Newcastle disease virus was found to contain three major proteins. The structure unit of the viral nucleocapsid appears to be monomeric and to consist of a single large protein of an approximate molecular weight of 62,000. Newcastle disease virus (NDV), a subgroup II myxovirus, consists of a helically arranged ribonucleic acid (RNA) containing nucleocapsid which is enclosed by a lipoprotein envelope (18, 24) . The viral envelope contains at least two distinct active proteins, a hemagglutinin

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... a viral neuraminidase, but the total number of the different proteins of the viral envelope is unknown. The structures of the nucleocapsids of several parainfluenza viruses including NDV are similar to that of tobacco mosaic virus (TMV) with regard to several physical and chemical properties. The nucleoproteins of parainfluenza viruses and of TMV have a diameter of about 20 nm (11, 24) and a density of about 1.3 g/ml (in sucrose or CsCl) (1, 21), and contain about 4 to 5% RNA and 95 to 96% protein (11, 13) . The lengths of the two kinds of nucleoproteins were found to be proportional to the size of the RNA molecules which they include. Tobacco mosaic virus has a length of 0.3 Am and contains an RNA of a molecule weight of 2 X 106 (11) and the nucleoprotein of parainfluenza viruses contains an RNA of about 6.5 X 105 (3, 18) and has a length of about 1 ,um (1, 12). Although it is known that the structure units of TMV consist of monomeric proteins, it is not known whether the capsomers of the nucleocapsids of parainfluenza viruses also consist of single proteins or whether they consist of several proteins. The subject of the present investigation was the isolation of the different proteins of the virus. Three distinct major proteins of NDV were isolated by polyacrylamide gel electrophoresis. A single protein was found to be associated with the viral nucleocapsid. Similar independent findings were recently reported by Evans and Kingsbury (6). METHODS AND MATERIALS Virus growth. The NDV strain, NDV-Beaudette, obtained from D. W. Kingsbury, St. Jude Hospital, Memphis, Tenn., was used in all experiments. The virus was grown on lung cultures of 15to 17-day-old chick embryos. The lungs were dispersed by stirring in tris(hydroxymethyl)aminomethane (Tris) saline containing Pronase (2.5 mg/ml) for 30 to 60 min at room temperature. Cultures were seeded at 2 X 107 to 4 X 107 cells per 10-cm plastic dish and cultured for 1 day prior to infection (19). After the medium was removed, the cells were incubated with about 3 ml of a twofold dilution of stock virus in Tris-saline. Stock virus consisted of allantoic fluid of infected chick embryos containing about 109 plaque-forming units (PFU)/ml (4). After incubation for 30 min at 37 C, the inoculum was replaced by 6 ml of amino acid-free (except for glutamine) medium 199 supplemented with 0.1% (w/v) lactalbumin hydrolysate, 0.1% (w/v) glucose, and 0.2 pAg of Fungazone per ml. Incubation was continued for 24 hr in the presence of 10 to 50,uc/ plate of 3H-amino acids (specific activity, 5 c/mmole), or 14C-amino acids (specific activity, 0.3 c/mmole) or 50 to 100 p&c/plate of 3H-uridine. At the end of this period, the medium was removed for virus purification. The cells had become rounded and partly detached from the plate. The hemagglutinin (HA) titer of such medium was usually about 160 HA units per ml. Virus purification. This process was a modification of the procedure described previously (4). Virus was purified in essentially two steps. First, the virus was concentrated from the medium of virus-producing cells by precipitation with an equal volume of saturated ammonium sulfate. The pellet was then redissolved in standard buffer [0.1 M NaCl, 0.01 M Tris (pH 7.4), 1 mt ethylenediaminetetraacetate (EDTA)] and concentrated by sedimentation on a sucrose cushion of a greater density than that of the virus. The concentrated virus was then transferred from the density interface and after appropriate dilution layered on a preformed sucrose density gradient in the same buffer. After centrifugation, viral infectivity coincided with radioactivity and optical density in a density range from 1.20 to 1.25 g/ml. From 50 to 100% of the 388 on May 9, 2020 by guest