Capsid Targeting Sequence Targets Foreign Proteins into Bacteriophage T4 and Permits Proteolytic Processing

• Published in: Journal of molecular biology Vol. 261; no. 3; pp. 372 - 385
• Main Authors: Mullaney, Julienne M., Black, Lindsay W.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 23-08-1996
• Subjects: Amino Acid Sequence; bacteriophage; Bacteriophage T4 - genetics; Bacteriophage T4 - metabolism; Base Sequence; Capsid - genetics; Capsid - metabolism; Capsid Proteins; Genetic Vectors; leader sequence; Molecular Sequence Data; Mutagenesis; packaging; phage T4; processing; Recombinant Fusion Proteins - genetics; Sequence Analysis; targeting; leader sequence; processing; targeting; packaging; bacteriophage
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1996.0470

A membrane-independent morphogenetic viral signal peptide is identified within bacteriophage T4 internal protein III (IPIII). Utilizing a phage- derived expression-packaging-processing system, which packages foreign proteins fused with IPIII into the phage capsid, a synthetic cleavage site introduced at the C terminus of IPIII is demonstrated to be functional and permits processing of fusion proteins. IPIII, which possesses a native P21 cleavage site at its N terminus, is altered to possess a second P21 cleavage site at its C terminus where cleavage occurs by means of the scaffold proteinase P21 within the capsid. The altered IPIII was inserted into an expression vector to permit the creation of fusion proteins with staphylococcal nuclease,
EcoRI endonuclease, β-globin, and luciferase. Western immunoblot analysis of packaged T4
eG326indicates that the IPIII:fusion-proteins are packaged into phage and proteolytically processed, thus the synthetic P21 cleavage site positioned at the C terminus of IPIII is demonstrated to be functional, and 20 to 200 protein molecules are packaged per capsid. Truncation experiments identified the minimal portion of IPIII required to achieve targeting into the phage capsid as a ten amino acid residue from the N terminus, which includes the N-terminal methionine residue and the proteinase P21 cleavage site, designated the CTS (capsid targeting sequence). The addition of the CTS to a fragment of luciferase permits the protein to be packaged and processed, which demonstrates that the CTS is by itself sufficient to target foreign protein to the capsid. The imputed dual function of the CTS is supported by site-directed PCR mutagenesis, which reveals two functionally separate domains of the CTS for targeting and processing. The CTS appears to function in a core-related targeting mechanism that directs a polymorphic set of proteins into the T-even capsid or scaffold. Although structure formation is often assumed to involve extended protein interfaces, the analysis shows that a limited but specific sequence, the CTS, drives the interaction required to achieve targeting.