Structural Proteins of Reoviruses

P. C. Loh, A. J. Shatkin
1968 Journal of Virology  
Polyacrylamide gel electrophoresis of the solubilized proteins from the three serotypes of reovirus revealed that each contained three major and four minor components. Subviral particles were prepared by brief treatment of complete virions with urea. Electron microscopy, density-gradient centrifugation, and chemical analyses of these particles indicated that their outer capsid structure had been selectively removed. They contained only two proteins, but their ribonucleic acid composition was
more » ... ilar to that of complete virions. The subviral particles were not infectious. The reovirus group includes three serological types which share a common complement-fixing antigen but are distinguishable by neutralization and hemagglutination-inhibition reactions (15). Electron microscopic examination of purified reovirus particles has shown that all serotypes consist of three principal morphological components: (i) the outer capsid, (ii) an inner structural layer, and (iii) a core containing the viral ribonucleic acid (RNA; 9, 13, 26). In contrast to the extensive information available on the properties of the double-stranded, segmented genome and the adenine-rich RNA of reoviruses (1-3, 20, 28), their structural proteins have not been characterized. The present investigation was undertaken to analyze and compare the proteins of all three serotypes and to relate them to the morphological features of purified virions. MATERIALS AND METHODS Cells antd viruses. Mouse L-929 cells were grown in suspension or stationary culture in Eagle's medium supplemented with 5% fetal calf serum. Reovirus types 1 (Lang), 2 (D-5 Jones), and 3 (Abney) were obtained from the American Type Culture Collection. The procedures for reovirus infection of cells and virus purification have been described (18, 19) . Preparationt of radioactive virus. Monolayer cultures of L cells were infected at an input multiplicity of 1 to 10 plaque-forming units per cell. After a 3-hr adsorption period, fresh medium containing 2%77 fetal calf serum, one-tenth the usual concentration of amino acids, and 0.1 Ac of reconstituted 3H-labeled protein hydrolysate per ml (Schwarz Bio Research Inc., Orangeburg, N.Y.) were added. The cells were incubated at 37 C for 2 to 4 days, harvested, and the 1 Present address: Department of Microbiology, University of Hawaii, Honolulu. 1353 virus was purified. Similar procedures were employed for the preparation of 32P-labeled virions, except that phosphate-free medium was employed (20). Solubilization of virion2s. Purified virus suspended in phosphate-buffered saline at a protein concentration of 150 to 500 ,ug/ml was degraded by treatment with 2% sodium dodecylsulfate (SDS), 1%cYG 2-mercaptoethanol (ME), and 8 M urea for 2 to 3 hr at room temperature. The samples, dialyzed for 2 to 3 hr against 0.1% SDS, 0.1%7 ME, and 8 M urea in 0.01 M phosphate buffer (pH 7.2), were then applied to polyacrylamide gels. Polyacrylamide-gel electrophor0esis. Gels were made as described previously (24) and consisted of 10% acrylamide, 0.26%C0 N, N'-bis-methylene acrylamide, 0.1% SDS, and 0.1 M phosphate buffer (pH 7.2). Samples (100 to 300 ,ug) of viral protein in 0.05 to 0.2 ml of buffer containing 10% sucrose were electrophoresed for 16 to 24 hr at 4 mamp per gel at 20 C. After electrophoresis, the gels were fixed in 10% trichloroacetic acid, stained with 0.025% Coomassie blue, and destained in 7% acetic acid (11). Gels containing radioactive proteins were frozen, sliced, solubilized in H202, and counted (21). Preparation of subviral particles (S VP). Among the several methods tested, it was found that the outer capsid component could be selectively removed from the complete virion by treatment with 4 M urea in phosphate-buffered saline for 3 min at 4C (8, 25). The SVP were then separated by isopycnic sedimentation in CsCl solutions. Immunodiffusioni. The double gel-diffusion method of Ouchterlony was carried out as described by Campbell et al. (5). Antireovirus chick sera for all three serotypes were kindly supplied by Philip Coleman of the Communicable Disease Center, Atlanta, Ga. The antisera had titers of 1/1,280 by hemagglutinationinhibition reaction; with heterologouw serotypes, the titers were less than 1/10. RNA antd proteini anialysis. Orcinol (12) and Lowry (10) procedures were employed to measure RNA and protein, respectively. The base composition of viral on May 9, 2020 by guest
doi:10.1128/jvi.2.11.1353-1359.1968 fatcat:xxqejsoqwrgf3oi4meygll7wau