Structural characterization of the Myxococcus xanthus encapsulin and ferritin-like cargo system gives insight into its iron storage mechanism.

Encapsulins are bacterial organelle-like cages involved in various aspects of metabolism, especially protection from oxidative stress. They can serve as vehicles for a wide range of medical applications. Encapsulin shell proteins are structurally similar to HK97 bacteriophage capsid protein and their function depends on the encapsulated cargos.

Cryo-Electron Tomography of the Herpesvirus Procapsid Reveals Interactions of the Portal with the Scaffold and a Shift on Maturation.

Herpes simplex virus 1 (HSV-1) requires seven proteins to package its genome through a vertex in its capsid, one of which is the portal protein, pUL6. The portal protein is also thought to facilitate assembly of the procapsid. While the portal has been visualized in mature capsids, we aimed to elucidate its role in the assembly and maturation of procapsids using cryo-electron tomography (cryoET).

The Mottled Capsid of the Giant Phage SPN3US, a Likely Maturation Intermediate with a Novel Internal Shell.

"Giant" phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid.

Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids.

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.

Identification of a Structural Element in HIV-1 Gag Required for Virus Particle Assembly and Maturation.

Late in the HIV-1 replication cycle, the viral structural protein Gag is targeted to virus assembly sites at the plasma membrane of infected cells. The capsid (CA) domain of Gag plays a critical role in the formation of the hexameric Gag lattice in the immature virion, and, during particle release, CA is cleaved from the Gag precursor by the viral protease and forms the conical core of the mature virion. A highly conserved Pro-Pro-Ile-Pro (PPIP) motif (CA residues 122 to 125) [PPIP(122-125)] in a loop connecting CA helices 6 and 7 resides at a 3-fold axis formed by neighboring hexamers in the immature Gag lattice.

The Primary Enveloped Virion of Herpes Simplex Virus 1: Its Role in Nuclear Egress.

Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein.

Internal Proteins of the Procapsid and Mature Capsids of Herpes Simplex Virus 1 Mapped by Bubblegram Imaging.

Unlabelled: The herpes simplex virus 1 (HSV-1) capsid is a huge assembly, ∼1,250 Å in diameter, and is composed of thousands of protein subunits with a combined mass of ∼200 MDa, housing a 100-MDa genome. First, a procapsid is formed through coassembly of the surface shell with an inner scaffolding shell; then the procapsid matures via a major structural transformation, triggered by limited proteolysis of the scaffolding proteins. Three mature capsids are found in the nuclei of infected cells.

Subassemblies and asymmetry in assembly of herpes simplex virus procapsid.

Unlabelled: The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein.

Distribution of DNA-condensing protein complexes in the adenovirus core.

Genome packing in adenovirus has long evaded precise description, since the viral dsDNA molecule condensed by proteins (core) lacks icosahedral order characteristic of the virus protein coating (capsid). We show that useful insights regarding the organization of the core can be inferred from the analysis of spatial distributions of the DNA and condensing protein units (adenosomes). These were obtained from the inspection of cryo-electron tomography reconstructions of individual human adenovirus particles.

A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress.

Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely primarily on protein-bound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear.

Three-dimensional structure of the toxin-delivery particle antifeeding prophage of Serratia entomophila.

The Serratia entomophila antifeeding prophage (Afp) is a bullet-shaped toxin-delivery apparatus similar to the R-pyocins of Pseudomonas aeruginosa. Morphologically it resembles the sheathed tail of bacteriophages such as T4, including a baseplate at one end. It also shares features with the type VI secretion systems.

Clathrin-coated vesicles from brain have small payloads: a cryo-electron tomographic study.

Clathrin coats, which stabilize membrane curvature during endocytosis and vesicular trafficking, form highly polymorphic fullerene lattices. We used cryo-electron tomography to visualize coated particles in isolates from bovine brain. The particles range from ∼66 to ∼134nm in diameter, and only 20% of them (all ⩾80nm) contain vesicles.

Sample preparation induced artifacts in cryo-electron tomographs.

We investigated the effects of sample preparation and of the exposure to an electron beam on particles in cryo-electron tomographs. Various virus particles with icosahedral symmetry were examined, allowing a comparison of symmetrically related components that should be identical in structure but might be affected differently by these imaging artifacts. Comparison of tomographic reconstructions with previously determined structures established by an independent method showed that neither freezing nor electron beam exposure produced a significant amount of shrinkage along the z axis (thickness).

Visualization of the two-step fusion process of the retrovirus avian sarcoma/leukosis virus by cryo-electron tomography.

Retrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as avian sarcoma/leukosis virus (ASLV), employ a two-step mechanism in which receptor binding precedes low-pH activation and fusion.

Structure and assembly of a paramyxovirus matrix protein.

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays.

Cryo-electron tomography of rubella virus.

Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus. The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface.

The role of capsid maturation on adenovirus priming for sequential uncoating.

Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity.

Structural changes in Influenza virus at low pH characterized by cryo-electron tomography.

Influenza virus enters host cells by endocytosis. The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. We have used cryo-electron tomography to visualize influenza A virus at pH 4.

Maturation of flaviviruses starts from one or more icosahedrally independent nucleation centres.

Flaviviruses assemble as fusion-incompetent immature particles and subsequently undergo conformational change leading to release of infectious virions. Flavivirus infections also produce combined 'mosaic' particles. Here, using cryo-electron tomography, we report that mosaic particles of dengue virus type 2 had glycoproteins organized into two regions of mature and immature structure.

Irregular and Semi-Regular Polyhedral Models for Rous Sarcoma Virus Cores.

Whereas many viruses have capsids of uniquely defined sizes that observe icosahedral symmetry, retrovirus capsids are highly polymorphic. Nevertheless, they may also be described as polyhedral foldings of a fullerene lattice on which the capsid protein (CA) is arrayed. Lacking the high order of symmetry that facilitates the reconstruction of icosahedral capsids from cryo-electron micrographs, the three-dimensional structures of individual retrovirus capsids may be determined by cryo-electron tomography, albeit at lower resolution.

RSV capsid polymorphism correlates with polymerization efficiency and envelope glycoprotein content: implications that nucleation controls morphogenesis.

We used cryo-electron tomography to visualize Rous sarcoma virus, the prototypic alpharetrovirus. Its polyprotein Gag assembles into spherical procapsids, concomitant with budding. In maturation, Gag is dissected into its matrix, capsid protein (CA), and nucleocapsid moieties.

Computational resources for cryo-electron tomography in Bsoft.

The Bsoft package [Heymann, J.B., Belnap, D.

Visualization of the herpes simplex virus portal in situ by cryo-electron tomography.

Herpes simplex virus type 1 (HSV-1), the prototypical herpesvirus, has an icosahedral nucleocapsid surrounded by a proteinaceous tegument and a lipoprotein envelope. As in tailed bacteriophages, the icosahedral symmetry of the capsid is broken at one of the 12 vertices, which is occupied by a dodecameric ring of portal protein, UL6, instead of a pentamer of the capsid protein, UL19. The portal ring serves as a conduit for DNA entering and exiting the capsid.

Influenza virus pleiomorphy characterized by cryoelectron tomography.

Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging many identical particles.