Isolation and comparative study of the nucleocapsids of measles and canine distemper viruses from infected cells.
Nucleocapsids were isolated and purified from cells infected with measles and canine distemper virus (CDV). Electron microscopy of negatively stained nucleocapsids revealed the mean outside diameter was approximately 17-18 nm in each case and was not significantly different from similar measurements obtained with SV5 nucleocapsids and tobacco mosaic virus (TMV). All nucleocapsids also revealed central hollow cores which ranged from 4.3 to 5.2 nm. Direct chemical determinations showed that both CDV and measles virus nucleocapsids contained about 5% RNA. UV absorption spectra of measles and CDV nucleocapsids were typical for nucleoproteins and 280 260 nm ratios gave estimates of 5% RNA in each case. Buoyant density determinations in CsCl with both unlabeled and 32P-labeled nucleocapsids gave figures of 1.29-1.30 g/cc. Sedimentation analysis by analytical and sucrose gradient centrifugation revealed considerable heterogeneity, with nucleocapsids ranging from about 200-300 S. This heterogeneity was shown by electron microscopy to be due to the highly fragmented nature of the nucleocapsids. Analysis of CDV and measles nucleocapsids by SDS-polyacrylamide gel electrophoresis revealed a single major polypeptide when infected cells were harvested in the absence of proteolytic enzymes. These had estimated molecular weight of 54,000 (CDV) and 61,000 (measles). However, nucleocapsids prepared from cells harvested with the aid of trypsin or pronase contained two polypeptides (38,000 and 24,000 for measles; 27,000 and 22,500 for CDV), while similarly prepared SV5 nucleocapsid contained only a single polypeptide (44,500). These differences in polypeptide composition were correlated with differences in morphologic appearance of the nucleocapsids. Nucleocapsids harvested from cells in the absence of proteolytic enzymes had a loosely coiled, flexible appearance, while those harvested from cells treated with trypsin or pronase had a tightly coiled, rigid appearance. RNA was extracted from 32P-labeled measles and CDV nucleocapsids. Treatment with RNase A converted most of the 32P to a TCA-soluble form and DNase had no effect. Base compositions were determined on the 32P-labeled RNA and both viruses were rich in uracil, the molar ratios for the four bases were similar for each virus and were not significantly different from values reported in the literature for the better-characterized members of the Paramyxovirus group (NDV, Sendai and SV5).
Waters
D J
DJ
Department of Microbiology, University of Kansas, Lawrence, Kansas 66045, USA.
Bussell
R H
RH