Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with approximately 90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.     

二十面体病毒衣壳自组装的热力学参数

球状病毒衣壳采用二十面体对称。本文就二十面体病毒蛋白质骨架的自组装提出一个统计热力学模型。统计结果表明病毒衣壳二十面体对称不是自由能最小化的结果，而是衣壳内部蛋白结构参数最优化的结果。     

蛋白亚基局部相互作用模拟构筑二十面体病毒衣壳

二十面体病毒衣壳结构蛋白具有不同的构象。装配过程中,正在附着的蛋白亚基构象与位置在先前蛋白亚基构象的指令下,发生相应的调整后装配到衣壳骨架上。局部相互作用指导下的装配反复进行,最终形成规整的二十面体病毒衣壳的立体结构。对T=1、3、4、7的病毒衣壳,分别建立了蛋白亚基构象局部相互作用的连接方式与增长方式,并构筑了T=4、7的立体模型。     

Different rhinovirus serotypes neutralized by antipeptide antibodies

Recently, Rossman et al. have described the three-dimensional structure of a human rhinovirus. A possible host cell surface receptor binding site was identified with a cleft on each icosahedral face. Two highly conserved amino-acid sequences found in rhino-, polio-, and foot-and-mouth disease (FMD) viruses are located near the base of this site and could be important in maintaining its topology. We have prepared site-specific antibodies to two synthetic peptides which include these sequences. The antibodies bind to the predicted capsid proteins of rhinovirus and neutralize approximately 60% of 48 rhinovirus serotypes tested. These results could provide a route to a rhinovirus vaccine effective against most of the numerous serotypes of this virus.     

Structural changes of envelope proteins during alphavirus fusion

Alphaviruses are enveloped RNA viruses that have a diameter of about 700?? and can be lethal human pathogens. Entry of virus into host cells by endocytosis is controlled by two envelope glycoproteins, E1 and E2. The E2-E1 heterodimers form 80 trimeric spikes on the icosahedral virus surface, 60 with quasi-three-fold symmetry and 20 coincident with the icosahedral three-fold axes arranged with T = 4 quasi-symmetry. The E1 glycoprotein has a hydrophobic fusion loop at one end and is responsible for membrane fusion. The E2 protein is responsible for receptor binding and protects the fusion loop at neutral pH. The lower pH in the endosome induces the virions to undergo an irreversible conformational change in which E2 and E1 dissociate and E1 forms homotrimers, triggering fusion of the viral membrane with the endosomal membrane and then releasing the viral genome into the cytoplasm. Here we report the structure of an alphavirus spike, crystallized at low pH, representing an intermediate in the fusion process and clarifying the maturation process. The trimer of E2-E1 in the crystal structure is similar to the spikes in the neutral pH virus except that the E2 middle region is disordered, exposing the fusion loop. The amino- and carboxy-terminal domains of E2 each form immunoglobulin-like folds, consistent with the receptor attachment properties of E2.     

Self-assembly of nanoscale cuboctahedra by coordination chemistry

Self-assembled polyhedral structures are common in biology. The coats of many viruses, for example, have a structure based on icosahedral symmetry. The preparation of synthetic polyhedral molecular assemblies represents a challenging problem, but supramolecular chemistry has now advanced to the point where the task may be addressed. Macromolecular and supramolecular entities of predefined geometric shape and with well-defined internal environments are potentially important for inclusion phenomena, molecular recognition and catalysis. Here we report the use of self-assembly of molecular units driven by coordination to transition-metal ions to prepare a cuboctahedron from 20 tridentate and bidentate subunits in a single step. The cuboctahedron is an archimedean semiregular polyhedron that combines square and triangular faces. Our self-assembled polyhedral capsules, characterized by NMR and electrospray mass spectrometry, are around 5 nanometres in diameter.     

