To check this idea, we have infected non-permissive YB886 cells with SPP1and SPP1that do not produce gp15 and gp16, respectively (Figure?2; Becker et al., 1997). but it is closed by gp16 at the bottom of the complex. Gp16 acts as a valve whose closure prevents DNA leakage, while its opening is required for DNA release upon interaction of the virus with its host. (Earnshaw and Casjens, 1980; Tavares NCR2 et al., 1996; and references therein). To avoid chromosome leakage, the portal channel has to be closed shortly after GNA002 encapsidation of the DNA. This can be achieved by a conformational change in the portal protein (29; Hagen et al., 1976; Donate et al., 1988) or by binding of head completion proteins that plug the portal pore to form the connector structure, i.e. T4 (Coombs and Eiserling, 1977), (Perucchetti et al., 1988), P22 (Strauss and King, 1984) and SPP1 (Lurz et al., 2001; this work). We define the connector as the complete knob structure assembled at the capsid portal vertex prior to tail attachment, and distinct from the portal protein cyclical oligomer (gp6 in SPP1). The additional feature in bacteriophages T3 and T7 is an internal core that extends from the portal structure to the procapsid interior (Steven and Trus, 1986). Interestingly, the T3 portal protein pore appears partially closed after DNA packaging (Valpuesta et al., 1992). Closure of the portal channel involves a valve mechanism that is reversed for ejection (Bazinet and King, 1985; Tavares et al., 1996). Viral DNA delivery to the host cytoplasm is a complex and regulated process that probably engages a variety of phage and host factors (Molineux, 2001; and references therein). Open in a separate window Fig. 1. SPP1 morphogenesis. Current knowledge of the sequence of assembly reactions during SPP1 capsid assembly (Dr?ge bacteriophage SPP1 is composed of the portal protein gp6 (subunit molecular mass of 57.3?kDa) and the two head completion proteins gp15 (11.6?kDa) and gp16 (12.5?kDa; Lurz et al., 2001). It was found that the connector complex has 12-fold cyclical symmetry (Lurz et al., 2001), though isolated gp6 is a closed cyclical 13mer in equilibrium with a small population of open curvilinear oligomers (9, 10, 11, 12, 13mers; van Heel et al., 1996b). Reassociation and refoldingCreassociation experiments showed that formation of closed rings of 13 subunits is an intrinsic property of gp6 (Jekow et al., 1999; our unpublished results). The portal protein participates in the early reactions of procapsid assembly (Dr?ge et al., 2000). Co-production of gp6 with the two other essential procapsid proteins of SPP1 in the same strain that is used to produce gp6 13mers led to formation of biologically active procapsids (Dr?ge and Tavares, 2000; Dr?ge et al., 2000). These procapsids contain a functional portal protein that is a 12mer at late stages of morphogenesis (Figure?1; Lurz et al., 2001). To reconcile the finding of the two different symmetries of the SPP1 portal protein, it was suggested that the gp6 oligomers competent for the procapsid assembly reaction are open curvilinear forms found in equilibrium with isolated 13mers. These open oligomers would form closed 12mers when they are surrounded by the major capsid protein and interact with the scaffolding protein (Lurz et al., 2001). After procapsid assembly, gp6 participates in the reactions required for viral DNA packaging. Packaging is terminated by cleavage of the DNA concatemer, generating unit-length virus chromosome molecules (Tavares et al., 1995). Encapsidation of the DNA is followed by binding of gp15 and gp16 to the portal vertex, leading to formation of the connector (Figure?1). The whole complex consists of gp6, gp15 and gp16 annular oligomers (Lurz et al., 2001; this work). The phage tail attaches to the gp16 ring, whereas the DNA extremity, which is packaged last, remains attached to the connector structure (Tavares et al., 1996). Initiation of phage infection requires the opening of the connector to enable the release of the viral chromosome through the tail channel into the host cytoplasm. Here GNA002 we present a structural analysis by cryo-electron microscopy and angular reconstitution of the 900?kDa connector complex and its comparison with the isolated portal protein. The new structure provides a framework for understanding GNA002 how the connector controls the final stages of DNA encapsidation and DNA release at the onset of viral infection. Results and discussion Gp15 and gp16 are required to prevent release of packaged DNA The portal protein gp6 is necessary for SPP1 DNA packaging, but the additional components of the connector, which prevent the release of the DNA that is held at high pressure inside the capsid, were not identified. Electron microscopy studies suggested that gp15 and gp16 present in the portal vertex of SPP1 capsids might serve to lock the connector base (Lurz et al.,.
