Stan Schein

Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks.

Human Adenovirus Structures Human adenoviruses may be a common cause of acute infections in humans, but they can also be used as vectors for vaccine and therapeutic gene transfer. Rational engineering of safe adenovirus vectors has been hampered by a lack of high-resolution structural information. Two papers now describe the structure of human adenovirus using complementary techniques. Reddy et al. (p. 1071; see the Perspective by Harrison) have determined the crystal structure at 3.5 angstrom resolution, while Liu et al. (p. 1038; see the Perspective by Harrison) solved the structure to 3.6 angstrom resolution by electron microscopy. Together the structures provide insights into viral assembly, stabilization, and cell entry mechanisms. High-resolution structures provide a basis for optimizing adenovirus as a vaccine and gene-therapy vector. Construction of a complex virus may involve a hierarchy of assembly elements. Here, we report the structure of the whole human adenovirus virion at 3.6 angstroms resolution by cryo–electron microscopy (cryo-EM), revealing in situ atomic models of three minor capsid proteins (IIIa, VIII, and IX), extensions of the (penton base and hexon) major capsid proteins, and interactions within three protein-protein networks. One network is mediated by protein IIIa at the vertices, within group-of-six (GOS) tiles—a penton base and its five surrounding hexons. Another is mediated by ropes (protein IX) that lash hexons together to form group-of-nine (GON) tiles and bind GONs to GONs. The third, mediated by IIIa and VIII, binds each GOS to five surrounding GONs. Optimization of adenovirus for cancer and gene therapy could target these networks.

Cryo-EM structure of the mature dengue virus at 3.5-Å resolution

Regulated by pH, membrane-anchored proteins E and M function during dengue virus maturation and membrane fusion. Our atomic model of the whole virion from cryo–electron microscopy at 3.5-Å resolution reveals that in the mature virus at neutral extracellular pH, the N-terminal 20-amino-acid segment of M (involving three pH-sensing histidines) latches and thereby prevents spring-loaded E fusion protein from prematurely exposing its fusion peptide. This M latch is fastened at an earlier stage, during maturation at acidic pH in the trans-Golgi network. At a later stage, to initiate infection in response to acidic pH in the late endosome, M releases the latch and exposes the fusion peptide. Thus, M serves as a multistep chaperone of E to control the conformational changes accompanying maturation and infection. These pH-sensitive interactions could serve as targets for drug discovery.

Cryo-EM Model of the Bullet-Shaped Vesicular Stomatitis Virus

VSV in 3D Rhabdoviruses are a family of negative-stranded RNA viruses that includes rabies virus, which have a characteristic bullet shape. Though structures of individual rhabdovirus proteins have been reported, how these are organized into a bullet shape has remained unclear. Now, Ge et al. (p. 689) report a cryo-electron microscopy structure of a model rhabdovirus, vesicular stomatitis virus. The structural data and examination of mutants allows modeling of virion assembly. The structure of a negative-strand RNA virus suggests how bullet-shaped rhabdoviruses assemble. Vesicular stomatitis virus (VSV) is a bullet-shaped rhabdovirus and a model system of negative-strand RNA viruses. Through direct visualization by means of cryo–electron microscopy, we show that each virion contains two nested, left-handed helices: an outer helix of matrix protein M and an inner helix of nucleoprotein N and RNA. M has a hub domain with four contact sites that link to neighboring M and N subunits, providing rigidity by clamping adjacent turns of the nucleocapsid. Side-by-side interactions between neighboring N subunits are critical for the nucleocapsid to form a bullet shape, and structure-based mutagenesis results support this description. Together, our data suggest a mechanism of VSV assembly in which the nucleocapsid spirals from the tip to become the helical trunk, both subsequently framed and rigidified by the M layer.

