virus was treated with neuraminidase. P A G E analysis of tritiated hog cholera virus revealed to peaks (Fig. 2A). I n addition to the polypeptide 46,000 (gp46) which was earlier shown to be a glycoprotein by PAS-staining (5) the polypeptide 55,.000 (gp 55) was demonstrated to contain also carbohydrate chains. In the virus-free supernatant of infected cells gp46 was present as a main polypeptide (Fig. 2B). I n the P A G E profile of tritiated virus some radioactivity was distributed in the molecular weight region of about 10,000 to 30,000 daltons. This m a y be due to partial degradation of labeled structural proteins since it is described that the enzyme galactose oxidase contains proteolytic activities (20) and the location of these products was not reproducible. The results do not mean unequivocally, however, that the demonstrated polypeptides are the only structural constituents of hog cholera virus. A minor polypeptide could also be present in the virion but be masked in the P A G E profile by the proteolytic cleavage products. I t was shown also with other "nonarbo" Togaviruses (2, t7, 21) t h a t the polypeptide profiles in polyacrylamde gels are not as clear cut as those of A1phaviruses and Flaviviruses. With the aid of radioimmune precipitation followed b y eleetrophoresis of viral proteins we have shown that the polypeptides detected in P A G E were of
3~6
P.-J. ESIZI~A~
and
F. WEILAND
:
Fig. 5. E l e c t r o n m i e r o g r a p h s of h o g cholera, v i r u s particles fixed w i t h f o r m a l d e h y d e T h e infectious cell c u l t u r e fluid was fixed w i t h 0.5 p e r c e n t f o r m a l d e h y d e . T h e v i r u s was p u r i f i e d as described. Selected p a r t i c l e s are d e m o n s t r a t e d e x h i b i t i n g surface p r o j e c t i o n s (bar = 50 n m ) a - - c : V i r u s s t a i n e d w i t h u r a n y l a c e t a t e ; d - - g : V i r u s s t a i n e d w i t h p h o s p h o t u n g s t d e acid
Structure of Hog Cholera Virus
347
viral origin. After autoradiography of dried slab gels it was demonstrated that several proteins obviously derived from celhflar material were eliminated by precipitation of viral proteins with immune serum (Fig. 3). To avoid coprecipitation of nonviral proteins possibly enclosed within the virion, virus was disrupted with Triton X-100. The immune precipitates were allowed to form in the cold since it was found t h a t rapid precipitation at 37 ° C resulted in coprecipitation of cellular proteins (data not shown). Our system of purification of hog cholera virus seems to be efficient. Material of cellular origin which has the same buoyant density as the virus (13) was removed b y the first step of virus purification, i.e. eentrifugation of concentrated virus through a cushion of 20 per cent sucrose. In Figure 3 (left panel) several nonviral proteins were demonstrated after electrophoresis of virus which was not pelleted through the sucrose cushion, yet these proteins were removed b y our system of radioimmune precipitation. I t has been shown earlier b y DALRYM-PLE et al. (3) t h a t radioimmune precipitation of Alphaviruses is a suitable system for specific determination of viral antigens and analysis of the immunological relationship among these viruses. Combined with earlier results (5) demonstrating that similar virM pol}'peptides were also found when hog cholera virus was grown in another cell system, that these polypeptides were always absent from uninfected control cells, and t h a t radioactivity was never found in virus grown in prelabeled cells, the polypeptide structure of hog cholera virus has been reestablished and confirmed b y our data. The structural polypeptides of hog cholera virus are different from those of Flaviviruses, Rubiviruses (for a review see 10), and two other possible members of the family Togaviridae, Equine arteritis virus, and lactic dehydrogenase virus (21, 2) but are similar to those of Alphaviruses. Electron microscopically hog cholera virus appeared as a spherical particle (Fig. 5). I t was heterogeneous in size. The diameter of the virion was determined to be 4 2 ~ 8 nm. After fixation of the infections cell culture fluid with formaldehyde followed b y concentration and purification most of the particles revealed a fringe of projections with a length of about 6 - - 8 nm (Figs. 5a and b). Similar particles have also been found repeatedly in preparations without fixation but not as frequently as in fixed preparations. Morphologically hog cholera virus which is a member of the genus Pestiviruses resembles more closely the Flaviviruses than the Alphaviruses within the family Togaviridae. Together with the above mentioned similarities in the polypeptide structure with Alphaviruses and the previously published data on the viral R N A (5) hog cholera virus and Togaviruses share the major structural properties.
