JOURNAL OF VIROLOGY, Feb. 1971, p. 241-249 Vol. 7, No. 2 Copyright ( 1971 American Society for Microbiology Printed in U.S.A. Structural Proteins of Rabies Virus FRANTISEK SOKOL, DANIEL STANCEK, AND HILARY KOPROWSKI The Wistar Institute of Anatomy and Biology, The World Health Organization International Reference Centerfor Rabies at The Wistar Institute, anid The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 Received for publication 23 November 1970 Purified rabies virions, unlabeled or labeled with radioactive amino acids or D-glucosamine, were dissociated into their polypeptides by treatment with sodium dodecyl sulfate in a reducing environment and fractionated by electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gel. The molecular weights of individual polypeptides were estimated by comparison of their rate of migration with that of protein markers of known molecular weight. Purified viral nucleocapsid and a mixture of envelope components, isolated from virions disrupted by sodium deoxycholate, were analyzed by the same procedure. The number of molecules per virion of each polypeptide was estimated from the proportions of the separated components, the known molecular weight of the viral ribonucleic acid, and the chemical composition of the nucleocapsid. The protein moiety of the nucleocapsid particle was estimated to consist of 1,713 molecules of a major polypeptide (molecu- lar weight, 62,000 daltons) and 76 molecules of a minor polypeptide (molecular weight, 55,000 daltons). In addition to 1,783 molecules of a glycoprotein com- ponent (molecular weight, 80,000 daltons), the viral envelope contains 789 and 1,661 molecules, respectively, of two other polypeptides (molecular weight, 40,000 and 25,000 daltons). The structural complexity of rabies virus (9) MATERIALS AND METHODS indicates that it is composed of several distinctive Virus. The 1210/A clone of the high-egg-passage proteins. It was reported recently that treatment (HEP) Flury strain (21) and the W clone of the ERA with sodium deoxycholate (DOC) causes disrup- strain (14) of rabies virus, isolated by T. J. Wiktor, tion of the protein-lipid envelope of rabies virions were used in all experiments. and release of the helical nucleocapsid, a nucleo- Tissue culture. BHK/13S cells were propagated protein containing the viral ribonucleic acid in roller cultures. The 2-liter bottles were seeded with reference The virions were 4 X 107 cells in 100 ml of BHK cell growth medium (RNA; 22). disrupted (17) supplemented with 10% fetal calf serum and fractionated into nucleocapsid and a mixture of were incubated at 37 C. The cell cultures became coat components on the basis of markedly differ- confluent in about 3 days and contained, at the time ent rates of sedimentation and buoyant density. of infection, approximately 2.4 X 108 cells per bottle. Several properties of purified nucleocapsid prep- BHK-21 cells were grown in 1-liter Blake bottles as arations have been described, but properties of described previously (21). the individual coat components remain largely Infection of the cells and labeling of the virus. unknown. Cultures of BHK/13S or BHK-21 cells were infected In the present study, purified rabies virions or with 1 plaque-forming unit of virus per cell, as their dissociated treatment with described previously (21). After 1 hr of adsorption components, by at 35 C, the inoculum was removed and the cells sodium dodecyl sulfate (SDS) and 2-mercapto- were refed with 50 ml of Eagle's minimum essential ethanol (2-ME), were analyzed by electrophoresis medium supplemented with 0.2% bovine serum in polyacrylamide gel to determine the number, albumin (BSA). For labeling of the virus, the medium the molecular weight, and the structural identity contained 2.5 ,Ci of a mixture of 16 3H-amino acids of the polypeptides of rabies virions. Of the four per ml (average specific activity, 17.6 Ci/mmole; major polypeptides of different molecular weights, Schwarz/Mann mixture no. 3130-08; Schwarz Bio- the second largest in molecular size is associated Research, Orangeburg, N.Y.), 0.5 MACi of a mixture with the viral RNA in the nucleocapsid, whereas of 13 14C-amino acids per ml (average specific ac- tivity, 250 mCi/mmole; Schwarz/Mann mixture no. the remaining three form the viral envelope. The 3122-08, Schwarz BioResearch), or 1 ,uCi of 3H-D- envelope polypeptide with the largest molecular glucosamine per ml (specific activity, 850 mCi/ weight is a glycoprotein. mmole; New England Nuclear Corp., Boston, Mass.). 