Temperature-sensitive mutations in the phage P22 coat protein which interfere with polypeptide chain folding

Area of Science:

• Virology
• Molecular Biology
• Protein Folding

Background:

• Bacteriophage P22 is a model system for studying viral capsid assembly.
• Temperature-sensitive (ts) mutations in viral coat proteins can disrupt particle formation.
• Understanding these mutations provides insight into protein structure-function relationships.

Purpose of the Study:

• To investigate the molecular basis of temperature-sensitive mutations in the phage P22 coat protein.
• To determine how specific amino acid substitutions affect coat protein assembly and particle formation.
• To classify the nature of these temperature-sensitive mutations.

Main Methods:

• Genetic analysis of 25 temperature-sensitive P22 strains.
• DNA sequencing to identify single amino acid substitutions in the coat gene.
• Analysis of mutant coat protein synthesis, stability, and assembly at permissive and restrictive temperatures.
• Morphological analysis of protein aggregates.

Main Results:

• 17 distinct sites of single amino acid substitutions were identified.
• Mutant coat proteins synthesized at permissive temperatures were functional but failed to assemble infectious particles at restrictive temperatures.
• Mutant coat proteins were synthesized at normal rates but accumulated as insoluble aggregates, resembling inclusion bodies.
• These aggregates indicated a failure in proper protein folding and subunit-subunit or subunit-scaffolding interactions.

Conclusions:

• The identified mutations are temperature-sensitive folding mutations.
• These mutations destabilize an intermediate in the intracellular folding pathway of the coat protein.
• Proper protein conformation is essential for phage P22 capsid assembly and infectivity.