Variable electrostatic interaction between DNA and coat protein in filamentous bacteriophage assembly

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Filamentous bacteriophages, like fd, infect Escherichia coli and utilize coat proteins for capsid formation.
• The major coat protein (gene VIII) plays a critical role in the assembly of bacteriophage particles.
• Understanding coat protein-DNA interactions is key to elucidating viral assembly mechanisms.

Purpose of the Study:

• To investigate the role of electrostatic interactions between bacteriophage fd DNA and its major coat protein.
• To determine the impact of specific charge mutations in the coat protein on viral assembly and DNA packaging.
• To explore the mechanism of viral capsid formation and DNA encapsulation.

Main Methods:

• Excised and cloned the bacteriophage fd gene VIII into expression plasmids (pKK223-3 and pEMBL9+).
• Utilized site-directed mutagenesis to alter a lysine residue (K48) to glutamic acid (E48) in the coat protein.
• Expressed wild-type and mutated coat proteins in E. coli and analyzed their assembly and DNA encapsidation capabilities.

Main Results:

• Wild-type and mutated coat proteins were expressed and inserted into the E. coli inner membrane.
• The mutated coat protein (E48) failed to encapsidate bacteriophage DNA, indicating a defect in assembly.
• Hybrid particles with mixed wild-type and mutant subunits were formed, suggesting an initiation defect in assembly.
• Other charge mutations at K48 also resulted in longer bacteriophage particles, supporting electrostatic interactions.

Conclusions:

• A net positive charge in the C-terminal region of the coat protein is essential for the initial stages of virus assembly.
• Electrostatic interactions between DNA and coat protein subunits are critical for bacteriophage assembly.
• The data support a model of non-specific DNA-coat protein binding with variable stoichiometry during assembly.