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Isolation and characterization of murine coronavirus mutants resistant to neutralization by soluble receptors
K Saeki1, N Ohtsuka, F Taguchi
1Division of Animal Models for Human Diseases, National Institute of Neuroscience, Tokyo, Japan.
Abstract:
Murine coronavirus mutants resistant to neutralization with soluble receptors were isolated to study the receptor-binding site on the S proteins since such mutants were expected to have mutations in an important site for receptor-binding. We have isolated five soluble receptor-resistant (srr) mutants which had mutations of a single amino acid at 3 different positions in S protein. Srr mutant 11 with an amino acid change at position 65 (Leu to His) in the S1 subunit showed an extremely reduced binding by virus overlay protein blot assay. However srr mutants with a mutation at 1114 (Leu to Phe) (srr mutants 3, 4 and 7) or 1163 (Cys to Phe) (srr mutant 18) in the S2 subunit had receptor-binding activity similar to that of wild type cl-2. These results suggest that an amino acid at position 62 located in a conserved region among MHV strains is in particular important for receptor binding. We also discuss why srr mutants with a mutation in S2 showed high resistance to neutralization by soluble receptor, irrespective of their binding to MHV receptors.
Insights
Researchers identified specific mutations in the murine coronavirus S protein that confer resistance to neutralization by soluble receptors. These findings pinpoint key amino acids crucial for viral receptor binding and neutralization resistance.
Area of Science:
- Virology
- Molecular Biology
- Protein Engineering
Background:
- Murine coronavirus (MHV) uses its S protein to bind host cell receptors, mediating viral entry.
- Understanding the S protein's receptor-binding site is crucial for developing antiviral strategies.
- Soluble receptor-resistant mutants can help map critical regions within the S protein.
Purpose of the Study:
- To identify mutations in the MHV S protein affecting receptor binding and neutralization resistance.
- To investigate the role of specific amino acid positions in the S protein's receptor-binding site.
- To elucidate the mechanisms behind neutralization resistance in soluble receptor-resistant mutants.
Main Methods:
- Isolation and characterization of soluble receptor-resistant (srr) MHV mutants.
- Site-directed mutagenesis to introduce specific amino acid changes in the S protein.
- Virus overlay protein blot assay to assess receptor-binding activity.
Main Results:
- Five srr mutants with single amino acid changes at three distinct positions were isolated.
- A mutation at position 65 (Leu to His) in the S1 subunit significantly reduced receptor binding.
- Mutations at positions 1114 and 1163 in the S2 subunit did not affect receptor binding affinity.
- Mutations in the S2 subunit conferred high resistance to neutralization by soluble receptors despite intact binding.
Conclusions:
- An amino acid at position 62 in the S protein is critical for MHV receptor binding.
- Mutations in the S2 subunit can confer neutralization resistance independently of receptor-binding affinity.
- These findings provide insights into the structural and functional domains of the MHV S protein relevant to viral entry and immune evasion.