CAPSID TARGETING SEQUENCE TARGETS FOREIGN PROTEINS INTO BACTERIOPHAGE-T4 AND PERMITS PROTEOLYTIC PROCESSING

A membrane-independent morphogenetic viral signal peptide is identifie d within bacteriophage T4 internal protein III (IPIII). Utilizing a ph age-derived expression-packaging-processing system, which packages for eign proteins fused with IPIII into the phage capsid, a synthetic clea vage site introduced at the C terminus of IPIII is demonstrated to be functional and permits processing of fusion proteins. IPIII, which pos sesses a native P21 cleavage site at its N terminus, is altered to pos sess a second P21 cleavage site at its C terminus where cleavage occur s by means of the scaffold proteinase P21 within the capsid. The alter ed IPIII was inserted into an expression vector to permit the creation of fusion proteins with staphylococcal nuclease, EcoRI endonuclease, beta-globin, and luciferase. Western immunoblot analysis of packaged T 4eG326 indicates that the IPIII:fusion-proteins are packaged into phag e 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 achiev e 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 seq uence). The addition of the CTS to a fragment of luciferase permits th e protein to be packaged and processed, which demonstrates that the CT S is by itself sufficient to target foreign protein to the capsid. The imputed dual function of the CTS is supported by site-directed-PCR mu tagenesis, which reveals two functionally separate domains of the CTS for targeting and processing. The CTS appears to function in a core-re lated targeting mechanism that directs a polymorphic set of proteins i nto the T-even capsid or scaffold. Although structure formation is oft en assumed to involve extended protein interfaces, the analysis shows that a limited but specific sequence, the CTS, drives the interaction required to achieve targeting. (C) 1996 Academic Press Limited.