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1.
Distribution of virus structural proteins and protein-protein interactions in plasma membrane of baby hamster kidney cells infected with Sindbis or vesicular stomatitis virus. 总被引:14,自引:1,他引:14
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H Scheefers U Scheefers-Borchel J Edwards D T Brown 《Proceedings of the National Academy of Sciences of the United States of America》1980,77(12):7277-7281
The plasma membrane of baby hamster kidney (BHK-21) cells infected with either Sindbis or vesicular stomatitis virus was isolated by a technique involving the ingestion of latex beads by the cells. Plasma membrane isolated from Sindbis virus-infected cells contained only one (E1) of the three (E1, E2, and C) structural proteins of this virus. When the latex beads were pretreated with either polylysine or DEAE-dextran, plasma membrane obtained from Sindbis virus-infected cells contained all three structural proteins and PE2, a precursor to one of the structural proteins. In pulse-chase radiolabeling experiments with Sindbis virus-infected cells, it was possible to follow the appearance of the precursor protein (PE2) i the plasma membrane and its eventual conversion to E2. The appearance of Sindbis virus membrane proteins PE2 and E1 in the purified plasma membrane was not affected by the drug tunicamycin, an inhibitor of glycosylation. These experiments imply the following: (i) Cleavage of the Sindbis virus precursor polypeptide PE2 to E2 is not a prerequisite for its transport to the cell plasma membrane; (ii) transport of virus membrane proteins to the cell surface does not depend on glycosylation; and (iii) although all Sindbis virus structural proteins are associated with the plasma membrane, a generally accepted pairing of PE2-E1 or E2-E1 in the plasma membrane either does not exist or, if it does exist, involves a very weak interaction. The procedures used in this study also resulted in the successful isolation of plasma membrane from vesicular stomatitis virus-infected cells containing the glycoprotein, the matrix protein, and the nucleocapsid protein, a result that suggests that these proteins are located on the media side of baby hamster kidney cells grown in monolayer. 相似文献
2.
David Veesler Thiam-Seng Ng Anoop K. Sendamarai Brian J. Eilers C. Martin Lawrence Shee-Mei Lok Mark J. Young John E. Johnson Chi-yu Fu 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(14):5504-5509
Sulfolobus turreted icosahedral virus (STIV) was isolated in acidic hot springs where it infects the archeon Sulfolobus solfataricus. We determined the STIV structure using near-atomic resolution electron microscopy and X-ray crystallography allowing tracing of structural polypeptide chains and visualization of transmembrane proteins embedded in the viral membrane. We propose that the vertex complexes orchestrate virion assembly by coordinating interactions of the membrane and various protein components involved. STIV shares the same coat subunit and penton base protein folds as some eukaryotic and bacterial viruses, suggesting that they derive from a common ancestor predating the divergence of the three kingdoms of life. One architectural motif (β-jelly roll fold) forms virtually the entire capsid (distributed in three different gene products), indicating that a single ancestral protein module may have been at the origin of its evolution. 相似文献
3.
S D London A L Schmaljohn J M Dalrymple C M Rice 《Proceedings of the National Academy of Sciences of the United States of America》1992,89(1):207-211
Random insertion mutagenesis has been used to construct infectious Sindbis virus structural protein chimeras containing a neutralization epitope from a heterologous virus, Rift Valley fever virus. Insertion sites, permissive for recovery of chimeric viruses with growth properties similar to the parental virus, were found in the virion E2 glycoprotein and the secreted E3 glycoprotein. For the E2 chimeras, the epitope was expressed on the virion surface and stimulated a partially protective immune response to Rift Valley fever virus infection in vivo. Besides providing a possible approach for developing live attenuated vaccine viruses, insertion of peptide ligands into virion surface proteins may ultimately allow targeting of virus infection to specific cell types. 相似文献
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5.
