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 .apprx. 200 .mu.g (from .apprx. 5 .times. 108 infected human cervical carcinoma HeLa cells). Electrophoretic analysis of this RNA under denaturing conditions revealed a single species whose mass was estimated at .apprx. 4.8 .times. 106 daltons. EM 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 2-3 genome polarity molecules for each antigenome RNA. Hybridization studies established that all of the virus-specific polyadenylated RNA 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 9 distinct RNA species (rather than the 6 predicted measles mRNA). The significance of these observations is discussed.