VIROLOGY

94,

4’74-478 (1979)

Heterogeneity

of Envelope Polypeptides among Strains of Venezuelan Encephalitis Virus1 M. E. WIEBE2

Department

of Microbiology,

Cornell

AND W. F. SCHERER

University

New

York

Medical 10021

Accepted December

College,

1300

York

Avenue,

New

York,

2.4, 1978

Comparative virion polypeptide analysis of 29 strains of purified Venezuelan encephalitis virus by electrophoresis side by side on discontinuous, slab polyacrylamide gels revealed heterogeneity in electrophoretic mobility and apparent numbers of polypeptides. All strains had two common proteins: a capsid protein of 36,000 molecular weight, and an envelope glycoprotein of 50,000-51,000 molecular weight. Another glycoprotein ranged in molecular weight between 51,000 and 55,000. Eight strains yielded an additional glycoprotein peak in the range of 56,000-58,000; two others showed a small glycoprotein peak at 45,00046,000. These polypeptide patterns were not influenced by the cell species (chick, hamster, monkey) in which the virus was cultured.

Strains of the alphavirus Venezuelan encephalitis (VE) are biologically diverse. They are distributed geographically as antigenie subtypes and varieties, and some strains differ remarkably with respect to virulence for people, equines, hamsters, and guinea pigs, epidemiologic behavior, vector competence, and plaque morphology (1-8). In an attempt to understand the molecular basesfor this biologic diversity, biochemical composition of virions, and synthesis of viral components are being studied. We now report on the initial phase of this investigation in which comparative eleetrophoresis of virion polypeptides on discontinuous SDSpolyacrylamide slab gels has demonstrated a strain heterogeneity. Primary chicken embryo cells (CEC) and Vero African green monkey kidney cells were cultured as described previously (9, 10). Primary hamster embryo cell (HEC) cultures were made from 13-day-old embryos by the method used for CEC except 1 This study was sponsored by the U. S. Army Medical Research and Development Command, Fort Detrick, Frederick, Maryland 21701, under Contract DADA 17-72-C-2140. 2 To whom requests for reprints should be addressed. 004%6822/79/060474-05$02.00/0

Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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culture medium contained 5% newborn calf serum. Of the 29 VE virus strains initially examined the six selected for further study were 69Zl (11), CBSI-9 (3), 68U.201 (IO), BeAr35645 (Pixuna) (Z), Fe5-47et (12), and 3880 (1.3). All were at low passagelevel (< 14) sinceoriginal isolation and three strains (69Z 1, 68U201, and BeAr35645) were cloned by three successive plaque selections before use. VE virus strains were propagated by inoculating l-day monolayers of CEC or 5-day monolayers of HEC at multiplicities ranging from 10e3to 10-l CEC plaque-forming units (PFU)/cell. Culture fluid was harvested 16-48 hr after infection at 37” and development of cytopathology in 5% or more of the cells. Virion polypeptides were labeled with [14C]amino acids by adding [14C]-labeled reconstituted protein hydrolysate (Schwarz/ Mann, Algal profile) to culture fluid [amino acid- and serum-free maintenance solution (9)] at a concentration of 30 $X1.5 X 10’ cells (7.5 pCi/ml culture fluid) 6 hr after infection; the approximate virus multiplicity was 10 PFU/cell and incubation was at 3’7”. Culture fluid was harvested 16-24 hr after infection. Virion glycoproteins were labeled with D-[l-J4C]glucosamine (Schwarz/Mann,

