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Similar structural basis for membrane localization and protein priming by an RNA-dependent RNA polymerase
John M Lyle1, Amy Clewell, Kathryn Richmond
1Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.
Abstract:
Protein primers are used to initiate genomic synthesis of several RNA and DNA viruses, although the structural details of the primer-polymerase interactions are not yet known. Poliovirus polymerase binds with high affinity to the membrane-bound viral protein 3AB but uridylylates only the smaller peptide 3B in vitro. Mutational analysis of the polymerase identified four surface residues on the three-dimensional structure of poliovirus polymerase whose wild-type identity is required for 3AB binding. These mutants also decreased 3B uridylylation, arguing that the binding sites for the membrane tether and the protein primer overlap. Mutation of flanking residues between the 3AB binding site and the polymerase active site specifically decreased 3B uridylylation, likely affecting steps subsequent to binding. The physical overlap of sites for protein priming and membrane association should facilitate replication initiation in the membrane-associated complex.
Insights
Poliovirus polymerase uses protein primers for genomic synthesis. Researchers found that the binding sites for the membrane-associated protein 3AB and the protein primer 3B overlap on the polymerase, facilitating replication.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Protein primers initiate genomic synthesis for various viruses, but their interaction with polymerases remains unclear.
- Poliovirus polymerase binds viral protein 3AB and the protein primer 3B, but only uridylylates 3B in vitro.
Purpose of the Study:
- To investigate the structural basis of poliovirus polymerase interactions with protein 3AB and protein primer 3B.
- To elucidate the functional relationship between membrane association and protein priming during viral replication.
Main Methods:
- Utilized mutational analysis of poliovirus polymerase.
- Determined the impact of specific residue mutations on 3AB binding and 3B uridylylation.
- Analyzed the three-dimensional structure of poliovirus polymerase.
Main Results:
- Identified four surface residues on poliovirus polymerase critical for 3AB binding.
- Mutations in these residues reduced both 3AB binding and 3B uridylylation, suggesting overlapping binding sites.
- Mutations in residues between the 3AB binding site and active site impaired 3B uridylylation, indicating effects on later steps.
Conclusions:
- The binding sites for the membrane-associated protein 3AB and the protein primer 3B physically overlap on the poliovirus polymerase.
- This overlap likely facilitates efficient replication initiation within the membrane-associated viral replication complex.