Extensive investigation of a large Brazilian pedigree of 11778/haplogroup J Leber hereditary optic neuropathy

PURPOSE To conduct systematic epidemiologic, neuro-ophthalmologic, psychophysical, and mitochondrial DNA (mtDNA) genetic examinations on a newly identified pedigree with Leber hereditary optic neuropathy (LHON). DESIGN Observational population cohort study. METHODS A prospective investigation of an entire Brazilian LHON family. SETTING A field investigation by an international team conducted in a remote part of Brazil. STUDY POPULATION We evaluated 265 (both eyes) of the 328 living family members of this LHON pedigree. Only members of this pedigree were studied. Those entering the pedigree as spouses were used as controls. OBSERVATION PROCEDURES We conducted epidemiologic interviews emphasizing possible environmental risk factors, comprehensive neuro-ophthalmologic examinations, psychophysical tests, Humphrey visual field studies, fundus photography, and blood testing for mitochondrial genetic analysis. RESULTS We reconstructed a seven-generation maternal lineage descended from a common ancestor dating to the 1870s. All maternally related family members were invariably homoplasmic 11778 with a haplogroup J mtDNA, 33 being affected, of which 22 are still living. With each subsequent generation, there was a progressive decrease of penetrance, and only males were affected in the last two generations. A significant exposure (greater than 95% confidence intervals) to a variety of environmental risk factors characterized the affected individuals, with smoking as the most common (P <.01). Both affected and carriers (95% confidence intervals) presented with a significantly lower incidence of hypertension and high cholesterol compared with the control group (P <.05). CONCLUSIONS Almost 95% of a 328-living-member pedigree with LHON 11778/J haplogroup was comprehensively studied. Our initial results indicate the strong influence of environmental risk factors. The remarkably reduced incidence of cardiovascular risk in the maternal lineage is discussed. Further genetic analysis may reveal a role for the nuclear genome.

Bluetongue virus coat protein VP2 contains sialic acid-binding domains, and VP5 resembles enveloped virus fusion proteins

Bluetongue virus (BTV) is transmitted by blood-feeding insects (Culicoides sp.) and causes hemorrhagic diseases in livestock. BTV is a nonenveloped, double-stranded RNA (dsRNA) virus with two capsids: a well-studied, stable core enclosing the dsRNA genome and a highly unstable, poorly studied coat responsible for host cell attachment and entry. Here, based on cryo-electron microscopy (cryoEM), we report a 7-Å resolution structure of the infectious BTV virion, including the coat proteins. We show that unlike other dsRNA viruses, the VP2 attachment trimer has a triskelion shape composed of three tip domains branching from a central hub domain. We identify three putative sialic acid-binding pockets in the hub and present supporting biochemical data indicating sugar moiety binding is important for BTV infection. Despite being a nonenveloped virus, the putative VP5 membrane penetration trimer, located slightly inward of the VP2 attachment trimer, has a central coiled-coil α-helical bundle, similar to the fusion proteins of many enveloped viruses (e.g., HIV, herpesviruses, vesicular stomatitis virus, and influenza virus). Moreover, mapping of the amino acid sequence of VP5 to the secondary structural elements identified by cryoEM locates 15 amphipathic α-helical regions on the external surface of each VP5 trimer. The cryoEM density map also reveals few, weak interactions between the VP5 trimer and both the outer-coat VP2 trimer and the underlying core VP7 trimer, suggesting that the surface of VP5 could unfurl like an umbrella during penetration and shedding of the coat to release the transcriptionally active core particle.

