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Temperature-sensitive polioviruses containing mutations in RNA polymerase.
C C Burns1, O C Richards, E Ehrenfeld
1Department of Cellular, Viral and Molecular Biology, University of Utah School of Medicine, Salt Lake City 84132.
Virology
|August 1, 1992
Summary
Site-directed mutagenesis of poliovirus RNA polymerase (3Dpol) at residue 424 revealed temperature-sensitive mutations. These mutations impact viral RNA synthesis and polymerase function, affecting poliovirus replication.
Area of Science:
- Virology
- Molecular Biology
- Enzymology
Background:
- Poliovirus RNA polymerase (3Dpol) is essential for viral replication.
- Understanding 3Dpol's structure-function relationship is crucial for antiviral development.
Purpose of the Study:
- To investigate the role of amino acid 424 in poliovirus 3Dpol function.
- To characterize the impact of specific mutations at this site on viral replication and polymerase activity.
Main Methods:
- Site-directed mutagenesis was used to introduce mutations (aspartate, histidine, tyrosine) at residue 424 of 3Dpol.
- Mutant viruses were generated and analyzed in HeLa cells.
- Viral RNA synthesis and polymerase activity of mutant enzymes were assessed in vitro and in infected cells.
Main Results:
- Mutant viruses exhibited small plaques at 32°C and reduced plaquing efficiency at 37°C.
- The histidine mutant (his424) showed defects in plus-strand RNA synthesis initiation.
- The aspartate mutant (asp424) displayed temperature-sensitive conformational changes affecting polymerase activity.
- The tyrosine mutant (tyr424) was completely inactive at 37°C.
Conclusions:
- Amino acid 424 of poliovirus 3Dpol is critical for enzyme function and viral replication.
- Mutations at this site can lead to temperature-sensitive phenotypes by affecting RNA synthesis initiation, enzyme conformation, or overall activity.
- These findings provide insights into poliovirus polymerase mechanisms and potential targets for antiviral strategies.