Abstract
Membrane-bound replication complexes containing transcriptionally active replicative intermediates (RI) can be isolated from poliovirus-infected HEp-2 cells and consist of rosette-like structures of virus-induced vesicles surrounding the replicating viral RNA. At low ionic strength and low temperature, the rosettes reversibly dissociate into individual tubulated vesicles. As determined by immunoelectron microscopy and immunoprecipitation, the vesicles carry a set of viral structural and nonstructural proteins as well as RI RNA. At 30 degrees C, the vesicles reassociate into rosettes synthesizing plus-strand RNA in the RI. The in vitro transcriptional activities of rosettes and vesicles kept separated by high dilution were assessed by an RNase protection assay. The synthesis of the first 178 nucleotides at the 5' end of the plus strand was considered to reflect initiation, and the detection of a 530-nucleotide fragment in the P2 genomic region was considered to reflect elongation. It could be shown that the initiation and elongation of plus strands on individual vesicles are comparable to those in rosettes, with initiation proceeding in de novo-assembled initiation complexes. By use of detergent treatment it was found that initiation, but not elongation, is dependent on vesicular membranes.