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A

abcdef

+

Origin

-

67,000

‘Origin

C

67,000

C

55,000

C

45,000

FIG. 1. Autoradiograms of electrophoretically separated virion polypeptides of five selected VE virus strains representing the spectrum of polypeptide diversity. Purified virus was dissociated into component polypeptides by boiling for 2 min in a solution containing SDS (LO%), 2-mercaptoethanol (l.O%), and glycerol (20%). Polypeptides were separated by electrophoresis at 25 mA on discontinuous vertical polyacrylamide slab gels consisting of a g-cm high resolving gel (10% acrylamide and 0.27% N,N’-methylene bisacrylamide in 375 m&f Tris buffer, pH 8.9, containing 0.1% SDS and 0.05% TEMED) covered with a l-em high stacking gel (6% aeryiamide and 0.16% N,N’-methylene bisacrylamide in 125 mM Tris buffer, pH 7.5, containing 0.1% SDS and 0.05% TEMED). The circulating electrode buffer contained 50 mM Tris, 0.1% SDS, and 270 mM glycine, pH 8.5. Following electrophoresis, gels were fixed in a solution containing 10% TCA and 25% 2-propanol for 1 hr, stained with Coomassie brilliant blue (0.1% Coomassie brilliant blue R-250 in 10% TCA and 25% 2-propanol) for 2 hr, and destained in 7% acetic acid 24-72 hr. Gels were then dried and autoradiograms prepared. Autoradiogram tracks in (A) were made from a single discontinuous SDS-polyacrylamide slab gel after electrophoresis for 3.25 hr, and represent different purified VE strains labeled with a [WIamino acid mixture and autoradiographic exposure times as follows: (a) 69Zl (7 days), (b) Fe5-47et (7 days), (c) CBSI-9 (7 days), (d) CBSI-9 (30 days), (e) 68U201 (3 days), and (f) BeAr35645 (30 days). (B) represents a different gel in which viral proteins were electrophoresed for 4 hr to resolve the polypeptides of (g) 68U201 and (h) BeAr35645. Positions of molecular weight standards (bovine albumin, 67,000; human y-globulin heavy chain, 55,000; and ovalbumin, 45,000) are indicated. Direction of electrophoresis was from top to bottom.

57 mCi/mmol) by the addition of 5 &i to 1.5 x lo7 cells (1.25 &i/ml culture fluid) immediately following infection at a multiplicity of 10 PFU/cell; 5 mM fructose was substituted for glucose in the medium and culture fluid was harvested 16-24 hr after infection. Virus in culture fluid was purified by a sequence of differential centrifugation (10,000 g for 1 hr at 5” to pellet cell debris; 80,000 g for 4 hr at 5” to pellet virus), rate-zonal eentrifugation [SO,000 g for 2 hr at 5" in 10 to 30% (w/v> sucrose], and equilibrium centrifugation [SO,000 g for 14 hr at 5” in 30 to 60% (w/v) sucrose]. Purified virus, concentrated by pelleting (80,000 g, 5 hr, 5”), was

resuspended in TNE buffer (Tris, 50 mM; NaCl, 100 m&f; EDTA, 1.0 n-&f, pH 7.4). Twenty-nine VE virus strains encompassing all HI subtypes and various geographic and host sources were studied. All strains had two common polypeptides with molecular weights of approximately 36,000 and 50,000-51,000. A third polypeptide was found in all strains and ranged in molecular weight from 51,000 to 55,000. Eight strains yielded an additional polypeptide peak of 56,000 to 58,000; two others showed a small peak at 45,000-46,000. Autoradiograms of representative polypeptide patterns are presented in Fig. 1. Although the 45,000 to 46,000 molecular weight protein was dif-

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ficult to see in autoradiograms, it was clearly demonstrated by absorbency scans (Fig. 2, strain CBSI-9). To assess the influence of host cells on virion structural polypeptide composition, virus cultured in chicken embryo and hamster embryo cells (strains 69Z1, CBSI-9, 3880, and BeAr35645), and Vero African green monkey kidney cells (strain BeAr35645) were compared by polyacrylamide gel electrophoresis. Results showed that the polypeptide electrophoretic patterns were not influenced by the cell species in which the virus was cultured. VE virion envelope polypeptides were identified by treating purified [14C]amino acid-labeled virus (strain 69Zl or 68U201) with the nonionic detergent, Triton X-100 (37” for 1 hr) to release envelope associated polypeptides; removing nucleocapsids by rate-zonal centrifugation [lo to 30% (w/v> sucrose gradient, 80,000 g, 3 hr, 57; and detecting soluble proteins by polyacrylamide gel electrophoresis and autoradiography. Results (not shown) revealed the presence of all virion polypeptides in the 50,00056,000 molecular weight range, but the polypeptide of 36,000 molecular weight was absent. This indicates that the virion polypeptides of 50,000 to 56,000 molecular weight are envelope associated, whereas the 36,000-molecular weight polypeptide is presumably associated with nucleocapsids. These results are similar to those obtained for other alphaviruses (18 -16). Five strains of virus representing the spectrum of polypeptide patterns were labeled with D-[lJ4C]glucosamine and electrophoresed side by side with virus labeled with [14C]amino acids. All proteins were found to be glycosylated except the one of 36,000 molecular weight (Fig. 2). An additional minor glycoprotein of about 86,000 molecular weight was detected in strains 69Z1, CBSI-9, and 68U201. However, lengthy autoradiography (2-4 weeks) of gels containing [ 14C]amino acid-labeled virion polypeptides was required in order to detect this minor protein. Variability of envelope glycoprotein composition among strains of alphaviruses other than VE, has not yet been studied extensively. Protein compositions of only two

