Replication of bacteriophage ribonucleic acid: alterations in polyribosome patterns and association of double-stranded RNA with polyribosomes in Escherichia coli infected with bacteriophage R17.

Double-stranded RNA isolated from cells infected with the RNA bacteriophage R17 has been implicated in the replicative mechanism of viral RNA.I Two types of double-stranded RNA have been described in cells infected with RNA viruses: the intact double strand of molecular weight 2 X 106,2, 3 knowA as replicative form,4 and the population of double-stranded molecules containing nascent single strands, known as replicative intermediate.5The description of replicative intermediate iso- lated from infected cells by phenol extraction and its relation to in vitro replicating RNA structures has been discussed.'-3Besides removal of the RNA-dependent RNA polymerase from the replicating structure, phenol could possibly dissociate some of the hydrogen-bonded single strands from the double strand.3Therefore it was necessary to investigate the localization of intracellular viral RNA-replicating structures prior to phenol extraction.These structures could be associated with cell membranes, or with ribosomes, or not associated with any recognizable cellular structures.In order to obtain meaningful data it was first necessary to obtain re- producible patterns of polyribosomes in uninfected cells.This was accomplished by adding poly-L-ornithine to the media during preparation of spheroplasts and lysate.This polyamino acid has been used to enhance titers of infectious RNA from polio- virus,6 presumably by protecting the viral RNA from degradation by nucleases.The authors are particularly grateful to Dr. S. Dales of this institute for suggesting the use of this compound.