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Manganese-dependent polioviruses caused by mutations within the viral polymerase
Shane Crotty1, David Gohara, Devin K Gilligan
1Department of Microbiology and Immunology, University of California, San Francisco 94143-0414, USA.
Abstract:
Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3D(pol), we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn(2+)-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.
Insights
Researchers found that poliovirus RNA-dependent RNA polymerase can replicate with manganese instead of magnesium. This discovery highlights viral polymerase flexibility and potential drug resistance.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Viral RNA-dependent RNA polymerases (RdRp) are crucial for viral replication.
- Only six amino acids are conserved across eukaryotic positive-strand RNA virus polymerases.
- The conserved asparagine 297 (N297) in poliovirus 3D(pol) is essential for polymerase function.
Purpose of the Study:
- To investigate the functional flexibility of conserved residues in viral RNA-dependent RNA polymerases.
- To explore the impact of altering N297 in poliovirus 3D(pol) on viral replication.
- To identify potential alternative cation dependencies for viral RNA replication.
Main Methods:
- Site-directed mutagenesis was used to create poliovirus mutants with noncanonical amino acids at position 297.
- Viral RNA replication and growth were assessed in the presence of different divalent cations.
- Analysis of conserved residues in the nucleotide binding pocket of viral polymerases.
Main Results:
- Three viable poliovirus mutants with glycine or alanine at position 297 were generated.
- These mutant polioviruses demonstrated manganese (Mn2+)-dependent RNA replication and viral growth.
- This represents the first reported instance of viruses with an alternative cation requirement for RNA replication.
Conclusions:
- Viral RNA-dependent RNA polymerases exhibit significant functional flexibility, even at highly conserved positions.
- The ability to alter N297 suggests that drugs targeting the polymerase nucleotide binding pocket may face challenges from drug-resistant mutants.
- These findings have implications for understanding viral evolution and developing antiviral strategies.