Large biological structures are assembled from smaller, often symmetric, sub-structures. However, asymmetry among sub-structures is fundamentally important for biological function. An extreme form of ...asymmetry, a 12-fold-symmetric dodecameric portal complex inserted into a 5-fold-symmetric capsid vertex, is found in numerous icosahedral viruses, including tailed bacteriophages, herpesviruses, and archaeal viruses. This vertex is critical for driving capsid assembly, DNA packaging, tail attachment, and genome ejection. Here, we report the near-atomic in situ structure of the symmetry-mismatched portal vertex from bacteriophage T4. Remarkably, the local structure of portal morphs to compensate for symmetry-mismatch, forming similar interactions in different capsid environments while maintaining strict symmetry in the rest of the structure. This creates a unique and unusually dynamic symmetry-mismatched vertex that is central to building an infectious virion.
Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells and carry out molecular repairs will have broad applications. Here, we describe an assembly-line ...approach to build AVVs by engineering the well-characterized structural components of bacteriophage T4. Starting with a 120 × 86 nm capsid shell that can accommodate 171-Kbp DNA and thousands of protein copies, various combinations of biomolecules, including DNAs, proteins, RNAs, and ribonucleoproteins, are externally and internally incorporated. The nanoparticles are then coated with cationic lipid to enable efficient entry into human cells. As proof of concept, we assemble a series of AVVs designed to deliver full-length dystrophin gene or perform various molecular operations to remodel human genome, including genome editing, gene recombination, gene replacement, gene expression, and gene silencing. These large capacity, customizable, multiplex, and all-in-one phage-based AVVs represent an additional category of nanomaterial that could potentially transform gene therapies and personalized medicine.
The recent Zika virus (ZIKV) epidemic has been linked to unusual and severe clinical manifestations including microcephaly in fetuses of infected pregnant women and Guillian-Barré syndrome in adults. ...Neutralizing antibodies present a possible therapeutic approach to prevent and control ZIKV infection. Here we present a 6.2 Å resolution three-dimensional cryo-electron microscopy (cryoEM) structure of an infectious ZIKV (strain H/PF/2013, French Polynesia) in complex with the Fab fragment of a highly therapeutic and neutralizing human monoclonal antibody, ZIKV-117. The antibody had been shown to prevent fetal infection and demise in mice. The structure shows that ZIKV-117 Fabs cross-link the monomers within the surface E glycoprotein dimers as well as between neighbouring dimers, thus preventing the reorganization of E protein monomers into fusogenic trimers in the acidic environment of endosomes.
Bacteriophage T4 consists of a head for protecting its genome and a sheathed tail for inserting its genome into a host. The tail terminates with a multiprotein baseplate that changes its conformation ...from a “high-energy” dome-shaped to a “low-energy” star-shaped structure during infection. Although these two structures represent different minima in the total energy landscape of the baseplate assembly, as the dome-shaped structure readily changes to the star-shaped structure when the virus infects a host bacterium, the dome-shaped structure must have more energy than the star-shaped structure. Here we describe the electron microscopy structure of a 3.3-MDa in vitro-assembled star-shaped baseplate with a resolution of 3.8 Å. This structure, together with other genetic and structural data, shows why the high-energy baseplate is formed in the presence of the central hub and how the baseplate changes to the low-energy structure, via two steps during infection. Thus, the presence of the central hub is required to initiate the assembly of metastable, high-energy structures. If the high-energy structure is formed and stabilized faster than the low-energy structure, there will be insufficient components to assemble the low-energy structure.
Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure, portal vertex, and genome ...packaging add a significant body of new literature to phage biology. Head structures in unexpanded and expanded conformations show dramatic domain movements, structural remodeling, and a ~70% increase in inner volume while creating high-affinity binding sites for the outer decoration proteins Soc and Hoc. Small changes in intercapsomer interactions modulate angles between capsomer planes, leading to profound alterations in head length. The in situ cryo-EM structure of the symmetry-mismatched portal vertex shows the remarkable structural morphing of local regions of the portal protein, allowing similar interactions with the capsid protein in different structural environments. Conformational changes in these interactions trigger the structural remodeling of capsid protein subunits surrounding the portal vertex, which propagate as a wave of expansion throughout the capsid. A second symmetry mismatch is created when a pentameric packaging motor assembles at the outer "clip" domains of the dodecameric portal vertex. The single-molecule dynamics of the packaging machine suggests a continuous burst mechanism in which the motor subunits adjusted to the shape of the DNA fire ATP hydrolysis, generating speeds as high as 2000 bp/s.
The 3.3-Å cryo-EM structure of the 860-Å-diameter isometric mutant bacteriophage T4 capsid has been determined. WT T4 has a prolate capsid characterized by triangulation numbers (T numbers) Tend = 13 ...for end caps and Tmid = 20 for midsection. A mutation in the major capsid protein, gp23, produced T=13 icosahedral capsids. The capsid is stabilized by 660 copies of the outer capsid protein, Soc, which clamp adjacent gp23 hexamers. The occupancies of Soc molecules are proportional to the size of the angle between the planes of adjacent hexameric capsomers. The angle between adjacent hexameric capsomers is greatest around the fivefold vertices, where there is the largest deviation from a planar hexagonal array. Thus, the Soc molecules reinforce the structure where there is the greatest strain in the gp23 hexagonal lattice. Mutations that change the angles between adjacent capsomers affect the positions of the pentameric vertices, resulting in different triangulation numbers in bacteriophage T4. The analysis of the T4 mutant head assembly gives guidance to how other icosahedral viruses reproducibly assemble into capsids with a predetermined T number, although the influence of scaffolding proteins is also important.
