The genetic diversity observed among bacteriophages remains a major obstacle for the identification of homologs and the comparison of their functional modules. In the structural module, although several classes of homologous proteins contributing to the head and tail structure can be detected, proteins of the head-to-tail connection (or neck) are generally more divergent. Yet, molecular analyses of a few tailed phages belonging to different morphological classes suggested that only a limited number of structural solutions are used in order to produce a functional virion. To challenge this hypothesis and analyze proteins diversity at the virion neck, we developed a specific computational strategy to cope with sequence divergence in phage proteins. We searched for homologs of a set of proteins encoded in the structural module using a phage learning database.

Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X‐ray structure of the bacteriophage SPP1 portal protein in its isolated 13‐subunit form and the pseudoatomic structure of a 12‐subunit assembly. The first defines the DNA‐interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal–DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a ‘mexican wave’ of positional and conformational changes propagating sequentially along this belt.

In many bacterial viruses and in certain animal viruses, the double-stranded DNA genome enters and exits the capsid through a portal gatekeeper. We report a pseudoatomic structure of a complete portal system. The bacteriophage SPP1 gatekeeper is composed of dodecamers of the portal protein gp6, the adaptor gp15, and the stopper gp16. The solution structures of gp15 and gp16 were determined by NMR. They were then docked together with the X-ray structure of gp6 into the electron density of the ≈1-MDa SPP1 portal complex purified from DNA-filled capsids. The resulting structure reveals that gatekeeper assembly is accompanied by a large rearrangement of the gp15 structure and by folding of a flexible loop of gp16 to form an intersubunit parallel β-sheet that closes the portal channel. This stopper system prevents release of packaged DNA. Disulfide cross-linking between β-strands of the stopper blocks the key conformational changes that control genome ejection from the virus at the beginning of host infection.

The majority of known bacteriophages have long noncontractile tails ( Siphoviridae ) that serve as a pipeline for genome delivery into the host cytoplasm. The tail extremity distal from the phage head is an adsorption device that recognises the bacterial receptor at the host cell surface. This interaction generates a signal transmitted to the head that leads to DNA release. We have determined structures of the bacteriophage SPP1 tail before and after DNA ejection. The results reveal extensive structural rearrangements in the internal wall of the tail tube. We propose that the adsorption device–receptor interaction triggers a conformational switch that is propagated as a domino‐like cascade along the 1600 Å‐long helical tail structure to reach the head‐to‐tail connector. This leads to opening of the connector culminating in DNA exit from the head into the host cell through the tail tube.

In tailed bacteriophages and herpes viruses, the viral DNA is packaged through the portal protein channel. Channel closure is essential to prevent DNA release after packaging. Here we present the connector structure from bacteriophage SPP1 using cryo‐electron microscopy and single particle analysis. The multiprotein complex comprises the portal protein gp6 and the head completion proteins gp15 and gp16. Although we show that gp6 in the connector has a fold similar to that of the isolated portal protein, we observe conformational changes in the region of gp6 exposed to the DNA‐packaging ATPase and to gp15. This reorganization does not cause closure of the channel. The connector channel traverses the full height of gp6 and gp15, 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.

The irreversible binding of bacteriophages to their receptor(s) in the host cell surface triggers release of the naked genome from the virion followed by transit of viral DNA to the host cell cytoplasm. We have purified, for the first time, a receptor from a Gram-positive bacterium that is active to trigger viral DNA ejection in vitro. This extracellular region ("ectodomain") of the Bacillus subtilis protein YueB (YueB780) was a 7 S elongated dimer forming a 36.5-nm-long fiber. YueB780 bound to the tail tip of bacteriophage SPP1. Although a stable receptor-phage interaction occurred between 0 and 37 degrees C, complete blocking of phage DNA release or partial ejection events were observed at temperatures below 15 degrees C. We also showed that the receptor was exposed to the B. subtilis surface. YueB differed structurally from phage receptors from Gram-negative bacteria. Its properties revealed a fiber spanning the full length of the 30-nm-thick peptidoglycan layer. The fiber is predicted to be anchored in the cell membrane through transmembrane segments. These features, highly suitable for a virus receptor in Gram-positive bacteria, are very likely shared by a large number of phage receptors.