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Primer-dependent synthesis of covalently linked dimeric RNA molecules by poliovirus replicase
Abstract:
Poliovirus-specific RNA-dependent RNA polymerase (replicase, 3Dpol) was purified from HeLa cells infected with poliovirus. The purified enzyme preparation contained two proteins of apparent molecular weights 63,000 and 35,000. The 63,000-Mr polypeptide was virus-specific RNA-dependent RNA polymerase, and the 35,000-Mr polypeptide was of host origin. Both polypeptides copurified through five column chromatographic steps. The purified enzyme preparation catalyzed synthesis of covalently linked dimeric RNA products from a poliovirion RNA template. This reaction was absolutely dependent on added oligo(U) primer, and the dimeric product appeared to be made of both plus- and minus-strand RNA molecules. Experiments with 5' [32P]oligo(U) primer and all four unlabeled nucleotides suggest that the viral replicase elongates the primer, copying the poliovirion RNA template (plus strand), and the newly synthesized minus strand snaps back on itself to generate a template-primer structure which is elongated by the replicase to form covalently linked dimeric RNA molecules. Kinetic studies showed that a partially purified preparation of poliovirus replicase contains a nuclease which can cleave the covalently linked dimeric RNA molecules, generating template-length RNA products.
Insights
Researchers purified poliovirus RNA-dependent RNA polymerase (3Dpol) and found it synthesizes dimeric RNA products. A co-purifying nuclease activity can cleave these dimeric products into template-length RNA.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Poliovirus replication relies on its RNA-dependent RNA polymerase (3Dpol).
- Understanding the enzymatic mechanism of 3Dpol is crucial for antiviral strategies.
Purpose of the Study:
- To purify and characterize the poliovirus RNA-dependent RNA polymerase (3Dpol).
- To elucidate the mechanism of viral RNA synthesis catalyzed by 3Dpol.
Main Methods:
- Purification of poliovirus 3Dpol from infected HeLa cells using multiple chromatographic steps.
- In vitro enzymatic assays to study RNA synthesis and product formation.
- Kinetic analysis to identify associated enzymatic activities.
Main Results:
- Purified 3Dpol preparation contained viral 63,000-Mr and host 35,000-Mr polypeptides.
- The enzyme synthesized covalently linked dimeric RNA products from a poliovirus RNA template, dependent on an oligo(U) primer.
- Evidence suggests a mechanism involving primer elongation, minus-strand synthesis, template-primer formation, and subsequent elongation to dimeric RNA.
- A nuclease activity associated with the replicase preparation cleaved dimeric RNA into template-length products.
Conclusions:
- The purified poliovirus 3Dpol can synthesize dimeric RNA intermediates.
- The viral replicase possesses both polymerase and nuclease activities, potentially involved in RNA replication and processing.