Jumbo phages are a group of tailed bacteriophages with large genomes and capsids. As a prototype of jumbo phage, ΦKZ infects Pseudomonas aeruginosa, a multi-drug-resistant (MDR) opportunistic ...pathogen leading to acute or chronic infection in immunocompromised individuals. It holds potential to be used as an antimicrobial agent and as a model for uncovering basic phage biology. Although previous low-resolution structural studies have indicated that jumbo phages may have more complicated capsid structures than smaller phages such as HK97, the detailed structures and the assembly mechanism of their capsids remain largely unknown. Here, we report a 3.5-Å-resolution cryo-EM structure of the ΦKZ capsid. The structure unveiled ten minor capsid proteins, with some decorating the outer surface of the capsid and the others forming a complex network attached to the capsid’s inner surface. This network seems to play roles in driving capsid assembly and capsid stabilization. Similar mechanisms of capsid assembly and stabilization are probably employed by many other jumbo viruses.Here, the authors present the 3.5-Å-resolution cryo-EM structure of the jumbo phage ΦKZ capsid, revealing ten minor capsid proteins that are hypothesized to contribute to capsid assembly and stabilization.
Marine bacteriophage TW1 belongs to the Siphoviridae family and infects Pseudoalteromonas phenolica. Mass spectrometry analysis has identified 16 different proteins in the TW1 virion. Functions of ...most of these proteins have been predicted by bioinformatic methods. A 3.6 Å resolution cryoelectron microscopy map of the icosahedrally averaged TW1 head showed the atomic structures of the major capsid protein, gp57∗, and the capsid-stabilizing protein, gp56. The gp57∗ structure is similar to that of the phage HK97 capsid protein. The gp56 protein has two domains, each having folds similar to that of the N-terminal part of phage λ gpD, indicating a common ancestry. The first gp56 domain clamps adjacent capsomers together, whereas the second domain is required for trimerization. A 6-fold-averaged reconstruction of the distal part of the tail showed that TW1 has six tail spikes, which are unusual for siphophages but are similar to the podophages P22 and Sf6, suggesting a common evolutionary origin of these spikes.
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•Structure of the marine siphovirus TW1 has been determined using cryo-EM•Atomic structures of the two proteins forming the capsid shell have been built•The capsid-stabilizing protein has two domains that each are similar to phage λ gpD•The TW1 tail has six spikes, similar to the spikes of podophages P22 and Sf6
Wang et al. describe the structure of the marine siphovirus TW1 determined by cryo-EM. The structure shows evolutionary linkage between the capsid-stabilizing proteins of TW1 and phage λ. The TW1 tail spikes share a common evolutionary origin with the spikes of podophages P22 and Sf6.
Tailed bacteriophages (phages) are one of the most abundant life forms on Earth. They encode highly efficient molecular machines to infect bacteria, but the initial interactions between a phage and a ...bacterium that then lead to irreversible virus attachment and infection are poorly understood. This information is critically needed to engineer machines with novel host specificities in order to combat antibiotic resistance, a major threat to global health today. The tailed phage T4 encodes a specialized device for this purpose, the long tail fiber (LTF), which allows the virus to move on the bacterial surface and find a suitable site for infection. Consequently, the infection efficiency of phage T4 is one of the highest, reaching the theoretical value of 1. Although the atomic structure of the tip of the LTF has been determined, its functional architecture and how interactions with two structurally very different Escherichia coli receptor molecules, lipopolysaccharide (LPS) and outer membrane protein C (OmpC), contribute to virus movement remained unknown. Here, by developing direct receptor binding assays, extensive mutational and biochemical analyses, and structural modeling, we discovered that the ball-shaped tip of the LTF, a trimer of gene product 37, consists of three sets of symmetrically alternating binding sites for LPS and/or OmpC. Our studies implicate reversible and dynamic interactions between these sites and the receptors. We speculate that the LTF might function as a "molecular pivot" allowing the virus to "walk" on the bacterium by adjusting the angle or position of interaction of the six LTFs attached to the six-fold symmetric baseplate.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bacteriophage T4 is decorated with 155 180 Å-long fibers of the highly antigenic outer capsid protein (Hoc). In this study, we describe a near-atomic structural model of Hoc by combining ...cryo-electron microscopy and AlphaFold structure predictions. It consists of a conserved C-terminal capsid-binding domain attached to a string of three variable immunoglobulin (Ig)-like domains, an architecture well-preserved in hundreds of Hoc molecules found in phage genomes. Each T4-Hoc fiber attaches randomly to the center of gp23* hexameric capsomers in one of the six possible orientations, though at the vertex-proximal hexamers that deviate from 6-fold symmetry, Hoc binds in two preferred orientations related by 180° rotation. Remarkably, each Hoc fiber binds to all six subunits of the capsomer, though the interactions are greatest with three of the subunits, resulting in the off-centered attachment of the C-domain. Biochemical analyses suggest that the acidic Hoc fiber (pI, ~4-5) allows for the clustering of virions in acidic pH and dispersion in neutral/alkaline pH. Hoc appears to have evolved as a sensing device that allows the phage to navigate its movements through reversible clustering-dispersion transitions so that it reaches its destination, the host bacterium, and persists in various ecological niches such as the human/mammalian gut.
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