Aeknowledfments We thank Prof. M. Mussgay for comments and discussion. The competent technical assistance of Miss M. Pfeifer is gratefully acknowledged. This work was supported by the Commission of the European Communities (Swine fever research program).
References I..~kVIGAD,G., A~tAtCAL,D., AS~NSIO, C., HOt~ECKEI~,B. L. : The I)-galactose oxidase of Polyporus circinatus. J. biol. Chem. 237, 2736--2743 (1962), 24 Arch.Virol. 57/4
348
P.-J. ENZ~ANSr
and F. W~5~ND
: Structure of Hog
Cholera Virus
2. BRINTON-1)ARNELL, M., P~AGE~ANN, P. G. W. : Structure and chemical-physical characteristics of lactate dehydrogcnase-elevating virus and its 1%NA. J. Virol. 16, 420--433 (1975). 3. 1)AZltYMI'LE, J. M., T~A~O~O, A. Y., C~DIFF, R. D., 1%VSSEL, P. K. : 1%adioimmune precipitation of group A arboviruses. J. Immunol. 109, 426-433 (1972). 4. DrNTE]~, Z. : l:~elationship between bovine virus diarrhoea virus and hog cholera virus. Zbl. Bakt. I. Abt. Orig. 188, 475---486 (1963). 5. ENZIVIANN, P.-J., 1%EIIBERG, l-I.: The structural components of hog cholera virus. Z. Naturforsch. 32 c, 456---458 (1977). 6. FEnNEl, F. : Classification and nomenclature of viruses. Intervirology 7, 44--47 (1976). 7. GAI~IB~G, C. G., HAtCO.n~O~I, S.-I. : External labeling of cell surface galaetose and galactosamine in glycolipid and glycoprotein of h u m a n erythrocytes. J. biol. Chem. 248, 431i--4317 (1973). 8. GAaOFF, ]ft., SIgo~s, K., 1%EN~ONE~~, O. : Isolation and characterization of the membrane proteins of Semliki Forest virus. Virolog~ 61, 493---503 (t974). 9. Hogzr~EK, M., MAESS, J., LavFS, 1%.: Studies on the substructure of Togaviruses. II. Analysis of equine arteritis, 1%ubella, bovine viral diarrhoea and hog cholera viruses. Arch. ges. Virusforseh. 33, 306--318 (197t). 10. Ho:azINEy:, M. C. : The structure of Togaviruses. Progr. reed. Virol. 16, 109--156 (1973). t l . IHLE, J. iN'., HANR'/k, M. (~., 1%OBERSON, E., KE1N-2qJ!]Y,F. T. : Autogenous i m m u n i t y to endogeneous RNA tumor viruses. J. exp. Med. 139, t568--158] (t974). t2. Ko~N, G., MATTK~.E~'S,W. : On the selection of pig immune sera for conjugation in swine fever diagnosis. Commission of the European Communities. Coordination of Agricultural tgesearch. EUI~ 5486. Sere. 1)iag. Epiz. Swine fever. Amsterdam (1975). 13. LA.UDE, H. : Improved method for the purification of hog cholera virus grown in tissue culture. Arch. Virol. 54, 4 t - - 5 1 (1977). 14. 5IAT~IEKA, H. 1)., ENZi~ANN, P.-J., BaCIIIgACH, N. L., MIGL, B.: The influence of sodium dodecylsulfate from different sources on the separation of virus proteins in polyaerylamide gel electrophoresis. Anal. Biochem. 81), 9--17 (1977). 15. MUSSGAY, M., ENZ~IA~N, P.-J., HO~Z~NEK, M. C., Wm~ANO, E. : Growth cycle of arboviruses in vertebrate and arthropod cells. Prog. reed. Virol. 19, 257---323 (1975). t6. t~IUSSGAY,M., ~ V ~ I ~ D , E., S ~ o ~ E ~ , K., U ~ s c ] ~ / 4 ~ , S., E?czsIA~, P.-J. : Properties of components obtained by treatment of Semliki Forest virus with Tween 80 and Tri(n-butyl)phosphate. J. gen. Virol. ]9, 89--101 (1973). 17. P~C~ET:C, 1~. F., ZEE, Y. C. : Structural proteins of bovine viral diarrhea virus. Amer. J. vet. Res. 36, 173t--1734 (1975). 18. Scm~'6B]~5, W., S~EI)ENTO]VF, V. : Charakterisierung eines drei Jahre alten Zellstammes aus der Schweineniere u n d Untersuchungen fiber sein Verhalten gegenfiber dem Virus der Maul- u n d I4]auenseuche. Zbl. Bakt. Abt. 1, Orig. 181, 3---].6 (1961). 19. S ~ P ~ o , A. L., V~NUE~, E., M~IzE~, J. V. : Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. biophys, t~es. Comm- 28, 815--820 (1967). 20. S~FF~E~), J. B., 1)~Y, T. M. : Extrinsic labeling of MuMTV with a galactose oxidase-tritiated borohydride method. Virology 70, 247--250 (1976). 21. ZEEeERS, J. J. W., VAN n ] ~ ZmXST, B. A. M., HO~Z~NEK, M. C. : The structural proteins of Equine arteritis virus. Virology 73, 200--205 (1976).
