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Determinants of membrane association for poliovirus protein 3AB
J S Towner1, T V Ho, B L Semler
1Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697, USA.
Abstract:
Poliovirus protein 3AB may serve as the lipophilic carrier of a protein primer (VPg or 3B) used for the initiation of genomic viral RNA synthesis. In order to study the membrane-protein interactions of 3AB required for its role in poliovirus RNA replication, we have developed an in vitro membrane association assay capable of distinguishing membrane-bound from non-membrane-bound proteins that are cotranslated together in the presence of canine microsomal membranes. This assay utilizes equilibrium sedimentation analysis in high density sucrose gradients to measure membrane association of both wild type and mutated forms of 3AB. Using this assay and other biochemical assays, we have identified the following properties of the 3AB-membrane interaction: (a) 3AB is able to post-translationally associate with microsomal membranes, (b) 3AB is able to associate with membranes in a manner consistent with that of an integral membrane protein, (c) 3AB contains a critical hydrophobic sequence within the carboxyl-terminal half of the protein that is required for membrane association, and (d) the introduction of charged residues into this hydrophobic sequence disrupts the 3AB membrane-protein interaction. Taken together, these studies indicate that poliovirus protein 3AB associates tightly with biological membranes de novo in a manner that would allow it to serve as a lipophilic anchor for the assembly of the poliovirus RNA replication complex.
Insights
Poliovirus protein 3AB acts as a lipophilic anchor for viral RNA synthesis. This protein tightly binds to cellular membranes via a hydrophobic sequence, essential for poliovirus replication complex assembly.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Poliovirus protein 3AB is implicated as a lipophilic carrier for protein primers (VPg or 3B) in viral RNA synthesis.
- Understanding the membrane-protein interactions of 3AB is crucial for elucidating its role in poliovirus RNA replication.
Purpose of the Study:
- To investigate the membrane-protein interactions of poliovirus protein 3AB.
- To characterize the properties of 3AB's association with biological membranes.
Main Methods:
- Development of an in vitro membrane association assay using canine microsomal membranes.
- Utilized equilibrium sedimentation analysis in high-density sucrose gradients.
- Employed biochemical assays to analyze wild-type and mutated forms of 3AB.
Main Results:
- Poliovirus protein 3AB exhibits post-translational association with microsomal membranes.
- 3AB associates with membranes similarly to an integral membrane protein.
- A critical hydrophobic sequence in the carboxyl-terminal half of 3AB is essential for membrane association.
- Disruption of this hydrophobic sequence by charged residues abolishes membrane interaction.
Conclusions:
- Poliovirus protein 3AB tightly associates with biological membranes.
- This interaction is mediated by a specific hydrophobic sequence, crucial for its function.
- 3AB likely serves as a lipophilic anchor for assembling the poliovirus RNA replication complex.