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1.
J Virol ; 83(23): 12101-7, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19759134

ABSTRACT

Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.


Subject(s)
Dengue Virus/physiology , Furin/metabolism , Virus Assembly , Virus Internalization , Animals , Cell Line , Cryoelectron Microscopy , Culicidae , Dengue Virus/ultrastructure , Hydrogen-Ion Concentration , Image Processing, Computer-Assisted , Models, Biological , Models, Molecular
2.
Nat Struct Biol ; 8(10): 874-8, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11573093

ABSTRACT

Group B coxsackieviruses (CVB) utilize the coxsackievirus-adenovirus receptor (CAR) to recognize host cells. CAR is a membrane protein with two Ig-like extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmic domain. The three-dimensional structure of coxsackievirus B3 (CVB3) in complex with full length human CAR and also with the D1D2 fragment of CAR were determined to approximately 22 A resolution using cryo-electron microscopy (cryo-EM). Pairs of transmembrane domains of CAR associate with each other in a detergent cloud that mimics a cellular plasma membrane. This is the first view of a virus-receptor interaction at this resolution that includes the transmembrane and cytoplasmic portion of the receptor. CAR binds with the distal end of domain D1 in the canyon of CVB3, similar to how other receptor molecules bind to entero- and rhinoviruses. The previously described interface of CAR with the adenovirus knob protein utilizes a side surface of D1.


Subject(s)
Adenoviridae/metabolism , Enterovirus B, Human/metabolism , Receptors, Virus/metabolism , Adenoviridae/chemistry , HeLa Cells , Humans , Microscopy, Electron/methods , Models, Molecular , Receptors, Virus/chemistry , Viral Plaque Assay
3.
J Clin Microbiol ; 39(7): 2412-7, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11427547

ABSTRACT

From 1997 to 1999 seven isolates of Campylobacter-like organisms from five patients that were exhibiting symptoms of gastroenteritis, including fever, stomach malaise, and diarrhea, were investigated. The organisms were isolated from stool samples and found to exhibit a diverse colony morphology; hence multiple isolates were submitted from one of the patients. All isolates were found to be identical. The organisms were catalase, urease, alkaline phosphatase, and nitrate negative but oxidase and indoxyl acetate positive. They grew at 37 degrees C but not at 42 degrees C, and three of the isolates from two different patients were sensitive to nalidixic acid and cephalothin. Full 16S rRNA sequence analysis not only grouped these organisms within the Helicobacter genus but also differentiated them from previously identified Helicobacter species. The closest relative by phylogenetic analysis was Helicobacter sp. flexispira taxon 1. Electron microscopy showed that these isolates had one or two bipolar flagella; however, the periplasmic fibers, a characteristic of the known Helicobacter sp. flexispira taxa, were not observed. The present isolates also lacked a flagellar sheath, a trait shared with four other Helicobacter spp., H. canadensis, H. mesocricetorum, H. pullorum, and H. rodentium. On the basis of the unique phenotypic properties of these isolates and 16S rRNA sequence analysis, we propose the classification of a new Helicobacter species, Helicobacter winghamensis sp. nov.


Subject(s)
Gastroenteritis/microbiology , Helicobacter Infections/microbiology , Helicobacter/classification , Helicobacter/isolation & purification , Adult , Bacterial Typing Techniques , Child , DNA, Ribosomal/analysis , DNA, Ribosomal/genetics , Genes, rRNA , Genotype , Helicobacter/ultrastructure , Humans , Microscopy, Electron , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
4.
J Virol ; 73(8): 6882-91, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10400786

ABSTRACT

The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.


