Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor
Min-Suk Song1, Gyanendra Kumar2, William R Shadrick3
1Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105;
Abstract:
The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.
Insights
Influenza endonuclease inhibitors are promising antiviral drugs. Mutations in the endonuclease active site confer resistance, guiding the development of new influenza therapeutics with lower resistance potential.
Area of Science:
- Virology
- Drug Discovery
- Structural Biology
Background:
- The influenza endonuclease is a key viral enzyme essential for replication.
- It is a validated target for anti-influenza drug development.
- Endonuclease inhibitors like L-742,0001 show therapeutic potential.
Purpose of the Study:
- To investigate the mechanisms of resistance to influenza endonuclease inhibitors.
- To characterize mutations affecting inhibitor binding and viral fitness.
- To inform the development of novel influenza therapeutics with reduced resistance.
Main Methods:
- Influenza virus passaging and random mutagenesis to generate resistant strains.
- In vitro and in vivo assays to assess drug sensitivity and viral fitness.
- Biochemical and crystallographic analyses of wild-type and mutant endonuclease domains.
Main Results:
- Repeated passaging did not yield resistant strains; point mutations were required.
- Mutations conferring resistance mapped to the endonuclease active site, impacting inhibitor binding.
- Engineered viruses with mutations showed in vitro and in vivo resistance to L-742,001 with modest fitness costs.
- Mutations transferred resistance to a second influenza virus strain.
Conclusions:
- Influenza endonuclease mutations can confer resistance to inhibitors.
- Understanding these resistance mechanisms is crucial for designing effective antivirals.
- These findings support the development of influenza therapeutics with a lower propensity for resistance.
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