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Isolation and preliminary characterization of temperature-sensitive mutants of encephalomyocarditis virus
Abstract:
Thirty temperature-sensitive mutants of encephalomyocarditis virus have been isolated and partially characterized. Fifteen of these mutants are phenotypically RNA+ thirteen are RNA-, and two are RNA +/-. Six RNA + mutants, one RNA- mutants, and one RNA +/- mutant have virions which are more thermosensitive at 56 degree C than the wild-type virions. Hela cells infected at the nonpermissive temperature with any of the RNA+ mutants produced neither infective nor noninfective viral particles. The cleavage of the precursor polypeptides in cells infected with 11 of the RNA+ mutants was defective at the nonpermissive temperature. This defect in cleavage occurred only in those precursor polypeptides leading to capsid proteins.
Insights
Researchers isolated thirty temperature-sensitive mutants of encephalomyocarditis virus (EMCV). RNA+ mutants showed defects in viral precursor polypeptide cleavage, preventing viral particle formation at nonpermissive temperatures.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Encephalomyocarditis virus (EMCV) is a picornavirus with a positive-sense single-stranded RNA genome.
- Temperature-sensitive (ts) mutants are valuable tools for studying viral replication and gene function.
- Understanding EMCV's genetic requirements for replication is crucial for virology research.
Purpose of the Study:
- To isolate and characterize temperature-sensitive mutants of encephalomyocarditis virus (EMCV).
- To investigate the effects of these mutations on viral replication and virion properties.
- To identify specific defects in viral protein processing caused by these mutants.
Main Methods:
- Isolation and phenotypic characterization of thirty temperature-sensitive mutants of EMCV.
- Classification of mutants based on RNA replication phenotype (RNA+, RNA-, RNA+/-).
- Thermosensitivity analysis of viral particles at 56°C.
- Assessment of viral particle production and precursor polypeptide cleavage in infected HeLa cells at nonpermissive temperatures.
Main Results:
- Fifteen mutants were phenotypically RNA+, thirteen RNA-, and two RNA+/-.
- Six RNA+ mutants, one RNA-, and one RNA+/- mutant exhibited increased virion thermosensitivity compared to wild-type.
- Infection with RNA+ mutants at the nonpermissive temperature resulted in no infectious or non-infectious viral particles.
- Eleven RNA+ mutants displayed defective cleavage of precursor polypeptides into capsid proteins at the nonpermissive temperature.
Conclusions:
- Temperature-sensitive mutations in EMCV can significantly impair viral replication.
- Specific RNA+ mutants disrupt the processing of viral precursor polypeptides essential for capsid formation.
- These findings highlight the importance of proper protein cleavage for EMCV assembly and infectivity.