241 242 SOKOL, STANCEK, AND KOPROWSKI J. VIROL. Also for protein labeling, the concentration of the (New England Nuclear), in a Packard liquid scintil- cold amino acids in the medium was reduced to one- lation spectrometer. fifth of the regular level. The cultures were incubated Protein standards for determination of molecular at 33 C. Extracellular virus was harvested approxi- weight. The following proteins, purchased from Mann mately 72 hr after infection. Research Laboratories, New York, N.Y., were used Purification of the virus and isolation of viral com- as markers for molecular weight determination: ponents. The virus purification scheme included human gamma globulin (160,000 daltons), BSA precipitation by zinc acetate, desalting on a Sepha- (67,000 daltons), ovalbumin (45,000 daltons), whale dex column, treatment with ribonuclease and deoxy- sperm myoglobin (17,800 daltons), and horse heart ribonuclease, pelleting by high-speed centrifugation, cytochrome c (12,400 daltons). and banding of the virus by centrifugation in a sucrose density gradient (21). Procedures for the RESULTS fractionation by velocity centrifugation in a sucrose density gradient of DOC-disrupted virions into nu- Polypeptides of rabies virions. When purified cleocapsid and a mixture of coat components, as rabies virions, labeled with radioactive amino well as the purification of the nucleocapsid by iso- acids, were disrupted by treatment with SDS and pycnic centrifugation in CsCl solution, have been 2-ME and analyzed by electrophoresis in poly- previously described (22). Protein determination. Protein was assayed as acrylamide gel, four major peaks of radioactivity described by Lowry et al. (16) by using BSA as were repeatedly observed (Fig. la). Similarly, the standard. optical density tracing of virus polypeptides, frac- Standard procedure for the preparation and elec- tionated by the same procedure and stained by trophoresis of polypeptides. Proteins from diluted Amido Black, showed four main bands of ab- solutions were precipitated at 0 C with 10% trichloro- sorbance (Fig. lb). The peaks of the stainable acetic acid. The precipitate collected by low-speed bands coincided with those of the radioactive centrifugation was washed at 0 C with 2 ml of 2% bands (Fig. la, inset). The radioactivity or the trichloroacetic acid and then with the same volume absorbance recovered in the four main bands of ice-cold acetone. The sediment was then dried in a stream of warm air and dissolved in 0.01 M phosphate represented 85 to 90% of the total radioactivity or buffer (pH 7.2) containing 3.33% (w/v) SDS and absorbance in the gel. A variable amount (5 to 0.67%7o (v/v) 2-ME. If necessary, the pH of the solu- 12% of the total) of polypeptides of a molecular tion was adjusted to 7.2 by addition of a few micro- size larger than the slowest of the four major com- liters of 1 N NH40H. In all instances, more than 94%O ponents was detected in each preparation of dis- of the original amino acid or glucosamine label was assembled virus. For reasons which will be given recovered in the dissolved precipitate. Sufficiently later, we believe that most of these components concentrated samples were directly adjusted to 3.33% represent aggregates (dimers) of the major poly- in SDS and dialyzed at room temperature overnight peptides. Peaks of radioactivity observed in elec- against the SDS- and 2-ME-containing phosphate cellular buffer. The polypeptide samples were stored at 4 C. trophoretograms of dissociated proteins. Sucrose was then added to make a final concentration obtained from cell-free fractions of mock-infected of 15%. Samples not exceeding 0.2 ml in volume and cultures and subjected to the same sequence of containing 20 to 400 ,ug of polypeptides were heated fractionation procedures used for the purification at 100 C for 1 min (18) and analyzed. of the virus did not coincide with the peaks of Nine volumes of a solution containing 7.5% acryl- viral polypeptide bands (Fig. 2). Therefore, con- amide, 0.27% N,N'-methylenebisacrylamide, 0.1% taminating cellular proteins would be easily de- SDS, and 0.05% N,N,N',N'-tetramethylethylene- tected in electrophoretograms of insufficiently diamine in 0.1 M sodium phosphate buffer (pH 7.2), purified preparations of dissociated virions. Al- were mixed with 1 volume of 1%/ ammonium per- the sulfate and polymerized to form gels 20 cm in length though the material not contained in four and 0.6 cm in thickness. The gels were prerun in the large bands probably represents either aggregates electrophoresis buffer (0.1 M phosphate buffer, pH of the major viral polypeptides or cellular contam- 7.2, containing 0.1% SDS) for 4 hr at 2 ma per gel. inants, it is possible that minor structural proteins The sample was then placed on the top of the gel of the virus were not detected by the fractionation and overlaid with the electrophoresis buffer. Elec- procedure used. trophoresis was carried out at 2 ma per gel for 20 Other methods of releasing rabies virus poly- min and then at 5 ma per gel for 11.2 to 16.5 hr. peptides, such as treatment with SDS and 2-ME Some gels were stained for 2 hr at 20 C with 1% Amido Black B-10 in 7% acetic acid and then de- at 37 C with or without previous precipitation of stained in several changes of 7% acetic acid.