Senkevich TG White CL Koonin EV Moss B 《Proceedings of the National Academy of Sciences of the United States of America》2002,99(10):6667-6672
We show that three cytoplasmic thiol oxidoreductases encoded by vaccinia virus comprise a complete pathway for formation of disulfide bonds in intracellular virion membrane proteins. The pathway was defined by analyzing conditional lethal mutants and effects of cysteine to serine substitutions and by trapping disulfide-bonded heterodimer intermediates for each consecutive step. The upstream component, E10R, belongs to the ERV1/ALR family of FAD-containing sulfhydryl oxidases that use oxygen as the electron acceptor. The second component, A2.5L, is a small alpha-helical protein with a CxxxC motif that forms a stable disulfide-linked heterodimer with E10R and a transient disulfide-linked complex with the third component, G4L. The latter is a thioredoxin-like protein that directly oxidizes thiols of L1R, a structural component of the virion membrane with three stable disulfide bonds, and of the related protein F9L. These five proteins are conserved in all poxviruses, suggesting that the pathway is an ancestral mechanism for direct thiol-disulfide interchanges between proteins even in an unfavorable reducing environment. 相似文献
6.
Joyce Jose Jinghua Tang Aaron B. Taylor Timothy S. Baker Richard J. Kuhn 《Viruses》2015,7(12):6182-6199
Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examined the virions by transmission electron cryo-microscopy and determined the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, respectively. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry analysis, contributed to a 10-fold reduction in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking experiments. We used the FP-tagged virus for high-resolution live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close association with filopodial extensions. 相似文献
7.
Lesley J. Calder Sebastian Wasilewski John A. Berriman Peter B. Rosenthal 《Proceedings of the National Academy of Sciences of the United States of America》2010,107(23):10685-10690
Influenza is a lipid-enveloped, pleomorphic virus. We combine electron cryotomography and analysis of images of frozen-hydrated virions to determine the structural organization of filamentous influenza A virus. Influenza A/Udorn/72 virions are capsule-shaped or filamentous particles of highly uniform diameter. We show that the matrix layer adjacent to the membrane is an ordered helix of the M1 protein and its close interaction with the surrounding envelope determines virion morphology. The ribonucleoprotein particles (RNPs) that package the genome segments form a tapered assembly at one end of the virus interior. The neuraminidase, which is present in smaller numbers than the hemagglutinin, clusters in patches and are typically present at the end of the virion opposite to RNP attachment. Incubation of virus at low pH causes a loss of filamentous morphology, during which we observe a structural transition of the matrix layer from its helical, membrane-associated form to a multilayered coil structure inside the virus particle. The polar organization of the virus provides a model for assembly of the virion during budding at the host membrane. Images and tomograms of A/Aichi/68 X-31 virions show the generality of these conclusions to non-filamentous virions. 相似文献
8.
Sheng Cao Wei Zhang 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(33):13362-13367
The sequential steps in the alphavirus membrane fusion pathway have been postulated based on the prefusion and postfusion crystal structures of the viral fusion protein E1 in conjunction with biochemical studies. However, the molecular structures of the hypothesized fusion intermediates have remained obscure due to difficulties inherent in the dynamic nature of the process. We developed an experimental system that uses liposomes as the target membrane to capture Sindbis virus, a prototypical alphavirus, in its membrane-binding form at pH 6.4. Cryoelectron micrograph analyses and 3D reconstructions showed that the virus retains its overall icosahedral structure at this mildly acidic pH, except in the membrane-binding region, where monomeric E1 associates with the target membrane and the E2 glycoprotein retains its original trimeric organization. The remaining E2 trimers may hinder E1 homotrimerization and are a potential target for antiviral drugs. 相似文献
9.