Subject(s)
Aleutian Mink Disease Virus/chemistry , Aleutian Mink Disease/virology , Capsid/chemistry , Protein Structure, Secondary , Aleutian Mink Disease/pathology , Aleutian Mink Disease Virus/pathogenicity , Aleutian Mink Disease Virus/ultrastructure , Amino Acid Sequence , Animals , Capsid/ultrastructure , Capsid Proteins , Cats , Cell Line , Cryoelectron Microscopy , Dogs , Humans , Mice , Models, Molecular , Molecular Sequence Data , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/ultrastructure , Sequence Homology, Amino Acid , Spodoptera/cytology
5.
Structure ; 6(11): 1355-67, 1998 Nov 15.
Article in English | MEDLINE | ID: mdl-9817847

ABSTRACT

BACKGROUND: Parvoviruses infect vertebrates, insects and crustaceans. Many arthropod parvoviruses (densoviruses) are highly pathogenic and kill approximately 90% of the host larvae within days, making them potentially effective as selective pesticides. Improved understanding of densoviral structure and function is therefore desirable. There are four different initiation sites for translation of the densovirus capsid protein mRNA, giving rise to the viral proteins VP1 to VP4. Sixty copies of the common, C-terminal domain make up the ordered part of the icosahedral capsid. RESULTS: The Galleria mellonella densovirus (GMDNV) capsid protein consists of a core beta-barrel motif, similar to that found in many other viral capsid proteins. The structure most closely resembles that of the vertebrate parvoviruses, but it has diverged beyond recognition in many of the long loop regions that constitute the surface features and intersubunit contacts. The N termini of twofold-related subunits have swapped their positions relative to those of the vertebrate parvoviruses. Unlike in the vertebrate parvoviruses, in GmDNV there is no continuous electron density in the channels running along the fivefold axes of the virus. Electron density corresponding to some of the single-stranded DNA genome is visible in the crystal structure, but it is not as well defined as in the vertebrate parvoviruses. CONCLUSIONS: The sequence of the glycine-rich motif, which occupies each of the channels along the fivefold axes in vertebrate viruses, is conserved between mammalian and insect parvoviruses. This motif may serve to externalize the N-terminal region of the single VP1 subunit per particle. The domain swapping of the N termini between insect and vertebrate parvoviruses may have the effect of increasing capsid stability in GmDNV.


Subject(s)
Capsid/chemistry , Densovirus/chemistry , Amino Acid Sequence , Animals , Binding Sites , Biological Evolution , Capsid/metabolism , Cations, Divalent , Densovirus/genetics , Models, Molecular , Molecular Sequence Data , Moths/virology , Sequence Homology, Amino Acid , X-Ray Diffraction
6.
Structure ; 6(2): 135-45, 1998 Feb 15.
Article in English | MEDLINE | ID: mdl-9519405

ABSTRACT

BACKGROUND: Spiroplasma virus, SpV4, is a small, non-enveloped virus that infects the helical mollicute Spiroplasma melliferum. SpV4 exhibits several similarities to the Chlamydia phage, Chp1, and the Coliphages alpha 3, phi K, G4 and phi X174. All of these viruses are members of the Microviridae. These viruses have isometric capsids with T = 1 icosahedral symmetry, cause lytic infections and are the only icosahedral phages that contain single-stranded circular DNA genomes. The aim of this comparative study on these phages was to understand the role of their capsid proteins during host receptor recognition. RESULTS: The three-dimensional structure of SpV4 was determined to 27 A resolution from images of frozen-hydrated particles. Cryo-electron microscopy (cryo-EM) revealed 20, approximately 54 A long, 'mushroom-like' protrusions on the surface of the capsid. Each protrusion comprises a trimeric structure that extends radially along the threefold icosahedral axes of the capsid. A 71 amino acid portion of VP1 (the SpV4 capsid protein) was shown, by structural alignment with the atomic structure of the F capsid protein of phi X174, to represent an insertion sequence between the E and F strands of the eight-stranded antiparallel beta-barrel. Secondary structure prediction of this insertion sequence provided the basis for a probable structural motif, consisting of a six-stranded antiparallel beta sheet connected by small turns. Three such motifs form the rigid stable trimeric structures (mushroom-like protrusions) at the threefold axes, with hydrophobic depressions at their distal surface. CONCLUSIONS: Sequence alignment and structural analysis indicate that distinct genera of the Microviridae might have evolved from a common primordial ancestor, with capsid surface variations, such as the SpV4 protrusions, resulting from gene fusion events that have enabled diverse host ranges. The hydrophobic nature of the cavity at the distal surface of the SpV4 protrusions suggests that this region may function as the receptor-recognition site during host infection.


