Archaeal viruses with a spindle-shaped virion are abundant and widespread in extremely diverse environments. However, efforts to obtain the high-resolution structure of a spindle-shaped virus have been unsuccessful. Here, we present the structure of SSV19, a spindle-shaped virus infecting the hyperthermophilic archaeonSulfolobussp. E11-6. Our near-atomic structure reveals an unusual sevenfold symmetrical virus tail consisting of the tailspike, nozzle, and adaptor proteins. The spindle-shaped capsid shell is formed by seven left-handed helical strands, constructed of the hydrophobic major capsid protein, emanating from the highly glycosylated tail assembly. Sliding between adjacent strands is responsible for the variation of a virion in size. Ultrathin sections of the SSV19-infected cells show that SSV19 virions adsorb to the host cell membrane through the tail after penetrating the S-layer. The tailspike harbors a putative endo-mannanase domain, which shares structural similarity to aBacteroides thetaiotaomicroendo-mannanase. Molecules of glycerol dibiphytanyl glycerol tetraether lipid were observed in hydrophobic clefts between the tail and the capsid shell. The nozzle protein resembles the stem and clip domains of the portals of herpesviruses and bacteriophages, implying an evolutionary relationship among the archaeal, bacterial, and eukaryotic viruses.

T5 is a siphophage that has been extensively studied by structural and biochemical methods. However, the complete in situ structures of T5 before and after DNA ejection remain unknown. In this study, we used cryo-electron microscopy (cryo-EM) to determine the structures of mature T5 (a laboratory-adapted, fiberless T5 mutant) and urea-treated empty T5 (lacking the tip complex) at near-atomic resolutions. Atomic models of the head, connector complex, tail tube, and tail tip were built for mature T5, and atomic models of the connector complex, comprising the portal protein pb7, adaptor protein p144, and tail terminator protein p142, were built for urea-treated empty T5. Our findings revealed that the aforementioned proteins did not undergo global conformational changes before and after DNA ejection, indicating that these structural features were conserved among most myophages and siphophages. The present study elucidates the underlying mechanisms of siphophage infection and DNA ejection.

It is estimated that over 60% of known tailed phages are siphophages, which are characterized by a long, flexible, and non-contractile tail. Nevertheless, entire high-resolution structures of siphophages remain scarce. Using cryo-EM, we resolved the structures of T-series siphophage T1, encompassing its head, connector complex, tail tube, and tail tip, at near-atomic resolution. The density maps enabled us to build the atomic models for the majority of T1 proteins. The T1 head comprises 415 copies of the major capsid protein gp47, arranged into an icosahedron with a triangulation number of seven, decorated with 80 homologous trimers and 60 heterotrimers along the threefold and quasi-threefold axes of the icosahedron. The T1 connector complex is composed of two dodecamers (a portal and an adaptor) and two hexamers (a stopper and a tail terminator). The flexible tail tube comprises approximately 34 hexameric rings of tail tube. The extensive disulfide bond network along the successive tail rings may mediate the flexible bending. The distal tip of T1, which is cone-shaped and assembled by proteins gp33, gp34, gp36, gp37, and gp38, displays structural similarity to that of phage lambda. In conjunction with previous studies of lambda-like siphophages, our structure will facilitate further exploration of the structural and mechanistic aspects of lambda-like siphophages.

Jumbo phage phiKZ, a key model for studying phage nucleus formation and bacterial defense mechanisms, possesses a highly complex tail machine that is essential for infection. Here, we present the structural atlas of the intact jumbo phage phiKZ by cryo-EM, thereby identifying 40 constituent proteins and unveiling its modular architecture. The virion, with a length of approximately 360 nm, is comprised of an icosahedral capsid of 2520 polypeptide chains from 11 proteins, and a massive tail machine of over 900 polypeptide chains from 29 proteins. The tail features a unique, multi-layered neck and a highly elaborate baseplate. The neck is reinforced by whisker-like proteins and anchors the contractile tail, which terminates in the baseplate. The baseplate is constituted by a central hub, an inner periphery of interlocking wedge heterotrimers and hexagonal rings, and an outer periphery with a striking hexagonal star configuration. This intricate peripheral region of the baseplate serves as an extended platform for twelve peripheral fibers, which mediate host cell adsorption. Our findings provide a structural framework for understanding jumbo phage assembly and infection, thus contributing to the foundation for future functional studies and rational engineering of these phages for potential therapeutic applications.

Siphophages possess a long, flexible, and non-contractile tail that is responsible for host recognition, cell wall perforation, and genome delivery. Although the majority of structurally characterized siphophages target Gram-negative bacteria, those infecting Gram-positive bacteria remain elusive. Moreover, structural information concerning phage infection and genome release events in Gram-positive infecting siphophages is sparse. Here, we present a near-atomic resolution structure of Brochothrix thermosphacta bacteriophage NF5 determined by cryo-electron microscopy (cryo-EM). The structure comprises 11 proteins associated with the head, neck, tail tube, and baseplate, amounting to 643 polypeptides in total. Integration of cellular cryo-electron tomography (cryo-ET) showed the infection process of NF5, providing insights into the adsorption mechanism of siphophages targeting Gram-positive bacteria. Structural comparisons of baseplates from multiple siphophages targeting Gram-negative and Gram-positive bacteria reveal divergent compositional architectures and distinct assembly mechanisms. These disparities are evident in the domains across divergent baseplate proteins, suggesting evolutionary adaptations to host envelope architectures.