The Experts below are selected from a list of 78 Experts worldwide ranked by ideXlab platform
Paul Gottlieb - One of the best experts on this subject based on the ideXlab platform.
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Component tree analysis of Cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions.
PloS one, 2018Co-Authors: Lucas M. Oliveira, Hui Wei, Alexandra Alimova, Al Katz, Gabor T. Herman, Paul GottliebAbstract:The 3-dimensional structure of the nucleocapsid (NC) of bacteriophage φ6 is described utilizing component tree analysis, a topological and geometric image descriptor. The component trees are derived from density maps of cryo-electron microscopy single particle reconstructions. Analysis determines position and occupancy of structure elements responsible for RNA packaging and transcription. Occupancy of the hexameric nucleotide triphosphorylase (P4) and RNA polymerase (P2) are found to be essentially complete in the NC. The P8 protein lattice likely fixes P4 and P2 in place during maturation. We propose that the viral procapsid (PC) is a dynamic structural intermediate where the P4 and P2 can attach and detach until held in place in mature NCs. During packaging, the PC expands to accommodate the RNA, and P2 translates from its original site near the inner 3-fold axis (20 sites) to the inner 5-fold axis (12 sites) with excess P2 positioned inside the central region of the NC.
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the ϕ6 Cystovirus protein p7 becomes accessible to antibodies in the transcribing nucleocapsid a probe for viral structural elements
PLOS ONE, 2015Co-Authors: Alexandra Alimova, Hui Wei, Al Katz, Linda Spatz, Paul GottliebAbstract:Protein P7 is a component of the Cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the five-fold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of Vκ and VH genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein’s antigenic sites.
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Disassembly of the Cystovirus ϕ6 envelope by montmorillonite clay
MicrobiologyOpen, 2013Co-Authors: Karin A. Block, Paul Gottlieb, Hui Wei, William J. Rice, Alexandra Alimova, Adrianna Trusiak, Al Katz, Jorge Morales, Jeffrey C. SteinerAbstract:Prior studies of clay–virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT–φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments.
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Protein P7 of the Cystovirus φ6 is located at the three-fold axis of the unexpanded procapsid.
PloS one, 2012Co-Authors: Garrett E. Katz, Hui Wei, Alexandra Alimova, Al Katz, David Gene Morgan, Paul GottliebAbstract:The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the φ6 Cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the φ6 complete unexpanded procapsid, the protein P2-minus procapsid (P2 is the RNA directed RNA-polymerase), and the P7-minus procapsid, show that prior to RNA packaging the P7 protein is located near the three-fold axis of symmetry. Difference maps highlight the precise position of P7 and demonstrate that in P7-minus particles the P2 proteins are less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated.
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Cystovirus ϕ6 Structure Probed by Stokes Shift Fluorescence Spectroscopy
Frontiers in Optics 2011 Laser Science XXVII, 2011Co-Authors: Alvin Katz, Hui Wei, Alexandra Alimova, Garrett E. Katz, Paul GottliebAbstract:Fluorescence Stokes shift measurements of tryptophan in bacteriophage ϕ6 were used to determine solvent exposure. It is determined that the envelope proteins are in a more hydrophobic environment than internal proteins of the polymerase complex.
Hui Wei - One of the best experts on this subject based on the ideXlab platform.
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Component tree analysis of Cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions.
PloS one, 2018Co-Authors: Lucas M. Oliveira, Hui Wei, Alexandra Alimova, Al Katz, Gabor T. Herman, Paul GottliebAbstract:The 3-dimensional structure of the nucleocapsid (NC) of bacteriophage φ6 is described utilizing component tree analysis, a topological and geometric image descriptor. The component trees are derived from density maps of cryo-electron microscopy single particle reconstructions. Analysis determines position and occupancy of structure elements responsible for RNA packaging and transcription. Occupancy of the hexameric nucleotide triphosphorylase (P4) and RNA polymerase (P2) are found to be essentially complete in the NC. The P8 protein lattice likely fixes P4 and P2 in place during maturation. We propose that the viral procapsid (PC) is a dynamic structural intermediate where the P4 and P2 can attach and detach until held in place in mature NCs. During packaging, the PC expands to accommodate the RNA, and P2 translates from its original site near the inner 3-fold axis (20 sites) to the inner 5-fold axis (12 sites) with excess P2 positioned inside the central region of the NC.