Authors' address: Dr. P.-J. E~rZ~A~-N, Bundesforsehungsanstalt ffir Viruskrankheiten der Tiere, Waldh/iuser H6he, PauI-Ehrlich-Stral?e 28, 1)-7400 Tfibingen, Federal Republic of Germany. Received November 7, 1977
Archives of Virology 57, 339--348 (1978)
© by Springer-Verlag 1978
Structural Similarities of Hofl Cholera Virus With Toflaviruses By P.-J. E~ZMAI~~ and F. W~ILA~I) Federal Research Institute for Animal Virus Diseases, Tfibingen, Federal l~epublic of Germany With 5 Figures Accepted January 23, 1978
Summary Hog cholera virus grown in PK-15 cells was purified by eentrifugation through a sucrose cushion followed b y sucrose gradient centrifugation. Analysis of virus labeled externally with [3H]sodiuln borohydride on polyacrylamide gel electrophoresis revealed two glycoproteins, gp 55 and gp 46. A third structural polypeptide, p36, seems not to be glycosylated. The gp46 was also found in tile virus-free supernatant of infected cells. I t could be demonstrated by radioimmune precipitation of virus labeled with [asS]methionine that all three polypeptides are specific for hog cholera virions. Electron microscopically hog cholera virus appeared as a spherical particle with a diameter of 42=L8 rim. The virus particles frequently displayed a fringe of projections with a length of about 6 - - 8 rim. The similarities of hog cholera virus with Alphaviruses and Flaviviruses are discussed.
Introduetion Togaviruses are the simplest of the lipid-containing animal viruses that mature b y budding through the plasma membranes of infected cells. Virions contain single-stranded RNA of about 4 x 106 daltons and have icosahedral nucleocapsids surrounded b y a lipoprotein envelope including one or more glycopeptides (for reference see 6). Based on the susceptibility to lipid solvents (4) and on electron microscopic studies (9) hog cholera virus was considered earlier to be a member of the Togavirus family. I n an earlier report additional evidence was presented for the classification of the virus as a Togavirus (5). I t was shown t h a t hog cholera virus is composed of at least three potypeptides: p55, gp46, and p36. This polypeptide pattern of the virion is similar ot t h a t of Alphaviruses within the family Togaviridae which are composed of two glycoproteins in the viral membrane and of one core protein within the range of 50,000 and 30,000 daltons, respectively (for
0304-8608/78/0057/0339/$ 02.00
340
P..J. ENZ~IAN~ and F. WEtLAnD:
a r e v i e w see 15). A f u r t h e r gtycoprotein f o u n d in Semliki F o r e s t virus has a molecular weight of a b o u t 10,000 d a l t o n s (8). F u r t h e r s u p p o r t for t h e classification of hog cholera virus has been o b t a i n e d f r o m studies on t h e v i r a l I~NA w h i c h h a v e been show~a to h a v e a molecular weight of a b o u t 4× 106 d a l t o n s a n d a s e d i m e n t a t i o n coefficient of a b o u t 40 S (5). Because of t h e difficulties in discriminating b e t w e e n cellular a n d v i r a l polypeptides, t h e p r o t e i n composition of hog cholera virus has been r e i n v e s t i g a t e d a n d confirmed. These d a t a t o g e t h e r w i t h electron microscopic observations are p r e s e n t e d here.
