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Poliovirus protein 3AB forms a complex with and stimulates the activity of the viral RNA polymerase, 3Dpol
1Molecular Biology Research Section, Lederle Laboratories, American Cyanamid Company, Pearl River, New York 10965, USA.
Abstract:
Poliovirus protein 3B (also known as VPg) is covalently linked to the 5' ends of both genomic and antigenomic viral RNA. Genetic and biochemical studies have implicated protein 3AB, the membrane-bound precursor to VPg, in the initiation of genomic RNA synthesis. We have purified 3AB to near homogeneity following thrombin cleavage of purified glutathione S-transferase-3AB. When added to transcription reaction mixtures catalyzed by poliovirus RNA polymerase (3Dpol), 3AB stimulated RNA synthesis up to 75-fold with oligo(U)-primed virion RNA, globin mRNA, and unprimed synthetic, full-length minus-strand viral RNA as the templates. Synthetic VPg also stimulated RNA synthesis but was only 1 to 2% as effective as 3AB on a molar basis. The increased level of transcription was not the result of enhancing the elongation rate of the polymerase. No evidence was found for uridylylation of 3AB or for covalent linkage to RNA transcription products. 3AB sedimented as a multimer in glycerol gradients. In the presence of the polymerase, the sedimentation rate of both proteins increased, suggesting the formation of a complex. Detergent prevented both multimerization and complex formation. The polymerase also bound to immobilized glutathione S-transferase-3AB; this procedure was used to purify the polymerase to near homogeneity. These results suggest a mechanism for bringing together 3AB, 3Dpol (or its precursor 3CD), and viral RNA in host cell membranous vesicles in which all viral RNA synthesis occurs.
Insights
Poliovirus protein 3AB significantly enhances viral RNA synthesis by interacting with the poliovirus RNA polymerase (3Dpol). This interaction, occurring in cellular membranes, is crucial for initiating the replication of poliovirus RNA genomes.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Poliovirus protein 3B (VPg) is essential for viral RNA replication, covalently attached to RNA ends.
- Protein 3AB, the precursor to VPg, is implicated in initiating genomic RNA synthesis.
Purpose of the Study:
- To investigate the role of purified poliovirus protein 3AB in viral RNA synthesis.
- To elucidate the mechanism by which 3AB interacts with the viral RNA polymerase (3Dpol).
Main Methods:
- Purification of glutathione S-transferase-3AB and subsequent cleavage to obtain 3AB.
- In vitro transcription assays using purified 3AB and poliovirus RNA polymerase (3Dpol) with various RNA templates.
- Analysis of protein-protein interactions using glycerol gradient sedimentation and binding assays.
Main Results:
- Purified 3AB stimulated poliovirus RNA synthesis by up to 75-fold.
- 3AB was significantly more effective (100-fold) than synthetic VPg in stimulating RNA synthesis.
- 3AB formed multimers and a complex with 3Dpol, which was dependent on membrane association and disrupted by detergent.
- 3Dpol could be purified using immobilized 3AB, indicating a strong interaction.
Conclusions:
- Poliovirus protein 3AB plays a critical role in stimulating viral RNA synthesis, likely by facilitating the assembly of replication complexes.
- The interaction between 3AB and 3Dpol, potentially within cellular membranous vesicles, is essential for efficient poliovirus RNA replication initiation.