Replicative intermediate (RI) is a unique RNA structure found in cells infected with RNA viruses.' RI sediments as a broad band, is only partially digested by ribonuclease, and sediments as a sharp band at a lower S value (13S) after ribonuclease treatment.'-3 It is termed replicative intermediate, since it is labeled with an RNA precursor, such as H3-uridine, before any other viral-specific RNA,' since label from replicative intermediate can be chased into single-stranded viral RNA,' and since parental viral RNA is incorporated intact into RI.2 4 RI may be a double-stranded RNA template with nascent, partially completed, single-stranded viral RNA chains.3 The double-stranded RNA with no nascent chains (13S component) is termed replicative form.', Since it has not been possible to purify this material until now, the properties of RI haVe been inferred from various indirect experiments. In general, RI has usually fractionated with ribosomal RNA. Thus, in a sucrose gradient, RI sediments as a broad band between approximately 23 and 16S, i.e., in the same region as the two ribosomal RNA components. Also, RI is precipitable with 1 M NaCl (along with ribosomal RNA), due to the single-stranded component.' It also fractionates with ribosomal RNA on a column of calcium phosphate according to the procedure of Bernardi and Timasheff,6 and in a two-phase polymer system consisting of dextran sulfate and polyethylene glycol.7 Although it can be partially separated from ribosomal RNA by sedimentaton in sucrose gradients of low ionic strength, the method has not proved practical for analysis or for bulk purification.7 It was, therefore, particularly gratifying to effect a remarkable separation of RI by stepwise elution from a cellulose column. Exploiting the change in chemical activity of nucleic acids in buffers containing variable amounts of alcohol, Barber separated soluble RNA from ribosomal RNA on a cellulose column.8 By further fractionation it was possible to separate RI from ribosomal RNA, as will be shown in this paper.

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Actinomycin D inhibits the synthesis of ribonucleic acid in L cells and the yield of vaccinia virus containing deoxyribonucleic acid, but it does not inhibit cellular deoxyribonucleic acid synthesis or the multiplication of Mengo virus containing ribonucleic acid. These observations serve to distinguish the replication of viral ribonucleic from ribonucleic acid synthesis which is controlled by viral or cellular deoxyribonucleic acid.

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