Cell density ratios in a foveal patch in macaque retina

We examine the assumptions that the fovea contains equal numbers of inner (invaginating or ON) and outer (flat or OFF) midget bipolar cells and equal numbers of inner and outer diffuse bipolar cells. Based on reconstruction from electron photomicrographs of serial thin sections through the fovea of a macaque monkey, we reject both assumptions. First, every foveal L and M cone is presynaptic to one inner and one outer midget bipolar cell; however, S cones are presynaptic to one outer but no inner midget bipolar cell. Second, we measure the density of all foveal cells in the same patch of fovea, affording accurate cell density ratios. For each foveal cone pedicle, at a density of 26,500 mm(-2), there is close to one (0.88) outer diffuse bipolar cell but only 0.40 inner diffuse bipolar cells. This asymmetry may be related to differences in resolution and sensitivity for light increments and decrements. We also find one (1.01) Müller cell, one (1.01) amacrine cell in the inner nuclear layer, and close to one (0.83) horizontal cell for each cone pedicle. In addition, for each S cone, there are two inner S-cone bipolar cells and two small bistratified ganglion cells. In total, there are 3.4 cone bipolar cells per cone but only 2.6 ganglion cells per cone. The latter ratio is enough to accommodate one midget ganglion cell for each midget bipolar cell.

Two Ribbon Synaptic Units in Rod Photoreceptors of Macaque, Human, and Cat

The rod photoreceptors synaptic terminal (or spherule) uses an elaborate synaptic structure to signal absorption of one or more photons to its postsynaptic targets. This structure includes one or two synaptic ribbons inside the terminal and a pouch‐like “invagination” outside the terminal, into which enter a widely variable number of incoming fibers and postsynaptic targets—central elements supplied by rod bipolar cells and lateral elements supplied by horizontal cells. Nonetheless, our three‐dimensional reconstructions of this synaptic structure in foveal retina of macaque monkey and peripheral retina of human and cat reveal several features that are highly conserved across species and with eccentricity: 1) every spherule has one invagination; 2) with rare exceptions, every spherule has two ribbon synaptic units with these features: a) on the presynaptic side, each ribbon synaptic unit has a ribbon or part of a ribbon and one trough‐shaped arciform density that demarcates its active zone; b) on the postsynaptic side, each ribbon synaptic unit has two apposed lateral elements and one or more central elements; 3) the volume of the extracellular space in the single invagination is small, ∼0.1 μm3; and 4) the largest distance from active zone to receptor regions on bipolar cells is small, less than ∼1.5 μm. With such small dimensions, release of one quantum of transmitter can pulse glutamate to a concentration comparable to the EC50 of the metabotropic glutamate receptors on the central elements associated with both synaptic units. We speculate that two ribbon synaptic units are required to sustain the high quantal release rate needed to signal a single photon. J. Comp. Neurol. 455:100–112, 2003.

Inner S-Cone Bipolar Cells Provide All of the Central Elements for S Cones in Macaque Retina

Synaptic terminals of cones (pedicles) are presynaptic to numerous processes that arise from the dendrites of many types of bipolar cell. One kind of process, a central element, reaches deeply into invaginations of the cone pedicle just below an active zone associated with a synaptic ribbon. By reconstruction from serial electron micrographs, we show that L‐ and M‐cone pedicles in macaque fovea are presynaptic to ∼20 central elements that arise from two types of inner (invaginating) bipolar cell, midget and diffuse. In contrast, S‐cone pedicles, with more synaptic ribbons, active zones/ribbon, and central elements/active zone, are presynaptic to ∼33 central elements. Moreover, all of these arise from one type of bipolar cell, previously described by others, here termed an inner S‐cone bipolar cell. Each provides ∼16 central elements. Thirty‐three is twice 16; correspondingly, these bipolar cells are twice as numerous as S cones. (Specifically, each S cone is presynaptic to four inner S‐cone bipolar cells; in turn, each bipolar cell provides central elements to two S cones.) These bipolar cells are presynaptic to an equal number of small‐field bistratified ganglion cells, giving cell numbers in 2G:2B:1S ratios. Each ganglion cell receives input from two or more inner S‐cone bipolar cells and thereby collects signals from three or more S cones. This convergence, along with chromatic aberration of short‐wavelength light, suggests that S‐cone contributions to this ganglion cells coextensive blue‐ON/yellow‐OFF receptive field are larger than opponent L/M‐cone contributions via outer diffuse bipolar cells and that opponent L/M‐cone signals are conveyed mainly by inner S‐cone bipolar cells. J. Comp. Neurol. 457:185–201, 2003.