Materials and Methods Cells and Virus PK-15 cells were grown in Earle's salt solution containing twice the amount of Eagle's vitamins, 5.0g/liter of lactalbumin hydrolysate, antibiotics (100 I U / m t penicillin and 50 ~g/ml streptomycin) and 10 per cent fetal calf serum. The hog cholera virus strain A L D 970a (12), was obtained from Dr. ti:orn, Tfibingen. The virus had been isolated from the serum of a viraemic pig infected with the ALD strain of hog cholera virus. Material from the third passage of this virus in PK-15 cells was used throughout this study. The virus was propagated in PK-15 cells during 72 hours in the presence of the medium described above except that 5 per cent fetal calf serum was used.
Puri]ieation o] Virus Hog cholera virus was concentrated by sedimentation through a 5 ml cushion of 20 per cent (w/w) sucrose in TEN-buffer (16) for two hours at 27,000 r p m in a SW27 rotor (Spinco, Beckman). F u r t h e r purification was achieved in a linear 20 to 50 per cent (w/w) sucrose gradient in TEN-buffer. MateriM in the density region of 1.15 to 1.16 g/ml was collected. For electron microscopic studies specimens of formaldehydefixed virus were prepared by adding the fixative (10 per cent formaldehyde in boratebuffer p H 9.0) to the clarified culture fluid harvested after about 48 hours p.i. to give a final concentration of 0.5 per cent. The virus was stored for about 24 hours at 4 ° C. The virus was then concentrated and purified as described above.
Labeling o] Virus Proteins 24 hours after infection of PK-15 cells with hog cholera virus the medium was replaced by fresh medium which consisted of ~ mixture of VM 3 A ( 18 ) and Eagle (10 : 1), 1 ~g/ml uctinomycin D (Serva, Heidelberg), and 10 y.Ci/ml p S ] m e t h i o n i n e (Buchler, Braunschweig). The labeling period was for 12--16 hours. The culture fluid was then harvested and cleared by centrifugation at 2200 × g for 30 minutes.
Extrinsic Labeling o/Glycoproteins With Sodium Barohydride Galactose oxidase-tritiated borohydride labeling of hog cholera virus was performed by modification of the techniques of GA~MBEnG and HA~:OMOR~ (7). Hog cholera virus was purified as described and was then pelleted through 10 per cent glycerol in TEN-buffer. Following gentle resuspension in 0.2 ml TEN-buffer, the virus was incubated for 10 minutes at 37 ° C with 500 U neuraminidase (Behring). The virus was then repelleted as described above, resuspended in 0.2 ml TEN-buffer and treated for 15 minutes at 37°C with 500 U galactose oxidase (Sigma). Again the virus was repelleted and incubated for 30 minutes at 20°C with 1 mCi of tritiated sodium borohydride (Buchler, Braunschweig) dissolved in 10 ~I of 0.01 ~ ]~TaOH. The labeled virus was precipitated with 5 per cent trichloroacetic acid. F u r t h e r t r e a t m e n t was as described for P A G E analysis.
Structure of I-Iog Cholera Virus
341
Radioimmune Precipitation The method of radioimmune precipitation used was similar to that described by I~LE et al. (11). Hog cholera virus (0.1 ml) labeled with [ssS]methionine and purified b y density gradient centrifugation was mixed with an equal volume of TEN-buffer containing I per cent Triton X-100. The virus was disrupted at 20 ° C for 30 minutes and then diluted twofold with TEN-buffer. The extract was incubated with an equal volume of hog cholera antibody (pig gammaglobulin fraction from an immune serum, kindly provided b y Dr. Kern, Tfibingen) at 4 ° C overnight. To the immune complexes 0.1 ml of antiglobulin (Miles, antipig IgG) was added. The precipitates were allowed to form at 4 ° C for two hours, collected by centrifugation at 2200 × g for 30 minutes, az~d washed two times with TEN-buffer. After the last wash the precipitates were resuspended in 0.5 ml of TE:N-buffer containing 2 per cent SDS and t per cent 2-mercapteethanol, and the solutions were heated for 5 minutes at 95 ~ C.
