Capsid targeting sequence targets foreign proteins into bacteriophage T4 and permits proteolytic processing

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Bacteriophage T4 internal protein III (IPIII) plays a role in phage morphogenesis.
• Phage capsid packaging systems can be engineered to encapsulate foreign proteins.

Purpose of the Study:

• To identify and characterize a membrane-independent morphogenetic viral signal peptide within IPIII.
• To investigate the potential of IPIII fusions for targeting and processing foreign proteins within phage capsids.

Main Methods:

• Development of a phage-derived expression-packaging-processing system.
• Construction of IPIII fusion proteins with staphylococcal nuclease, EcoRI endonuclease, beta-globin, and luciferase.
• Western immunoblot analysis and site-directed PCR mutagenesis to assess packaging, processing, and targeting efficiency.

Main Results:

• A functional synthetic cleavage site was introduced at the C terminus of IPIII, enabling proteolytic processing of fusion proteins by scaffold proteinase P21 within the capsid.
• The N-terminal ten amino acid residue of IPIII, termed the capsid targeting sequence (CTS), was identified as the minimal domain sufficient for targeting foreign proteins into the phage capsid.
• The CTS demonstrated dual functionality for both targeting and processing, with distinct domains responsible for each function.

Conclusions:

• The CTS is a novel, minimal sequence capable of directing foreign proteins into the T-even phage capsid.
• The CTS functions in a core-related targeting mechanism, independent of membrane interactions.
• This finding has significant implications for protein engineering and the development of novel phage-based delivery systems.