Key result
Poliovirus transcripts with authentic 5' ends generated by a hammerhead ribozyme replicate substantially more rapidly in cell culture without a lag phase compared to transcripts with extra 5' nucleotides.
Poliovirus requires a precise 5' end for efficient positive-strand RNA synthesis, which can be achieved in vitro using a cis-active hammerhead ribozyme.
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Optimizes in vitro poliovirus models; leaves open translation to animal or clinical antiviral strategies.
Poliovirus infectious RNA can be synthesized in vitro using phage DNA-dependent RNA-polymerases. These synthetic transcripts contain several extra nucleotides at the 5' end, which are deleted during replication to generate authentic viral genomes. We removed those 5'-end extra nucleotides utilizing a hammerhead ribozyme to produce transcripts with accurate 5' ends. These transcripts replicate substantially more rapidly in cell culture, demonstrating no lag before replication; they also replicate more efficiently in Xenopus laevis oocytes and in in vitro translation-replication cell extracts. In both systems, an exact 5' end is necessary for synthesis of positive-strand RNA but not negative-strand RNA.
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Herold et al. (2000) studied Poliovirus replication (in vitro). Poliovirus transcripts with authentic 5' ends (via hammerhead ribozyme) vs. Poliovirus transcripts with extra 5' nucleotides was evaluated on RNA replication kinetics and positive-strand RNA synthesis. Poliovirus transcripts with authentic 5' ends generated by a hammerhead ribozyme replicate substantially more rapidly in cell culture without a lag phase compared to transcripts with extra 5' nucleotides.
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