Localization of VP4 neutralization sites in rotavirus by three-dimensional cryo-electron microscopy

Three-dimensional structures of several spherical viruses have been determined by electron microscopy and X-ray crystallography. We report here the first three-dimensional structure of the complex between an intact virus and Fab fragments of a neutralizing monoclonal antibody. The antibody is against VP4, one of the two outer capsid proteins of rotaviruses. These large icosahedral viruses cause gastroenteritis in children and young animals and account for over a million human deaths annually. VP4 in these viruses has been implicated in several important functions such as cell penetration, haemagglutination, neutralization and virulence. Here we demonstrate that the surface spikes on rotavirus particles are made up of VP4. Antigenic sites are located near the distal ends of the spikes and two Fab fragments bind to each of the sixty spikes. The mass of the spike indicates that it is a dimer of VP4. The bilobed structure at the distal end of the spike may be involved in both the attachment to the cell and in viral penetration. A novel feature in the virus-Fab complex is the structural difference between the two chemically equivalent Fab fragments on each spike, which could be indicative of variations in the Fab elbow angles.     

Structural basis of West Nile virus neutralization by a therapeutic antibody

West Nile virus is a mosquito-borne flavivirus closely related to the human epidemic-causing dengue, yellow fever and Japanese encephalitis viruses. In establishing infection these icosahedral viruses undergo endosomal membrane fusion catalysed by envelope glycoprotein rearrangement of the putative receptor-binding domain III (DIII) and exposure of the hydrophobic fusion loop. Humoral immunity has an essential protective function early in the course of West Nile virus infection. Here, we investigate the mechanism of neutralization by the E16 monoclonal antibody that specifically binds DIII. Structurally, the E16 antibody Fab fragment engages 16 residues positioned on four loops of DIII, a consensus neutralizing epitope sequence conserved in West Nile virus and distinct in other flaviviruses. The E16 epitope protrudes from the surface of mature virions in three distinct environments, and docking studies predict Fab binding will leave five-fold clustered epitopes exposed. We also show that E16 inhibits infection primarily at a step after viral attachment, potentially by blocking envelope glycoprotein conformational changes. Collectively, our results suggest that a vaccine strategy targeting the dominant DIII epitope may elicit safe and effective immune responses against flaviviral diseases.     

Membrane structure and interactions with protein and DNA in bacteriophage PRD1

Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 A resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 A resolution, allows the first detailed analysis of a membrane-containing virus. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.     

The virophage as a unique parasite of the giant mimivirus

Viruses are obligate parasites of Eukarya, Archaea and Bacteria. Acanthamoeba polyphaga mimivirus (APMV) is the largest known virus; it grows only in amoeba and is visible under the optical microscope. Mimivirus possesses a 1,185-kilobase double-stranded linear chromosome whose coding capacity is greater than that of numerous bacteria and archaea1, 2, 3. Here we describe an icosahedral small virus, Sputnik, 50 nm in size, found associated with a new strain of APMV. Sputnik cannot multiply in Acanthamoeba castellanii but grows rapidly, after an eclipse phase, in the giant virus factory found in amoebae co-infected with APMV4. Sputnik growth is deleterious to APMV and results in the production of abortive forms and abnormal capsid assembly of the host virus. The Sputnik genome is an 18.343-kilobase circular double-stranded DNA and contains genes that are linked to viruses infecting each of the three domains of life Eukarya, Archaea and Bacteria. Of the 21 predicted protein-coding genes, eight encode proteins with detectable homologues, including three proteins apparently derived from APMV, a homologue of an archaeal virus integrase, a predicted primase-helicase, a packaging ATPase with homologues in bacteriophages and eukaryotic viruses, a distant homologue of bacterial insertion sequence transposase DNA-binding subunit, and a Zn-ribbon protein. The closest homologues of the last four of these proteins were detected in the Global Ocean Survey environmental data set5, suggesting that Sputnik represents a currently unknown family of viruses. Considering its functional analogy with bacteriophages, we classify this virus as a virophage. The virophage could be a vehicle mediating lateral gene transfer between giant viruses.     

Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications.

