Purification and Characterization of Poliovirus-induced Infectious Double-stranded Ribonucleic Acid

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.