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Updated: May 27, 2025
Reverse Genetics Mediated Recovery of Infectious Murine Norovirus
Published on: June 24, 2012
Murine norovirus allosteric escape mutants mimic gut activation
Abstract:
Murine norovirus (MNV) undergoes large conformational changes in response to the environment. The T=3 icosahedral capsid is composed of 180 copies of ~58 kDa VP1 that has N-terminal (N), shell (S) and C-terminal protruding (P) domains. In phosphate buffered saline, the P domains are loosely tethered to the shell and float ~15Å above the surface. At conditions found in the gut (i.e. low pH with high metal ion and bile salt concentrations) the P domain rotates and drops onto the shell with intra P domain changes that enhance receptor interactions while blocking antibody binding. Two of our monoclonal antibodies (2D3 and 4F9) have broad strain recognition and the only escape mutants, V339I and D348E, are located on the C D loop and ~20 Å from the epitope. Here we determined the cryo-EM structures of V339I and D348E at neutral pH +/- metal ions and bile salts. These allosteric escape mutants are constitutively in the activated state without the addition of metal ions or bile salts, thus explaining how they escape neutralization. Dynamic simulations of the P domain further suggest that movement of the C D loop may be the rate limiting step in the conformational change and that V339I increases the motion of the A B /E F loops compared to wt, making it easier for the virus to transition to the activated state. These findings have important implications for norovirus vaccine design since they uncover a form of the viral capsid that should lend superior immune protection against subsequent challenge by wild type virus.
Insights
Murine norovirus (MNV) capsid mutations allow it to remain in an activated state, evading antibody neutralization. This discovery offers insights for designing more effective norovirus vaccines.
Area of Science:
- Virology
- Structural Biology
- Immunology
Background:
- Murine norovirus (MNV) exhibits significant conformational changes in its capsid (VP1 protein) in response to environmental conditions.
- The capsid's protruding (P) domain shifts from a loosely tethered state in neutral conditions to an activated state in simulated gut conditions, enhancing receptor binding and blocking antibodies.
Purpose of the Study:
- To investigate the structural basis of norovirus immune evasion by determining the cryo-electron microscopy (cryo-EM) structures of two escape mutants (V339I and D348E).
- To understand the allosteric mechanisms underlying capsid conformational changes and their impact on antibody neutralization.
Main Methods:
- Determined cryo-EM structures of MNV escape mutants V339I and D348E at neutral pH, with and without metal ions and bile salts.
- Performed dynamic simulations of the MNV P domain to analyze conformational flexibility and transition dynamics.
Main Results:
- The V339I and D348E mutants are constitutively in the activated capsid state, even without environmental triggers like metal ions or bile salts.
- Dynamic simulations indicate that C D loop movement is crucial for the conformational transition, and the V339I mutation facilitates this transition by increasing loop motion.
Conclusions:
- Allosteric escape mutants of MNV are locked in an activated conformation, explaining their resistance to neutralization.
- Understanding these conformational dynamics is key for developing norovirus vaccines that elicit broadly protective immunity against wild-type strains.
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