Polyaerylamide Gel Eleetrophoresis (PAGE) Viral proteins were precipitated by 5 per cent trichloroacetic acid. The pellet was washed three times with ethanol and then dissolved in 0.3 ml of TEl~l-buffer containing 2 per cent SDS and 1 per cent 2-mercaptoethanol by heating for 5 minutes at about 95 ° C. Electrophoresis of viral proteins in polyacrylamide gels using a continuous system (0.1 y! phosphate buffer) was carried out as described earlier (16). Optimal separation of viral proteins in PAGE was achieved by using a SDS-mixture (MCB--Pierce) described earlier (14). Electrophoresis was performed in cyclindrieal gels (5 × 60 ram) and vertical slab gels (130 × 110 ram). Molecular weights were estimated b y the procedure of SHAPI~aO et al. (19). ]~adioactive labeled proteins were mixed with unlabeled marker proteins of defined molecular weights (bovine serl~m albumin, rabbit immune globulin G, ovalbumin, and chymotrypsinogen) and coelectrophoresed with each radioactive sample to determine the relative electrophoretic mobilities. After destaining, cylindrical gels were fractionated and counted as described earlier (5). Slab gels were dried and autoradiographed on Agfa-Gevaert Osray T 4 films. Exposure time varied from 3 to 9 days.
Electron Microscopic Preparation Negative staining of the particles was done with an 1 per cent unbuffered solution of uranyt acetate or with phosphotungstic acid (2 per cent) at p H 6.8.
Results P A G E o/ Hog Cholera Virus Structural Proteins Purified hog cholera virus, labeled with [ssS]methionine was disrupted a n d the s t r u c t u r a l proteins were f r a c t i o n a t e d b y electrophoresis in polyacrylamide gels. I n Figure 1 a polypeptide p a t t e r n is shown which is u s u a l l y o b t a i n e d after SDSt r e a t m e n t of viral proteins followed b y electrophoretic s e p a r a t i o n i n P A G E using a c o n t i n u o u s buffer-system. Three m a i n proteins with molecu]ar weights of 54,000--56,000; 45,000--47,000; a n d 35,000--37,000 were f o u n d in all experim e n t s performed. However a n o t h e r polypeptide with a molecular weight of a b o u t 65,000 d a l t o n s also m i g h t be of viral origin a l t h o u g h it was n o t consistently e n c o u n t e r e d in the polypeptide p a t t e r n . I t was t h o u g h t t h a t this p r o t e i n m i g h t be a n u n c l e a r e d precursor of one of the s t r u c t u r a l proteins which is only sometimes i n c o r p o r a t e d into the virion. Several other proteins with molecular weights in the range of a b o u t 30,000--35,000 a n d 18,000--25,000 were f o u n d sometimes. I t appeared t h a t these polypeptides were d e g r a d a t i o n products which arose from proteolytie cleavage because t h e y occurred m a i n l y i n older preparations.
342
P.-J. E~z~A~ 55 46
and
F. WEILAND
:
36
~5 ¸
%
10 ¸
×
E
¢:L U
5
to
15
20
25
30
~
~5
50
5s
60
fractions
Fig'. 1. PAGE of hog" cholera virus proteins Hog cholera virus labeled with [3aS]methionine was disrupted with SDS, mixed with unlabeled marker proteins and eleetrophoresed in polyacrylamide gels
Viral Glycoprotein8 By labeling of purified hog cholera virus with [3H]sodium borohydride it was shown that the two polypeptides 54,000--56,000 and 45,000--47,000 contained carbohydride chains (Fig. 2A). These proteins were therefore designated gp55 and gp46. Ill addition to the gp55 and gp46 several larger and minor glycoproteins with molecular weights above 70,000 and below 30,000 were labeled also. It seems that these products originated either from the enzymatic system used for the labeling procedure or arose from proteolytie cleavage of larger proteins possibly by the proteolytic activities described for the enzyme galactose oxidase (20). Only the labeled glycopeptides gp55 and gp46 coincided with the peptides shown in Figure 1. Of the three main peaks gp55, g p ¢ 6 , and p36, gp46 was usually most prominent (Fig. 1). It was also shown that this main structural protein was present in the supernatant of infected cells. After pelleting the virus labeled with [35S]methionine the proteins of the supernatant were precipitated by ammonium sulfate (30 per cent, w/w). In PAGE it was shown that gp46 was the main labeled product in the supernatant (Fig. 2B). In the supernatant of uninfected cells, this glycopeptide was never found. Radioimmune Precipitation o/Hog Cholera Virus It seemed, further necessary to prove that the demonstrated viral polypeptides were specific for hog cholera virions, because it has been shown by LAIJDE (13) using ~ntracel]nlar virus harvested by disruption of the infected cells that hog cholera virions may be contaminated with cellular membranes after isopycnic gradient eentrifugation. Material from cells was found in the same density region as hog cholera virions. Therefore we tried to show by radioimmune precipitation
