Abstract
Abstract Ribonuclease-resistant infectious double-stranded RNA has been isolated in milligram quantities from poliovirus-infected HeLa cells. Purification was accomplished by eliminating high molecular weight single-stranded RNA by differential NaCl precipitation and chromatographing the remaining RNA on columns of methylated albumin-Kieselguhr. The product has the following physicochemical and biological properties: (a) Homogeneity with respect to size, as determined by sedimentation analysis; (b) an s20w, of 17.2, which is compatible with the molecular weight of 4 x 106 anticipated for poliovirus double-stranded RNA; (c) buoyant density in Cs2SO4 of 1.60 g cm-3; (d) abrupt hyperchromic shift of 25% on heating, the value of Tm (temperature at midpoint) being dependent upon ionic environment; (e) nucleotide composition consistent with a base-paired, double-stranded secondary structure; (f) infectivity which is partially resistant to RNase and resistant to formaldehyde; (g) specific infectivity 30-fold greater than that of poliovirus single-stranded RNA; (h) optimal conditions for assay of infectivity which differ from those of single-stranded RNA. It is concluded that the RNase-resistant infectivity derives from information contained in a double-stranded molecule and that the replicative cycle initiated by double-stranded RNA must differ from the cycle initiated by single-stranded RNA. The fact that double-stranded RNA is infectious has definite implications regarding the possible functional role of this material in the virus growth cycle.
Highlights
MethodsPropagation of Cells and Virus-Strain S3 HeLa cells were grown in suspension at cell densities between 2 and 5 x lo cells per ml in Eagle’s medium supplemented with 5% horse serum
Buoyant density in Cs2S04 of 1.60 g cmF3; (d) abrupt hyperchromic shift of 25% on heating, the value of T, being dependent upon ionic environment; (e) nucleotide composition consistent with a base-paired, double-stranded secondary structure; (f) infectivity which is partially resistant to RNase and resistant to formaldehyde; (g) specific infectivity 30-fold greater than that of poliovirus single-stranded RNA; (h) optimal conditions for assay of infectivity which differ from those of single-stranded RNA
Animal virus replicative form is of unique interest because, in contradistinction to that of bacterial
Summary
Propagation of Cells and Virus-Strain S3 HeLa cells were grown in suspension at cell densities between 2 and 5 x lo cells per ml in Eagle’s medium supplemented with 5% horse serum. Labeling of Infected Cells with 32P-Ceils concentrated in suspension (4 x lo cells per ml) were incubated with actinomycin D (gift of Merck Sharp and Dohme), 5 pg per ml, for 1 hour at 37”, infected in phosphate-free medium in the presence of carrier-free 32P-orthophosphate (International Chemical and Nuclear Corporation City of Industry, California) at 25 PC per ml. Under these conditions, transfer RNA is the only form of host cell RNA that is labeled [10]. The cell suspension was stirred with an equal volume of phenol (Mallinckrodt, reagent grade) at 60” for 10 min and cooled in an
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