Isolation and characterization of measles virus intracellular nucleocapsid RNA

Abstract

Protocols have been established for the preparation of large amounts of pure measles virus intracellular nucleocapsids. As a result, it has been possible to routinely achieve nucleocapsid RNA yields of approximately 200 micrograms (from approximately 5 X 10(8) infected cells). Electrophoretic analysis of this RNA under denaturing conditions revealed a single species whose mass was estimated at approximately 4.8 X 10(6) daltons. Electron microscopic assessment of nucleocapsid RNA contour lengths corroborated the electrophoretic size determination. Total nucleocapsid RNA was shown to contain both negative- and positive-stranded species distributed in a ratio of 2 to 3 genome polarity molecules for each antigenome RNA. Hybridization studies established that all of the virus-specified polyadenylated RNAs were encoded by the negative-stranded nucleocapsid RNA and, therefore, that this nucleocapsid RNA was the measles genome. Examination of the measles virus-specified, polyadenylated transcription products by HCHO-agarose gel electrophoresis revealed at least nine distinct RNA species (rather than the six predicted measles mRNAs). The significance of these observations is discussed.