Mechanism of in vitro synthesis of covalently linked dimeric RNA molecules by the poliovirus replicase.

Four RNA fragments of approximately 1,000 to 1,200 nucleotides, representing both the 5' and 3' termini of poliovirus plus- and minus-strand RNAs, were generated by transcription of poliovirus cDNA by using bacteriophage SP6 RNA polymerase. The copying of these templates by the poliovirus replicase invariably produced RNA products approximately twice the size of the templates. In experiments with templates uniformly labeled with 32P it was shown that some of the apparently double-length products were generated by extension from an internal site of the template. Filter hybridization of the labeled in vitro-synthesized products with various unlabeled templates suggested a second mechanism by which double-length molecules could be synthesized; the results can be best explained by de novo synthesis of the first strand by copying of the template RNA, followed by snap-back of the newly synthesized RNA, generating a template-primer structure for the synthesis of the second strand. Highly purified poliovirus replicase was able to support the synthesis of double-length RNA products in response to these templates. These reactions did not require host factor. In contrast, synthesis of genome-length copies of poliovirion RNA by the same replicase was absolutely dependent on added host factor. The synthesis of double-length RNA products did not require either the 3'-terminal poly(A) of plus RNA or sequences within the 3' termini of both plus- and minus-strand RNAs.