B Berger P W Shor L Tucker-Kellogg J King 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(16):7732-7736
A local rule-based theory is developed which shows that the self-assembly of icosahedral virus shells may depend on only the lower-level interactions of a protein subunit with its neighbors--i.e., on local rules rather than on larger structural building blocks. The local rule theory provides a framework for understanding the assembly of icosahedral viruses. These include both viruses that fall in the quasiequivalence theory of Caspar and Klug and the polyoma virus structure, which violates quasi-equivalence and has puzzled researchers since it was first observed. Local rules are essentially templates for energetically favorable arrangements. The tolerance margins for these rules are investigated through computer simulations. When these tolerance margins are exceeded in a particular way, the result is a "spiraling" malformation that has been observed in nature. 相似文献
10.
Hafenstein S Palermo LM Kostyuchenko VA Xiao C Morais MC Nelson CD Bowman VD Battisti AJ Chipman PR Parrish CR Rossmann MG 《Proceedings of the National Academy of Sciences of the United States of America》2007,104(16):6585-6589
Although many viruses are icosahedral when they initially bind to one or more receptor molecules on the cell surface, such an interaction is asymmetric, probably causing a breakdown in the symmetry and conformation of the original infecting virion in preparation for membrane penetration and release of the viral genome. Cryoelectron microscopy and biochemical analyses show that transferrin receptor, the cellular receptor for canine parvovirus, can bind to only one or a few of the 60 icosahedrally equivalent sites on the virion, indicating that either canine parvovirus has inherent asymmetry or binding of receptor induces asymmetry. The asymmetry of receptor binding to canine parvovirus is reminiscent of the special portal in tailed bacteriophages and some large, icosahedral viruses. Asymmetric interactions of icosahedral viruses with their hosts might be a more common phenomenon than previously thought and may have been obscured by averaging in previous crystallographic and electron microscopic structure determinations. 相似文献
11.
Reconstruction of the three-dimensional structure of simian virus 40 and visualization of the chromatin core. 总被引:19,自引:5,他引:19
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T S Baker J Drak M Bina 《Proceedings of the National Academy of Sciences of the United States of America》1988,85(2):422-426
The three-dimensional structure of the capsid and the nucleohistone core of simian virus 40 (SV40) has been reconstructed by image analysis of electron micrographs of frozen hydrated samples. The 72 prominent capsomere units that comprise the T = 7d icosahedral surface lattice of the capsid are clearly resolved. Both the pentavalent and hexavalent capsomeres appear with pentameric substructure, indicating that bonding specificity in the shell is not quasi-equivalent. There is a remarkable similarity between the structure of the SV40 virion capsid and the structure reported for the polyoma empty capsid. This result establishes that (i) the unexpected pentameric substructure of the hexavalent capsomeres is also present in virions and (ii) the arrangement of the 72 pentamers in the capsid lattice may be a characteristic feature of the entire papova family of viruses. The center of the SV40 reconstruction reveals electron density corresponding to the nucleohistone core. This density is smeared, suggesting that the minichromosome is not organized with icosahedral symmetry matching the capsid symmetry. The visualization of the virion chromatin provides a basis for invoking new models for the higher order structure of the encapsidated minichromosome. 相似文献
12.
Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus 总被引:36,自引:7,他引:29
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J. K. Rose W. J. Welch B. M. Sefton F. S. Esch N. C. Ling 《Proceedings of the National Academy of Sciences of the United States of America》1980,77(7):3884-3888
We have determined the COOH-terminal and NH(2)-terminal amino acid sequences of the vesicular stomatitis virus (VSV) glycoprotein (G). A sequence of 122 COOH-terminal amino acids was deduced from the complete sequence of a cloned DNA insert carrying 470 nucleotides derived from the 3' end of the G mRNA. Evidence presented indicates that this portion of the polypeptide includes the domains of G that reside inside the virion and span the lipid bilayer of the virion. This seems clear because a partial amino acid sequence of a fragment of G that remains associated with the membrane of the virion after exhaustive proteolytic digestions can be located unambiguously in the predicted sequence. This predicted sequence contains an uninterrupted hydrophobic domain beginning 49 amino acids and ending 30 amino acids from the COOH terminus. This region presumably spans the lipid bilayer. The COOH-terminal portion of 29 amino acids contains a high proportion of basic residues and resides inside the virion. The COOH-terminal portion of the VSV G protein therefore resembles in structure that of glycophorin, an erythrocyte membrane protein well characterized previously. The configuration of G in the viral membrane demonstrated here is probably similar for other viral glycoproteins, although this has not been tested as directly in any other case. From the sequence of a DNA primer extended on the RNA genome from the adjacent M protein gene into the G protein gene, we have deduced an NH(2)-terminal G protein sequence of 53 amino acids, including the leader sequence of 16 amino acids. Our sequence confirms, extends, and corrects two partial amino acid sequences reported for this region previously. 相似文献
13.