Subject(s)
Biological Evolution , Capsid/chemistry , Microviridae/chemistry , Protein Structure, Secondary , Spiroplasma/virology , Amino Acid Sequence , Crystallography, X-Ray , Genetic Variation , Image Processing, Computer-Assisted , Microscopy, Electron/methods , Microviridae/ultrastructure , Molecular Sequence Data , Phylogeny , Sequence Alignment , Virion/ultrastructure
7.
J Virol ; 71(10): 7911-6, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9311881

ABSTRACT

K. Fukuyama, S. S. Abdel-Meguid, J. E. Johnson, and M. G. Rossmann (J. Mol. Biol. 167:873-984, 1983) reported the structure of alfalfa mosaic virus assembled from the capsid protein as a T=1 icosahedral empty particle at 4.5-A resolution. The information contained in the structure included the particle size, protein shell thickness, presence of wide holes at the icosahedral fivefold axes, and a proposal that the capsid protein adopts a beta-barrel structure. In the present work, the X-ray diffraction data of Fukuyama et al. as well as the data subsequently collected by I. Fita, Y. Hata, and M. G. Rossmann (unpublished) were reprocessed to 4.0-A resolution, and the structure was solved by molecular replacement. The current structure allowed the tracing of the polypeptide chain of the capsid protein confirming the beta-sandwich fold and provides information on intersubunit interactions in the particle. However, it was not possible to definitively assign the amino acid sequence to the side chain density at 4-A resolution. The particle structure was also determined by cryoelectron microscopy and image reconstruction methods and found to be in excellent agreement with the X-ray model.


Subject(s)
Alfalfa mosaic virus/ultrastructure , Capsid/chemistry , Capsid/ultrastructure , Protein Conformation , Alfalfa mosaic virus/chemistry , Freezing , Medicago sativa/virology , Microscopy, Electron , Models, Molecular , Models, Structural , Models, Theoretical , Protein Structure, Secondary , Protein Structure, Tertiary , X-Ray Diffraction
8.
Proc Natl Acad Sci U S A ; 93(15): 7502-6, 1996 Jul 23.
Article in English | MEDLINE | ID: mdl-8755503

ABSTRACT

The three-dimensional structures of human parvovirus B19 VP2 capsids, alone and complexed with its cellular receptor, globoside, have been determined to 26 resolution. The B19 capsid structure, reconstructed from cryo-electron micrographs of vitrified specimens, has depressions on the icosahedral 2-fold and 3-fold axes, as well as a canyon-like region around the 5-fold axes. Similar results had previously been found in an 8 angstrom resolution map derived from x-ray diffraction data. Other parvoviral structures have a cylindrical channel along the 5-fold icosahedral axes, whereas density covers the 5-fold axes in B19. The glycolipid receptor molecules bind into the depressions on the 3-fold axes of the B19:globoside complex. A model of the tetrasaccharide component of globoside, organized as a trimeric fiber, fits well into the difference density representing the globoside receptor. Escape mutations to neutralizing antibodies map onto th capsid surface at regions immediately surrounding the globoside attachment sites. The proximity of the antigenic epitopes to the receptor site suggests that neutralization of virus infectivity is caused by preventing attachment of viruses to cells.


Subject(s)
Capsid/ultrastructure , Parvovirus B19, Human/ultrastructure , Receptors, Virus/ultrastructure , Carbohydrate Conformation , Carbohydrate Sequence , Crystallography, X-Ray , Freezing , Globosides/chemistry , Globosides/physiology , Humans , Microscopy, Electron/methods , Models, Structural , Molecular Sequence Data , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Parvovirus B19, Human/physiology , Receptors, Virus/chemistry , Receptors, Virus/physiology
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