Structure of I-Iog Cholera Virus
343
A
4055
46
30,
% x
20
E
c~
®
.... ® 5
I0
15
20
25
30
35
~0
45
50
55
60
fractions
g
Is46
5
10
15
20
25
30
35
/,0
/.5
50
55
60
fractions
Fig. 2. PAGE of hog cholera virus glyeoproteins A. Virus labeled externally with [sI-I]sodium borohydride; t3. Supernatant of hog cholera virus infected PK-15 celts from which virions are removed by ultraeentrifugation. Molecular weights were determined by internal standardization in both runs as deseribed
which proteins were specific for hog cholera virions. Concentrated hog cholera virus labeled with [35S]methionine and purified by density gradient centrifugation was treated with immune serum from pigs and then precipitated by a second antibody from rabbit against pig IgG. The precipitate was washed and redissolved in TEN-buffer containing SDS and electrophoresed in a slab gel. In Figure 3 (right panel) an autoradiogram of a slab gel is shown. The resultant eleetrophoretie profile containing the main peaks gp55, gp46, and p36 was comparable with that shown before (Fig. 1). As a control the left panel shows an autoradiogram of hog cholera virM proteins not precipitated by immune sera. Centrifugation of hog eholera virus through a cushion of 20 per cent sucrose as it was usually done
344
P.-J. ENZlVIANN
and
F. WEILAND
:
seemed therefore necessary for effective purification. By this step material derived from cells with the same buoyant density as the virus will be eliminated as a result of its lower sedimentation coefficient.
Fig. 3. PAGE of radioimmune precipitated hog cholera virus Autoradiograms of virus-specific proteins separal~ed by SDS-polyaerylamide slab gel eleetrophoresis. The derails of slab gel eleetrophoresis and autoradiography are described in Materials and Methods. Left panel: hog cholera virus labeled with [35S]methionine and purified by density gradient centrifugation. Right panel: The same virus as in the left panel, but precipitated by immune sofa
Electron Microscopic Studies Electron microscopy of purified hog cholera virus usually revealed the structures as seen in Figure 4. When virus concentrated and purified as described was used a population of roughly spherical particles varying in size was found. The mean diameter of the virions was determined to be 4~2~L8nm. In several preparations particles were found to contain surface projections but most of the virions always seemed to be disintegrated. After fixation of infectious cell culture fluid with formaldehyde followed by concentration and purification of the virus, the particles frequently displayed a fringe of projections with a length of about 6 to 8 nm (Fig. 5).
Structure of Hog Cholera Virus
345
Fig. 4. Electron micrographs of hog cholera virus particles The virus preparation was purified as described and stained with uranyl acetate (bar = 50 nm)
Discussion
I n order to determine the glycoproteins of hog cholera virus the galaetose oxidase-tritiated borohydride method of external labeling was applied. By this treatment terminal galactosyl and N-aeetylgalactosaminyl residues are oxidized to the corresponding aldehydes b y the enzyme galactose oxidase (1). These aldehydes are then reduced with tritiated borohydride b y the method of GA~I~I~I~G and HAKO~ORI (7). Because sialic acids are often linked to subterminal galactosyl residues, the virus was treated with neuraminidase. P A G E analysis of tritiated hog cholera virus revealed to peaks (Fig. 2A). I n addition to the polypeptide 46,000 (gp46) which was earlier shown to be a glycoprotein by PAS-staining (5) the polypeptide 55,.000 (gp 55) was demonstrated to contain also carbohydrate chains. In the virus-free supernatant of infected cells gp46 was present as a main polypeptide (Fig. 2B). I n the P A G E profile of tritiated virus some radioactivity was distributed in the molecular weight region of about 10,000 to 30,000 daltons. This m a y be due to partial degradation of labeled structural proteins since it is described that the enzyme galactose oxidase contains