African swine fever virus (ASFV) is a highly contagious pathogen which causes a lethal haemorrhagic fever in domestic pigs and wild boar. The large, double-stranded DNA virus replicates in perinuclear cytoplasmic replication sites known as viral factories. These factories are complex, multi-dimensional structures. Here we investigated the protein and membrane compartments of the factory using super-resolution and electron tomography. Click IT chemistry in combination with stimulated emission depletion (STED) microscopy revealed a reticular network of newly synthesized viral proteins, including the structural proteins p54 and p34, previously seen as a pleomorphic ribbon by confocal microscopy. Electron microscopy and tomography confirmed that this network is an accumulation of membrane assembly intermediates which take several forms. At early time points in the factory formation, these intermediates present as small, individual membrane fragments which appear to grow and link together, in a continuous progression towards new, icosahedral virions. It remains unknown how these membranes form and how they traffic to the factory during virus morphogenesis. 相似文献
14.
Amino-terminal sequence analysis of the structural proteins of Sindbis virus. 总被引:1,自引:0,他引:1
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J R Bell M W Hunkapiller L E Hood J H Strauss 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(6):2722-2726
The structural proteins of Sindbis virus, an enveloped virus which belongs to the Togavirus family, have been subjected to automated Edman degradation using improved techniques. Extensive NH2-terminal sequences of about 50 residues were determined for each of the two membrane glycoproteins. In both cases the NH2 terminus of the molecule was found to be similar in composition to typical water-soluble proteins. The viral capsid protein was found to have a blocked alpha-amino group. This is consistent with other observations that viral proteins derived from the NH2 terminus of precursor molecules are often blocked. 相似文献
15.
Xinzheng Zhang Siyang Sun Ye Xiang Jimson Wong Thomas Klose Didier Raoult Michael G. Rossmann 《Proceedings of the National Academy of Sciences of the United States of America》2012,109(45):18431-18436
“Sputnik” is a dsDNA virus, referred to as a virophage, that is coassembled with Mimivirus in the host amoeba. We have used cryo-EM to produce an electron density map of the icosahedral Sputnik virus at 3.5-Å resolution, sufficient to verify the identity of most amino acids in the capsid proteins and to establish the identity of the pentameric protein forming the fivefold vertices. It was also shown that the virus lacks an internal membrane. The capsid is organized into a T = 27 lattice in which there are 260 trimeric capsomers and 12 pentameric capsomers. The trimeric capsomers consist of three double “jelly-roll” major capsid proteins creating pseudohexameric capsomer symmetry. The pentameric capsomers consist of five single jelly-roll proteins. The release of the genome by displacing one or more of the pentameric capsomers may be the result of a low-pH environment. These results suggest a mechanism of Sputnik DNA ejection that probably also occurs in other big icosahedral double jelly-roll viruses such as Adenovirus. In this study, the near-atomic resolution structure of a virus has been established where crystallization for X-ray crystallography was not feasible. 相似文献
16.
