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Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A)
Abstract:
A template-dependent RNA polymerase has been isolated from poliovirus-infected cells by assaying for the ability of the enzyme to copy poly(A) complexed to an oligo(U) primer. The polymerase was solubilized with detergent, and RNA was removed by precipitation with 2 M LiCl. The solubilized polymerase required both poly(A) and oligo(U) for activity and was stimulated by Mg2+ but was inhibited by Mn2+. Poly(A)-oligo(U)-dependent poly(U) polymerase was not found in extracts of HeLa cells until about 2 hr after poliovirus infection, and then there was a linear increase in activity until about 5 hr. Analysis of the polymerase by glycerol gradient centrifugation showed that the majority of the activity sedimented at about 4 S, indicating that it was no longer complexed with high-molecular-weight RNA or cellular membranes. This poly(A)-oligo(U)-dependent polymerase activity could represent an important component of the poliovirus RNA-dependent RNA polymerase.
Insights
Researchers isolated a template-dependent RNA polymerase from poliovirus-infected cells. This enzyme copies poly(A) with an oligo(U) primer, suggesting a role in poliovirus RNA replication.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Poliovirus replication relies on RNA-dependent RNA polymerase (RdRp).
- Understanding the specific components of poliovirus RdRp is crucial for comprehending viral replication strategies.
Purpose of the Study:
- To isolate and characterize a template-dependent RNA polymerase from poliovirus-infected cells.
- To investigate the enzymatic activity and properties of the isolated polymerase.
Main Methods:
- Isolation of RNA polymerase from poliovirus-infected HeLa cells using detergent solubilization and LiCl precipitation.
- Assay for polymerase activity using poly(A) template and oligo(U) primer.
- Analysis of polymerase properties, including cofactor requirements (Mg2+, Mn2+) and sedimentation behavior via glycerol gradient centrifugation.
Main Results:
- A poly(A)-oligo(U)-dependent poly(U) polymerase activity was detected in poliovirus-infected cells, appearing 2 hours post-infection and increasing linearly until 5 hours.
- The polymerase activity was stimulated by Mg2+ and inhibited by Mn2+.
- Glycerol gradient centrifugation indicated the active polymerase sedimented at approximately 4 S, suggesting it was not associated with high-molecular-weight RNA or cellular membranes.
Conclusions:
- The isolated poly(A)-oligo(U)-dependent polymerase activity represents a significant component of the poliovirus RNA-dependent RNA polymerase.
- This finding contributes to understanding the molecular mechanisms of poliovirus RNA synthesis.