proteolytic activities (20) and the location of these products was not reproducible. The results do not mean unequivocally, however, that the demonstrated polypeptides are the only structural constituents of hog cholera virus. A minor polypeptide could also be present in the virion but be masked in the P A G E profile by the proteolytic cleavage products. I t was shown also with other "nonarbo" Togaviruses (2, t7, 21) t h a t the polypeptide profiles in polyacrylamde gels are not as clear cut as those of A1phaviruses and Flaviviruses. With the aid of radioimmune precipitation followed b y eleetrophoresis of viral proteins we have shown that the polypeptides detected in P A G E were of
3~6
P.-J. ESIZI~A~
and
F. WEILAND
:
Fig. 5. E l e c t r o n m i e r o g r a p h s of h o g cholera, v i r u s particles fixed w i t h f o r m a l d e h y d e T h e infectious cell c u l t u r e fluid was fixed w i t h 0.5 p e r c e n t f o r m a l d e h y d e . T h e v i r u s was p u r i f i e d as described. Selected p a r t i c l e s are d e m o n s t r a t e d e x h i b i t i n g surface p r o j e c t i o n s (bar = 50 n m ) a - - c : V i r u s s t a i n e d w i t h u r a n y l a c e t a t e ; d - - g : V i r u s s t a i n e d w i t h p h o s p h o t u n g s t d e acid
Structure of Hog Cholera Virus
347
viral origin. After autoradiography of dried slab gels it was demonstrated that several proteins obviously derived from celhflar material were eliminated by precipitation of viral proteins with immune serum (Fig. 3). To avoid coprecipitation of nonviral proteins possibly enclosed within the virion, virus was disrupted with Triton X-100. The immune precipitates were allowed to form in the cold since it was found t h a t rapid precipitation at 37 ° C resulted in coprecipitation of cellular proteins (data not shown). Our system of purification of hog cholera virus seems to be efficient. Material of cellular origin which has the same buoyant density as the virus (13) was removed b y the first step of virus purification, i.e. eentrifugation of concentrated virus through a cushion of 20 per cent sucrose. In Figure 3 (left panel) several nonviral proteins were demonstrated after electrophoresis of virus which was not pelleted through the sucrose cushion, yet these proteins were removed b y our system of radioimmune precipitation. I t has been shown earlier b y DALRYM-PLE et al. (3) t h a t radioimmune precipitation of Alphaviruses is a suitable system for specific determination of viral antigens and analysis of the immunological relationship among these viruses. Combined with earlier results (5) demonstrating that similar virM pol}'peptides were also found when hog cholera virus was grown in another cell system, that these polypeptides were always absent from uninfected control cells, and t h a t radioactivity was never found in virus grown in prelabeled cells, the polypeptide structure of hog cholera virus has been reestablished and confirmed b y our data. The structural polypeptides of hog cholera virus are different from those of Flaviviruses, Rubiviruses (for a review see 10), and two other possible members of the family Togaviridae, Equine arteritis virus, and lactic dehydrogenase virus (21, 2) but are similar to those of Alphaviruses. Electron microscopically hog cholera virus appeared as a spherical particle (Fig. 5). I t was heterogeneous in size. The diameter of the virion was determined to be 4 2 ~ 8 nm. After fixation of the infections cell culture fluid with formaldehyde followed b y concentration and purification most of the particles revealed a fringe of projections with a length of about 6 - - 8 nm (Figs. 5a and b). Similar particles have also been found repeatedly in preparations without fixation but not as frequently as in fixed preparations. Morphologically hog cholera virus which is a member of the genus Pestiviruses resembles more closely the Flaviviruses than the Alphaviruses within the family Togaviridae. Together with the above mentioned similarities in the polypeptide structure with Alphaviruses and the previously published data on the viral R N A (5) hog cholera virus and Togaviruses share the major structural properties.
Aeknowledfments We thank Prof. M. Mussgay for comments and discussion. The competent technical assistance of Miss M. Pfeifer is gratefully acknowledged. This work was supported by the Commission of the European Communities (Swine fever research program).