The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage phi X174. The F protein forms a T = 1 capsid whose major folding motif is the eight-stranded antiparallel beta barrel found in many other icosahedral viruses. Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density. Each G protein is a tight beta barrel with its strands running radially outwards and with a topology similar to that of the F protein. The 12 'pilot' H proteins per virion may be partially located in the putative ion channel. The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein. Tentatively, there are three regions of partially ordered DNA structure,     

Antigen chimaeras of poliovirus as potential new vaccines

Polioviruses occur as three distinct serotypes, 1, 2 and 3, and are composed of a single-stranded positive-sense RNA genome of approximately 7,450 nucleotides enclosed in an icosahedral particle of diameter 27 nm. The three-dimensional crystallographic structure of poliovirus type 1 has been determined at 2.9 A resolution, providing a detailed knowledge of the folding and arrangement of the individual virus proteins, VP1-VP4. From this and the characterization of monoclonal antibody-resistant mutants, the amino acids contributing to antigenic sites have been identified and located on the surface of the virus particle. Here we describe the construction and characterization of a poliovirus chimaera having a defined region of type 3 inserted into type 1. This virus has composite antigenicity and the substitute site is immunogenic in small animals and primates. The ability to construct such viruses has implications for the design of improved poliovirus vaccine strains or vaccines against other picornaviruses, such as hepatitis A.     

人和动物的多瘤病毒

多瘤病毒分类于乳多空病毒科，ＤＮＡ肿瘤病毒。病毒无囊膜，直径４０ｎｍ～４５ｎｍ，有３种～４种衣壳蛋白，基因组为约５０００对核苷酸组成的双链闭合环状ＤＮＡ。病毒在自然界分布广泛，目前已从人、兔子、小牛、鸟类、啮齿类和灵长类等动物分离到多种多瘤病毒。各病毒内部有共同的属特异性抗原，但大多数病毒表面蛋白无血清学交叉反应。病毒在容许细胞中增殖良好，能使非容许细胞发生转化，在转化细胞中病毒ＤＮＡ以整合到宿主染色体的方式存在。多瘤病毒感染有严格的种特异性，在自然宿主内大多数病毒呈隐性感染，但人和虎皮鹦鹉多瘤病毒对宿主有一定致病性。本文就有关多瘤病毒的感染和生物学特性研究进展作一概述     

Structural and serological evidence for a novel mechanism of antigenic variation in foot-and-mouth disease virus.

Changes resulting in altered antigenic properties of viruses nearly always occur on their surface and have been attributed to the substitution of residues directly involved in binding antibody. To investigate the mechanism of antigenic variation in foot-and-mouth disease virus (FMDV), variants that escape neutralization by a monoclonal antibody have been compared crystallographically and serologically with parental virus. FMDVs form one of the four genera of the Picornaviridae. The unenveloped icosahedral shell comprises 60 copies each of four structural proteins VP1-4. Representatives from each of the genera have similar overall structure, but differences in the external features. For example, human rhinovirus has a pronounced 'canyon' that is proposed to contain the cell attachment site, whereas elements of the attachment site for FMDV, which involves the G-H loop (residues 134-160) and C-terminus (200-213) of VP1, are exposed on the surface. Moreover, this G-H loop, which is a major antigenic site of FMDV, forms a prominent, highly accessible protrusion, a feature not seen in other picornaviruses. It is this loop that is perturbed in the variant viruses that we have studied. The amino acid mutations characterizing the variants are not at positions directly involved in antibody binding, but result in far-reaching perturbations of the surface structure of the virus. Thus, this virus seems to use a novel escape mechanism whereby an induced conformational change in a major antigenic loop destroys the integrity of the epitope.     

Insights into assembly from structural analysis of bacteriophage PRD1

The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.     