Assembly of silver Trigons into a buckyball-like Ag180 nanocage

Significance Here we present a striking outcome from the alliance between chemistry and mathematics in the design, synthesis, and characterization of a silver cage, Ag180. In principle, the design replaces each carbon atom of C60 with a triplet of argentophilicity-bonded silver atoms to produce a 3.4.6.4 (1,1) polyhedron with sixty 3-gons, ninety 4-gons, twelve 5-gons, and twenty 6-gons. Results from mass spectroscopy suggest an assembly mechanism in solution based on such triplets––the Silver-Trigon Assembly Road (STAR). Indeed, the STAR mechanism may be a general synthetic pathway toward even larger silver polyhedral cages. Besides its fundamental appeal, this synthetic cage may be considered for use as a molecular luminescent thermometer. Buckminsterfullerene (C60) represents a perfect combination of geometry and molecular structural chemistry. It has inspired many creative ideas for building fullerene-like nanopolyhedra. These include other fullerenes, virus capsids, polyhedra based on DNA, and synthetic polynuclear metal clusters and cages. Indeed, the regular organization of large numbers of metal atoms into one highly complex structure remains one of the foremost challenges in supramolecular chemistry. Here we describe the design, synthesis, and characterization of a Ag180 nanocage with 180 Ag atoms as 4-valent vertices (V), 360 edges (E), and 182 faces (F)––sixty 3-gons, ninety 4-gons, twelve 5-gons, and twenty 6-gons––in agreement with Euler’s rule V − E + F = 2. If each 3-gon (or silver Trigon) were replaced with a carbon atom linked by edges along the 4-gons, the result would be like C60, topologically a truncated icosahedron, an Archimedean solid with icosahedral (Ih) point-group symmetry. If C60 can be described mathematically as a curling up of a 6.6.6 Platonic tiling, the Ag180 cage can be described as a curling up of a 3.4.6.4 Archimedean tiling. High-resolution electrospray ionization mass spectrometry reveals that {Ag3}n subunits coexist with the Ag180 species in the assembly system before the final crystallization of Ag180, suggesting that the silver Trigon is the smallest building block in assembly of the final cage. Thus, we assign the underlying growth mechanism of Ag180 to the Silver-Trigon Assembly Road (STAR), an assembly path that might be further employed to fabricate larger, elegant silver cages.

Evidence That Each S Cone in Macaque Fovea Drives One Narrow-Field and Several Wide-Field Blue-Yellow Ganglion Cells

A rule of retinal wiring is that many receptors converge onto fewer bipolar cells and still fewer ganglion cells. However, for each S cone in macaque fovea, there are two S-cone ON bipolar cells and two blue-yellow (BY) ganglion cells. To understand this apparent rule reversal, we reconstructed synaptic patterns of divergence and convergence and determined the basic three-tiered unit of connectivity that repeats across the retina. Each foveal S cone diverges to four S-cone ON bipolar cells but contacts them unequally, providing 1–16 ribbon synapses per cell. Next, each bipolar cell diverges to two BY ganglion cells and also contacts them unequally, providing ∼14 and ∼28 ribbon synapses per cell. Overall, each S cone diverges to approximately six BY ganglion cells, dominating one and contributing more modestly to the others. Conversely, of each pair of BY ganglion cells, one is dominated by a single S cone and one is diffusely driven by several. This repeating circuit extracts blue/yellow information on two different spatiotemporal scales and thus parallels the circuits for achromatic, spatial vision, in which each cone dominates one narrow-field ganglion cell (midget) and contributes some input to several wider-field ganglion cells (parasol). Finally, because BY ganglion cells have coextensive +S and –(L+M) receptive fields, and each S cone contributes different weights to different BY ganglion cells, the coextensive receptive fields must be already present in the synaptic terminal of the S cone. The S-cone terminal thus constitutes the first critical locus for BY color vision.