A recently solved structure of the aquareovirus virion (Zhang, X; Jin, L.; Fang, Q; Hui, W.H.; Zhou Z.H. 3.3 Å Cryo-EM Structure of a Nonenveloped Virus Reveals a Priming Mechanism for Cell Entry. Cell 2010, 141, 472–482 [1]) provides new insights into the order of entry events, as well as confirming and refining several aspects of the entry mechanism, for aquareovirus and the related orthoreovirus. In particular, the structure provides evidence of a defined order for the progressive proteolytic cleavages of myristoylated penetration protein VP5 that prime the virion for membrane penetration. These observations reinforce the concept that, much like enveloped viruses, nonenveloped virions often undergo priming events that lead to a meta-stable state, preparing the virus for membrane penetration under the appropriate circumstances. In addition, this and other recent studies highlight the increasing power of electron cryomicroscopy to analyze large, geometrically regular structures, such as icosahedral viruses, at atomic resolution. 相似文献
17.
Chen DH Baker ML Hryc CF DiMaio F Jakana J Wu W Dougherty M Haase-Pettingell C Schmid MF Jiang W Baker D King JA Chiu W 《Proceedings of the National Academy of Sciences of the United States of America》2011,108(4):1355-1360
Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions. 相似文献
18.
V irus and lectin agglutination of erythrocytes: spin label study of membrane lipid-protein interactions. 总被引:2,自引:0,他引:2
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D S Lyles F R Landsberger 《Proceedings of the National Academy of Sciences of the United States of America》1976,73(10):3497-3501
Techniques of spin-label electron spin resonance have been used to prove changes in the structure of the lipid phase of erythrocyte membrane after agglutination by viruses and lectins. When chicken erythrocytes are agglutinated by Sendai and influenza viruses and by the lectins concanavalin A and wheat germ agglutinin, the membrane lipid phase becomes more fluid, as detected by three different lipophilic spin-laveled probes. Colchicine, vinblastine, and tetracaine inhibit the fluidization of chicken erythrocyte membrane by Sendai virus, whereas cytochalasin B has no effect. The effect of colchicine was time dependent, the initial inhibition decreasing with longer preincubation times. Extensive treatment of erythrocytes with proteases or neuraminidase, while not altering the bilayer structure, abolishes the effect of Sendai virus on the erythrocyte membrane, suggesting that a change in the interaction of the receptor protein with the lipid phase occurs upon virus attachment. Glutaraldehyde fixation increased the structural rigidity of the chicken erythrocyte membrane and inhibited the effect of viral agglutination. No change in bilayer structure was observed upon agglutination of human erythrocytes or the isolated plasma membranes of either human or chicken erythrocytes. This result is consistent with the drug sensitivity of the effects of agglutination upon chicken erythrocytes, since human erythrocytes and isolated membranes lack microtubule-like structures. 相似文献
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20.
Sun J DuFort C Daniel MC Murali A Chen C Gopinath K Stein B De M Rotello VM Holzenburg A Kao CC Dragnea B 《Proceedings of the National Academy of Sciences of the United States of America》2007,104(4):1354-1359
This study concerns the self-assembly of virus-like particles (VLPs) composed of an icosahedral virus protein coat encapsulating a functionalized spherical nanoparticle core. The recent development of efficient methods for VLP self-assembly has opened the way to structural studies. Using electron microscopy with image reconstruction, the structures of several VLPs obtained from brome mosaic virus capsid proteins and gold nanoparticles were elucidated. Varying the gold core diameter provides control over the capsid structure. The number of subunits required for a complete capsid increases with the core diameter. The packaging efficiency is a function of the number of capsid protein subunits per gold nanoparticle. VLPs of varying diameters were found to resemble to three classes of viral particles found in cells (T=1, 2, and 3). As a consequence of their regularity, VLPs form three-dimensional crystals under the same conditions as the wild-type virus. The crystals represent a form of metallodielectric material that exhibits optical properties influenced by multipolar plasmonic coupling. 相似文献