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The 134-residue phage L decoration protein (Dec) forms a capsid-stabilizing homotrimer that has an asymmetric tripod-like structure when bound to phage L capsids. The N-termini of the trimer subunits consist of spatially separated globular OB-fold domains that interact with the virions of phage L or the related phage P22. The C-termini of the trimer form a three-stranded intertwined spike structure that accounts for nearly all the interactions that stabilize the trimer. A Dec mutant with the spike residues 99-134 deleted (Dec 1-98 ) was used to demonstrate that the stable globular OB-fold domain folds independently of the C-terminal residues. However, Dec 1-98 was unable to bind phage P22 virions, indicating the C-terminal spike is essential for stable capsid interaction. The full-length Dec trimer is disassembled into monomers by acidification to pH <2. These monomers retain the folded globular OB-fold domain structure, but the spike is unfolded. Increasing the pH of the Dec monomer solution to pH 6 allowed for slow trimer formation in vitro over the course of days. The infectious cycle of phage L is only around an hour, however, implying Dec trimer assembly in vivo is templated by the phage capsid. The Thermodynamic Hypothesis holds that protein folding is determined by the amino acid sequence. Dec serves as an unusual example of an oligomeric folding step that is kinetically accelerated by a viral capsid template. The capsid templating mechanism could satisfy the flexibility needed for Dec to adapt to the unusual quasi-symmetric binding site on the mature phage L capsid., Competing Interests: CONFLICT OF INTEREST The authors declare no potential conflict of interest.

Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. Salmonella phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the c2 (repressor), gtrABC , sieA , and sieB genes. Here we report that the P22-encoded SieA protein is necessary and sufficient for exclusion by the SieA system and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants that overcome the SieA block were isolated, and they have amino acid changes in the C-terminal regions of the gene 16 and 20 encoded ejection proteins. Three different single-amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in phage target specificity. Our data strongly suggest a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.IMPORTANCEThe ongoing evolutionary battle between bacteria and the viruses that infect them is a critical feature of bacterial ecology on Earth. Viruses can kill bacteria by infecting them. However, when their chromosomes are integrated into a bacterial genome as a prophage, viruses can also protect the host bacterium by expressing genes whose products defend against infection by other viruses. This defense property is called "superinfection exclusion." A significant fraction of bacteria harbor prophages that encode such protective systems, and there are many different molecular strategies by which superinfection exclusion is mediated. This report is the first to describe the mechanism by which bacteriophage P22 SieA superinfection exclusion protein protects its host bacterium from infection by other P22-like phages. The P22 prophage-encoded inner membrane SieA protein prevents infection by blocking transport of superinfecting phage DNA across the inner membrane during injection., Competing Interests: The authors declare no conflict of interest.

The oligomerization and incorporation of the bacteriophage P22 portal protein complex into procapsids (PCs) depends upon an interaction with scaffolding protein, but the region of the portal protein that interacts with scaffolding protein has not been defined. In herpes simplex virus 1 (HSV-1), conserved tryptophan residues located in the wing domain are required for portal-scaffolding protein interactions. In this study, tryptophan residues (W) present at positions 41, 44, 207 and 211 within the wing domain of the bacteriophage P22 portal protein were mutated to both conserved and non-conserved amino acids. Substitutions at each of these positions were shown to impair portal function in vivo, resulting in a lethal phenotype by complementation. The alanine substitutions caused the most severe defects and were thus further characterized. An analysis of infected cell lysates for the W to A mutants revealed that all the portal protein variants except W211A, which has a temperature-sensitive incorporation defect, were successfully recruited into procapsids. By charge detection mass spectrometry, all W to A mutant portal proteins were shown to form stable dodecameric rings except the variant W41A, which dissociated readily to monomers. Together, these results suggest that for P22 conserved tryptophan, residues in the wing domain of the portal protein play key roles in portal protein oligomerization and incorporation into procapsids, ultimately affecting the functionality of the portal protein at specific stages of virus assembly.

Tailed double-stranded DNA bacteriophages inject some proteins with their dsDNA during infection. Phage P22 injects about 12, 12, and 30 molecules of the proteins encoded by genes 7 , 16 and 20 , respectively. After their ejection from the virion, they assemble into a trans-periplasmic conduit through which the DNA passes to enter the cytoplasm. The location of these proteins in the virion before injection is not well understood, although we recently showed they reside near the portal protein barrel in DNA-filled heads. In this report we show that when these proteins are missing from the virion, a longer than normal DNA molecule is encapsidated by the P22 headful DNA packaging machinery. Thus, the ejection proteins occupy positions within the virion that can be occupied by packaged DNA when they are absent.