6921

Fe5-47et

E CBSI-9

68U201

BeAr35645 iPixunal

FIG. 2. Absorbency scans of autoradiograms of proteins of five VE virus strains representing the spectrum of virion polypeptides observed by PAGE. Polyacrylamide gel electrophoresis and autoradiography were as described in legend to Fig. 1. Autoradiograms were scanned at 550 nm with a Gilford 250 recording spectrophotometer equipped with a linear transport. Solid lines signify [W]amino acid-labeled proteins and broken lines indicate proteins labeled with D-[lJ*C]glucosamine. Electrophoresis was from right to left.

strains of Sindbis virus have been reported although numerous strains have been isolated from natural sources in Africa, Asia, and Australia (17-19). Investigations of only one strain of Semliki Forest virus have been recorded. However, heterogeneity among serotypes in the electrophoretic mobility of virion polypeptides has been described for a number of other viruses. Poliovirus type 1 has a larger VP2, but a smaller VP3 than type 2 (2.2). Substantial heterogeneity among corresponding reovirus polypeptides

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was found by Ramig et al. (23). Electrophoretic analysis of 16 blue tongue virus serotypes showed variation in at least one of the capsid polypeptides (24). Examination of 53 strains of herpes simplex virus type 1 revealed seven electrophoretically variable polypeptides (25). Variation has also been reported for three viral polypeptides of the Indiana and New Jersey serotypes of vesicular stomatitis virus (26). The existence in some VE virus strains of three apparent glycoproteins in the 45,000 to 58,000 molecular weight range is unlike Sindbis (with only two envelope proteins) and Semliki Forest [where the third envelope protein has a molecular weight of 10,000 (20, 16)]. It has been estimated that the 26s mRNA of Sindbis virus has a coding capacity for proteins totaling approximately 130,000 daltons (21). Although some VE virus strains appear to contain proteins of molecular weights 36,000, 51,000, 53,000, and 56,000 (total 196,000), it seems unlikely that a presumed 26s mRNA could contain enough genetic information to code for all of these structural polypeptides. This paradox could be explained if one of the following were true: (1) the mRNA of these VE virus strains is larger than 26 S, (2) the virus preparations are mixtures of two genetically distinct virus populations, each with two glycoproteins, one common and one different, or (3) the virus populations are genetically homogeneous but two of the three glycoproteins are products of the same gene. The tirst possibility is currently being tested by characterizing molecular weights of VE viral RNAs synthesized during infection. The possibility of mixed virus populations seems unlikely since one of the strains having three glycoproteins (69Zl) was carefully cloned. Concerning the third possibility, glycoproteins of slightly different molecular weights could be products of the same gene if they underwent different post-translational modifications, e.g., ambiguous proteolytic post-translational cleavage or different glycosylations. Ambiguous cleavage of poliovirus-specific precursor proteins has been reported (27, 22). In support of ambiguous cleavage it should be noted that the three envelope glycoproteins were not found in equal molar quantities (Fig. 2) as

477

would be expected if they are unique gene products, derived post-translationally from the same polyprotein. The synthesis and processing of envelope glycoproteins of VE virus strains will be the subject of a subsequent paper. If two of the three glycoproteins are products of the same gene, a great deal of sequence homology should exist between them. This is being tested by peptide mapping. Whether differences in the glycoprotein composition of VE virus strains are partly responsible for their biologic diversity was beyond the scope of this investigation. However, there appears to be no simple correlation with virulence for equines or laboratory animals as described by others (1, 7’) or epizootic-epidemic potential (3). There seemed, however, to be some correlation between HI antigenic subtypes and electrophoretic patterns of virion glycoproteins. ACKNOWLEDGMENTS We thank W. J. Peick technical assistance.