References I..~kVIGAD,G., A~tAtCAL,D., AS~NSIO, C., HOt~ECKEI~,B. L. : The I)-galactose oxidase of Polyporus circinatus. J. biol. Chem. 237, 2736--2743 (1962), 24 Arch.Virol. 57/4
348
P.-J. ENZ~ANSr
and F. W~5~ND
: Structure of Hog
Cholera Virus
2. BRINTON-1)ARNELL, M., P~AGE~ANN, P. G. W. : Structure and chemical-physical characteristics of lactate dehydrogcnase-elevating virus and its 1%NA. J. Virol. 16, 420--433 (1975). 3. 1)AZltYMI'LE, J. M., T~A~O~O, A. Y., C~DIFF, R. D., 1%VSSEL, P. K. : 1%adioimmune precipitation of group A arboviruses. J. Immunol. 109, 426-433 (1972). 4. DrNTE]~, Z. : l:~elationship between bovine virus diarrhoea virus and hog cholera virus. Zbl. Bakt. I. Abt. Orig. 188, 475---486 (1963). 5. ENZIVIANN, P.-J., 1%EIIBERG, l-I.: The structural components of hog cholera virus. Z. Naturforsch. 32 c, 456---458 (1977). 6. FEnNEl, F. : Classification and nomenclature of viruses. Intervirology 7, 44--47 (1976). 7. GAI~IB~G, C. G., HAtCO.n~O~I, S.-I. : External labeling of cell surface galaetose and galactosamine in glycolipid and glycoprotein of h u m a n erythrocytes. J. biol. Chem. 248, 431i--4317 (1973). 8. GAaOFF, ]ft., SIgo~s, K., 1%EN~ONE~~, O. : Isolation and characterization of the membrane proteins of Semliki Forest virus. Virolog~ 61, 493---503 (t974). 9. Hogzr~EK, M., MAESS, J., LavFS, 1%.: Studies on the substructure of Togaviruses. II. Analysis of equine arteritis, 1%ubella, bovine viral diarrhoea and hog cholera viruses. Arch. ges. Virusforseh. 33, 306--318 (197t). 10. Ho:azINEy:, M. C. : The structure of Togaviruses. Progr. reed. Virol. 16, 109--156 (1973). t l . IHLE, J. iN'., HANR'/k, M. (~., 1%OBERSON, E., KE1N-2qJ!]Y,F. T. : Autogenous i m m u n i t y to endogeneous RNA tumor viruses. J. exp. Med. 139, t568--158] (t974). t2. Ko~N, G., MATTK~.E~'S,W. : On the selection of pig immune sera for conjugation in swine fever diagnosis. Commission of the European Communities. Coordination of Agricultural tgesearch. EUI~ 5486. Sere. 1)iag. Epiz. Swine fever. Amsterdam (1975). 13. LA.UDE, H. : Improved method for the purification of hog cholera virus grown in tissue culture. Arch. Virol. 54, 4 t - - 5 1 (1977). 14. 5IAT~IEKA, H. 1)., ENZi~ANN, P.-J., BaCIIIgACH, N. L., MIGL, B.: The influence of sodium dodecylsulfate from different sources on the separation of virus proteins in polyaerylamide gel electrophoresis. Anal. Biochem. 81), 9--17 (1977). 15. MUSSGAY, M., ENZ~IA~N, P.-J., HO~Z~NEK, M. C., Wm~ANO, E. : Growth cycle of arboviruses in vertebrate and arthropod cells. Prog. reed. Virol. 19, 257---323 (1975). t6. t~IUSSGAY,M., ~ V ~ I ~ D , E., S ~ o ~ E ~ , K., U ~ s c ] ~ / 4 ~ , S., E?czsIA~, P.-J. : Properties of components obtained by treatment of Semliki Forest virus with Tween 80 and Tri(n-butyl)phosphate. J. gen. Virol. ]9, 89--101 (1973). 17. P~C~ET:C, 1~. F., ZEE, Y. C. : Structural proteins of bovine viral diarrhea virus. Amer. J. vet. Res. 36, 173t--1734 (1975). 18. Scm~'6B]~5, W., S~EI)ENTO]VF, V. : Charakterisierung eines drei Jahre alten Zellstammes aus der Schweineniere u n d Untersuchungen fiber sein Verhalten gegenfiber dem Virus der Maul- u n d I4]auenseuche. Zbl. Bakt. Abt. 1, Orig. 181, 3---].6 (1961). 19. S ~ P ~ o , A. L., V~NUE~, E., M~IzE~, J. V. : Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. biophys, t~es. Comm- 28, 815--820 (1967). 20. S~FF~E~), J. B., 1)~Y, T. M. : Extrinsic labeling of MuMTV with a galactose oxidase-tritiated borohydride method. Virology 70, 247--250 (1976). 21. ZEEeERS, J. J. W., VAN n ] ~ ZmXST, B. A. M., HO~Z~NEK, M. C. : The structural proteins of Equine arteritis virus. Virology 73, 200--205 (1976).
Authors' address: Dr. P.-J. E~rZ~A~-N, Bundesforsehungsanstalt ffir Viruskrankheiten der Tiere, Waldh/iuser H6he, PauI-Ehrlich-Stral?e 28, 1)-7400 Tfibingen, Federal Republic of Germany. Received November 7, 1977