熔态急冷合金准晶Ⅰ相的形成

TEM和SEM观察表明,熔态Al_(86)Mn_(14)合金急冷时形成的二十面体准晶相,存在大量精细的亚结构;准晶体因形成时的熔体冷却速率的高低,可具有“菊花”状或规则枝晶状的形态,它们都是分形结构且分形维数相近,提出了一种基于熔体中二十面体相小颗粒无规扩散和聚集过程的准晶体形成模型,据此模型进行的计算机模拟计算得到的准晶体生长图形,其形态特征与分形维数都与实验观察结果基本相符。     

大块准晶材料的制备

给出了一种大块准晶材料的制取方法,并且对所得材料的性能进行了研究。采用快速凝固制取几乎完全为二十面体相的准晶粉末,通过爆炸成形得到大块准晶材料。研究结果表明,在Al-Mn,Al-Cr和Al-Mn-Cr合金系列中,调整合金成分和冷却速度能够制得几乎完全为二十面体相的Al_(77.5)Mn_(22.5),Al_(82)Cr_(18)和Al_(78)Mn_(18)Cr_4准晶粉末,经爆炸成形后,所得的大块准晶材料仍然保持其二十面体相结构。     

Self-assembly of regular hollow icosahedra in salt-free catanionic solutions

Self-assembled structures having a regular hollow icosahedral form (such as those observed for proteins of virus capsids) can occur as a result of biomineralization processes, but are extremely rare in mineral crystallites. Compact icosahedra made from a boron oxide have been reported, but equivalent structures made of synthetic organic components such as surfactants have not hitherto been observed. It is, however, well known that lipids, as well as mixtures of anionic and cationic single chain surfactants, can readily form bilayers that can adopt a variety of distinct geometric forms: they can fold into soft vesicles or random bilayers (the so-called sponge phase) or form ordered stacks of flat or undulating membranes. Here we show that in salt-free mixtures of anionic and cationic surfactants, such bilayers can self-assemble into hollow aggregates with a regular icosahedral shape. These aggregates are stabilized by the presence of pores located at the vertices of the icosahedra. The resulting structures have a size of about one micrometre and mass of about 1010 daltons, making them larger than any known icosahedral protein assembly or virus capsid. We expect the combination of wall rigidity and holes at vertices of these icosahedral aggregates to be of practical value for controlled drug or DNA release.     

Three-dimensional structure of the wild-type RHDV

The three-dimensional (3D) structure of the wild-type rabbit hemorrhagic disease virus (RHDV) has been determined to a resolution of 3.2 nm by electron cryomicroscopy and computer image reconstruction techniques. The 3D density map exhibits characteristic structural features of a calicivirus: a T=3 icosahedral capsid with 90 arch-like capsomeres at the icosahedral and local 2-fold axes and 32 large surface hollows at the icosahedral 5- and 3-fold axes. This result confirms that the RHDV isolated in China is a member of the Caliciviridae family. A rather continuous capsid shell was found without channels. However, our RHDV structure also reveals some distinct structural characteristics not observed in other caliciviruses, including interconnected capsomeres and the lack of protuberance on the base of each of the surface hollows. Two types of particles were identified with similar outer capsid structure but different density distributions inside the capsid shells, which could not be distinguished by conventional negative staining electron microscopy. As the genomic and subgenomic RNAs are both packaged into particles for RHDV, we suggest that the two types of particles identified correspond to those containing either the genomic or subgenomic RNAs, respectively.     

玻璃形成液体其内在结构中的二十面体

采用分子动力学模拟(molecular dynamics简称MD)的方法,并利用势能曲面(potential energy landscape简称PEL)理论对玻璃形成液体进行研究.在模拟中,键角分布序参数(An)被首先应用于描述二元玻璃形成液体其内在结构(Inherent Structure简称IS)中的结构序.通过计算An,得出了一般液体和IS液体中六面体、八面体和二十面体结构随温度的变化情况.在IS液体中二十面体序所占比例明显多于一般液体.在IS液体中,六面体、八面体的结构随温度的变化不是很明显,而二十面体结构随着温度的变化呈现出一定的规律.得出了二十面体是IS的主要变化的结构.玻璃转变是一般液体接近IS的过程,同时也是二十面体增多的过程.