and J. Chin

for their

expert

REFERENCES 1. JOHNSON, K. M., and MARTIN, D. H., Adv. Vet. Sci. Comp. Med. 18, 79-116 (1974). 2. SHOPE, R. E., CAUSEY, 0. R., DE ANDRADE, A. H. P., and THEILER, M., Anzer. J. Trap. Med. Hyg. 13, 723-727 (1964). S. YOUNG, N. A., and JOHNSON, K. M., Amer. J. Epidemiol. 89, 286-307 (1969). 4. SCHERER, W. F., and PANCAKE, B. A., Amer. J. Epidemiol. 91, 225-229 (1970). 5. SCHERER, W. F., and PANCAKE, B. A., J. Clin. Microbial. 6, 578-585 (1977). 6. FRANCK, P. T., and JOHNSON, K. M., Amer. J. Epidemiol. 94, 487-495 (1971). ‘i. SCHERER, W. F., and CHIN, J., Amer. J. Trop. Med. Hyg. 26, 307-312 (1977). 8. KRAMER, L. D., and SCHERER, W. F., Amer. J. Trap. Med. Hyg. 25, 336-346 (1976). 9. SCHERER, W. F., Amer. J. Pathol. 45, 393-411 (1964). 10. SCHERER, W. F., ANDERSON, K., PANCAKE, B. A., DICKERMAN, R. W., and ORDONEZ, J. V., Amer. J. Epidemiol. 103, 576-588 (1976). 11. SCHERER, W. F., “Venezuelan Encephalitis,” pp. 329-330. Pan American Health Organization Scientific Publication No. 243. Washington, D. C., 1972. 12. CHAMBERLAIN, R. W., SUDIA, W. D., WORK, T. H., COLEMAN, P. H., NEWHOUSE, V. H.,

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SHORT COMMUNICATIONS and JOHNSTON,J. G., Amer. J. Epidemiol. 89. 197-210 (1969). JOHNSON,K. M., SHELOKOV,A., PERALTA, P. H., DAMMIN, G. J., and YOUNG, N. A., Amer. J. Trop. Med. Hyg. 17, 432-440 (1968). PEDERSEN, C. E., JR., SLOCUM, D. R., and EDDY, G. A., Infect. Zmmun. 8,901-906 (1973). STRAUSS,J. H., JR., BURGE, B. W., PFEFFERKORN, E. R., and DARNELL, J. E., JR., Proc. Nat. Acad. Sci. USA 59, 533-537 (1968). GAROFF, H., SIMONS, K., and RENKONEN, D., Virology 61, 493-504 (1974). TAYLOR,R. M., HURLBUT, H. S., WORK, T. H., KINGSTON, J. R., and FROTHINGHAM, T. E., Amer. J. Trop. Med. Hyg. 4, 844-862 (1955). SHAH, K. V., JOHNSON,H. N., RAO, T. R., RAJAGOPALAN,P. K., and LAMBA, B. S., Ind. J. Med. Res. 48, 300-308 (1960).

19. DOHERTY, R. L., CARLEY, J. G., MACKERRAS, M. J., and MARKS, E. N., Aust. J. Exp. Biol. Med. Sci. 41, 17-40 (1963). 20, SCHLESINGER, M. J., SCHLESINGER, S., and BURGE, B. W., Virology 47, 539-541 (1972). 21. MOWSHOWITZ,D., J. Viral. 11, 535-543(1973). 22. BECKMAN,L. D., CALIGUIRI,L. A., and LJLLY, L. S. Virology 73, 216-217(1976). 23. RAMIG,R. F., CROSS, R. K., andFIELDS,B. N., J. Viral. 22, 726-733 (1977). 2.4. DEVILLERS, E. M., Intervirology 3,47-53 (1974). 25. PEREIRA,L. CASSA'I,E., HONESS, R. W., ROIZMAN,B., TERNI,M.,and NAHMIAS,A., Infect. Zmmun. 13, 211-220 (1976).

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Heterogeneity of envelope polypeptides among strains of venezuelan encephalitis virus.

VIROLOGY 94, 4’74-478 (1979) Heterogeneity of Envelope Polypeptides among Strains of Venezuelan Encephalitis Virus1 M. E. WIEBE2 Department of M...
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