Regulation of icosahedral virion capsid size by the in vivo activity of a cloned gene product.

Abstract

Determination of icosahedral virion capsid size can be directly studied during helper-dependent lytic development of satellite P4 because the assembly pathway specified by the P2 helper virus is altered to yield smaller-sized capsids. Size determination (sid) mutations identify a P4-encoded function regulating this process. To determine whether the sid gene product is necessary and sufficient to redirect the assembly pathway, we (i) cloned the sid structural gene in a plasmid vector (pMA30) under the control of an inducible promoter and (i) constructed a packaging substrate (pMA1), a P4 genome-sized plasmid containing only that region of P4, the cos site, necessary for encapsidation. Superinfection by P2 of a host carrying pMA30 under induced conditions resulted in a shift from large to small capsid production. P2 superinfection of a host carrying the cos plasmid pMA1 plus pMA30 under induced conditions yielded pMAl-transducing particles of P4 capsid size. These cloning-based analyses directly demonstrate that sid protein is the only P4 gene product required for small-capsid size determination. In the absence of the P2 0 gene product no capsids of any size are assembled during solo infection by P2. Nevertheless, P2 Oam mutant superinfection of a host carrying pMA1 and pMA30 under induced conditions yielded small P4-sized transducing particles. We therefore propose that (i) the sid gene product competes with the 0 gene product to determine the assembly of small vs. large capsid sizes and (U) both gene products probably function as temporary scaffolding proteins.Regular polyhedra composed of identical subunits were proposed by Crick and Watson (1) as the basic capsid structure of simple spherical viruses. To account for both the 5-and 6-fold clustering of such subunits necessary to the formation of icosahedral shells, Casper and Klug (2) conceived of a related model involving conformational flexibility of the identical subunit proteins based on the "quasi-equivalence" of their capsid shell environments. Such flexibility proved to be not an intrinsic property but to result from specific interactions with virally encoded scaffolding proteins (3-5).Thus, rather than the simple "self-assembly" of identical subunits, in vivo construction of an icosahedral virion shell typically involves a pathway ofordered steps that provide the perturbations necessary to induce the quasi-equivalence of the capsid subunits. A striking example of this phenomenon occurs during the lytic development of satellite virus P4. Here, the assembly pathway normally followed by a helper virus-encoded major subunit capsid protein is perturbed by the activity of a P4-encoded gene product (gp). The result of this interference is a distinct class of smaller-sized functional icosahedral capsids appropriate for encapsidation of the P4 genome (6). This report focuses on the gps that shift the helper virus-encoded capsid subunits into alternate assembly pathways.Although originally described as a satellite virus (7), P4 virl is a naturally occurri...

Staphylococcus aureus Pathogenicity Island DNA Is Packaged in Particles Composed of Phage Proteins

“…The staphylococcal phage-SaPI system provides an interesting contrast with the Escherichia coli P2/P4 phage system. In both cases, the parasitic element, SaPI or P4, encodes proteins that The remodeling protein, Sid, of P4 forms an external scaffold for small capsid assembly (1). As noted, the SaPIs encode three proteins that are required for capsid morphogenesis; however, the mechanism in this case has yet to be identified.…”

Scaffolding proteins and their role in viral assembly

“…Expression of both Sid and gpO together with gpN leads to increased fidelity of assembly, but results in a mixture of particles of P4-like and P2-like sizes [39]. This suggests that there is a competition between gpO and Sid in terms of size determination [39,142]. Clearly, additional control points must exist for efficient assembly and size determination in vivo.…”

“…Bacteriophage P2, the helper virus of satellite phage P4, encodes a protein, gpO, which is required for shell assembly in vivo [141] and is believed to comprise an internal scaffold [142]. gpO is associated with a subset of empty procapsid-like particles produced during both P2 and P4 infections and is absent from mature capsids [143], although a cleavage product of gpO, O*, remains inside the mature capsid [144].…”

Bacteriophage Protein–Protein Interactions

“…Presumably this is because its protease activity is essential, and gpO may also facilitate incorporation of the portal. Indeed, sid complements the P2 Oam 279 mutant, which produces a 237 residue N-terminal fragment of gpO that includes the protease domain but lacks scaffolding activity (Agarwal et al ., 1990). The P4 genome contains P2 cos sites and is packaged into small capsids using the P2 terminase (Ziermann and Calendar, 1990).…”