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Folding and processing of the capsid protein precursor P1 is kinetically retarded in neutralization site 3B mutants
1Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge 02139-4307.
Abstract:
Poliovirus mutants in neutralizing antigenic site 3B were constructed by replacing the glutamic acid residue at amino acid 74 of capsid protein VP2 (VP2074E), using site-specific mutagenesis methods. All viable mutants display small-plaque phenotypes. Characterization of these mutants indicates that capsid assembly is perturbed. Although the defect in capsid assembly reduces the yield of mutant virus particles per cell, the resultant assembled particle is wild-type-like in structure and infectivity. Analyses of capsid assembly intermediates show a transient accumulation of the unprocessed capsid protein precursor, P1, indicating that cleavage of the mutant P1 by the 3CD protease is retarded. The mutant VP0-VP3-VP1 complex generated upon P1 cleavage appears assembly competent, forming pentamer and empty capsid assembly intermediates and infectious virion particles. Although the structure of the infectious mutant virus is virtually identical with that of the wild-type virus, the thermal stability of the mutant virus is dramatically increased over that of the wild-type virus. Thus, mutations at this residue are pleiotropic, altering the kinetics of capsid assembly and generating a virus that is more thermostable and more resistant to neutralization by the site 3B monoclonal antibodies.
Insights
Poliovirus mutations in VP2 protein enhance thermal stability and antibody resistance. These genetic changes affect capsid assembly but yield infectious, wild-type-like virus particles.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Poliovirus capsid structure and assembly are critical for viral infectivity.
- Neutralizing antigenic sites, like site 3B on VP2, are key targets for antibody-mediated viral control.
Purpose of the Study:
- To investigate the impact of specific mutations in poliovirus VP2 on capsid assembly and viral properties.
- To characterize the functional consequences of altering residue 74 in VP2 (VP2-E74) on virus assembly, stability, and immunogenicity.
Main Methods:
- Site-specific mutagenesis was employed to replace glutamic acid at amino acid 74 of capsid protein VP2.
- Characterization of poliovirus mutants included phenotypic analysis (plaque size), capsid assembly studies, protease cleavage assays, and thermal stability assessments.
Main Results:
- Viable poliovirus mutants (VP2-E74) exhibited small-plaque phenotypes and perturbed capsid assembly kinetics.
- Mutant assembly defects led to reduced viral yield but produced particles with wild-type-like structure and infectivity.
- Mutant viruses displayed significantly increased thermal stability and resistance to neutralization by site 3B monoclonal antibodies.
Conclusions:
- Mutations at VP2 residue 74 are pleiotropic, affecting poliovirus capsid assembly kinetics.
- Altered poliovirus exhibits enhanced thermostability and reduced susceptibility to neutralization, offering insights into viral evolution and vaccine design.