Rapid isolation of morbillivirus nucleocapsid for genomic RNA cDNA cloning and the production of specific core protein antisera.
A procedure is described for the rapid isolation of canine distemper virus nucleocapsid, free from contaminating viral non-core and host cellular proteins. Nucleocapsid isolated in this manner is amenable to ultrastructural evaluation, protein isolation for the production of monospecific hyperimmune serum, and genomic RNA isolation for cDNA cloning. Nucleocapsid (NC) and a defective NC variant (Df-NC) isolated from 5.5 x 10(7) Vero cells infected with Ond-CDV is readily visualized on cesium gradients. The calculated density for NC is 1.2976 +/- 0.0033 g/ml and 1.2458 +/- 0.0056 g/ml for Df-NC. Ultrastructurally, NC appears as long uninterrupted strands, 1.6 +/- 0.1 microns in length, 21.2 +/- 1.7 nm in diameter, with well defined capsid subunits. Df-NC are truncated with a uniform length of 85.8 +/- 7.1 nm and a 24.5 +/- 1.3 nm diameter. A total of 2.1 +/- 0.2 mu of NC protein is obtained for every 1 x 10(6) cells infected; 89.7% of this mass is represented by a 61 kDa protein (N), 8.4% by a 75 kDa protein (P), and 1.9% by a 160-200 kDa protein (L), which is in agreement with the NC constituency of other paramyxoviruses. Viral N and P proteins, purified by 7.5% SDS-PAGE, were used in the production of hyperimmune serum. Specificity was demonstrated by Western blot analysis. Both antisera were capable of detecting viral antigen in persistently and lytically CDV infected cells by indirect immunofluorescence. A single high molecular weight species of nucleic acid was isolated from purified nucleocapsids compatible with a 14.6 kb morbillivirus genome. Although the efficiency of RNA extraction from purified NC was low (14.2%), sufficient RNA was obtained for gel analysis and the establishment of genomic RNA cDNA clones.
Oglesbee
M
M
Department of Veterinary Pathobiology, College of Veterinary Medicine, Ohio State University, Columbus 43210.
Tatalick
L
L
Ringler
S
S
Rice
J
J
Krakowka
S
S
D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000276:immunology; D015153:Blotting, Western; D002213:Capsid / Q000276:immunology / Q000302:isolation & purification* / Q000648:ultrastructure; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004217:Distemper Virus, Canine / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D014758:Viral Core Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000648:ultrastructure; D014764:Viral Proteins / Q000276:immunology
D000914:Antibodies, Viral; D004279:DNA, Viral; D012367:RNA, Viral; D014758:Viral Core Proteins; D014764:Viral Proteins