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Three-Dimensional Structure of the Enveloped Bacteriophage W12: An Incomplete T = 13 Lattice Is Superposed on an Enclosed T = 1 Shell
2015Co-Authors: Hui Wei, John Berriman, William J. Rice, David L. Stokes, Alvin Katz, R. Holl, David Gene, Morgan Paul GottliebAbstract:Background: Bacteriophage w12 is a member of the Cystoviridae, a unique group of lipid containing membrane enveloped bacteriophages that infect the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola. The genomes of the virus species contain three double-stranded (dsRNA) segments, and the virus capsid itself is organized in multiple protein shells. The segmented dsRNA genome, the multi-layered arrangement of the capsid and the overall viral replication scheme make the Cystoviridae similar to the Reoviridae. Methodology/Principal Findings: We present structural studies of Cystovirus w12 obtained using cryo-electron microscopy and image processing techniques. We have collected images of isolated w12 virions and generated reconstructions of both the entire particles and the polymerase complex (PC). We find that in the nucleocapsid (NC), the w12 P8 protein is organized on an incomplete T = 13 icosahedral lattice where the symmetry axes of the T = 13 layer and the enclosed T = 1 layer of the PC superpose. This is the same general protein-component organization found in w6 NC’s but the detailed structure of the entire w12 P8 layer is distinct from that found in the best classified Cystovirus species w6. In the reconstruction of the NC, the P8 layer includes protein density surrounding the hexamers of P4 that sit at the 5-fold vertices of the icosahedral lattice. W
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the ϕ6 Cystovirus protein p7 becomes accessible to antibodies in the transcribing nucleocapsid a probe for viral structural elements
PLOS ONE, 2015Co-Authors: Alexandra Alimova, Hui Wei, Al Katz, Linda Spatz, Paul GottliebAbstract:Protein P7 is a component of the Cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the five-fold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of Vκ and VH genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein’s antigenic sites.
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Disassembly of the Cystovirus ϕ6 envelope by montmorillonite clay
MicrobiologyOpen, 2013Co-Authors: Karin A. Block, Paul Gottlieb, Hui Wei, William J. Rice, Alexandra Alimova, Adrianna Trusiak, Al Katz, Jorge Morales, Jeffrey C. SteinerAbstract:Prior studies of clay–virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT–φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments.
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Protein P7 of the Cystovirus φ6 is located at the three-fold axis of the unexpanded procapsid.
PloS one, 2012Co-Authors: Garrett E. Katz, Hui Wei, Alexandra Alimova, Al Katz, David Gene Morgan, Paul GottliebAbstract:The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the φ6 Cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the φ6 complete unexpanded procapsid, the protein P2-minus procapsid (P2 is the RNA directed RNA-polymerase), and the P7-minus procapsid, show that prior to RNA packaging the P7 protein is located near the three-fold axis of symmetry. Difference maps highlight the precise position of P7 and demonstrate that in P7-minus particles the P2 proteins are less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated.
Ranajeet Ghose - One of the best experts on this subject based on the ideXlab platform.
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cystoviral polymerase complex protein p7 uses its acidic c terminal tail to regulate the rna directed rna polymerase p2
Journal of Molecular Biology, 2014Co-Authors: Sébastien Alphonse, Ranajeet Ghose, Shibani Bhattacharya, Jamie J Arnold, Hsin Wang, Brian Kloss, Craig E CameronAbstract:In bacteriophages of the Cystovirus family, the polymerase complex (PX) encodes a 75-kDa RNA-directed RNA polymerase (P2) that transcribes the double-stranded RNA genome. Also a constituent of the PX is the essential protein P7 that, in addition to accelerating PX assembly and facilitating genome packaging, plays a regulatory role in transcription. Deletion of P7 from the PX leads to aberrant plus-strand synthesis suggesting its influence on the transcriptase activity of P2. Here, using solution NMR techniques and the P2 and P7 proteins from Cystovirus ϕ12, we demonstrate their largely electrostatic interaction in vitro. Chemical shift perturbations on P7 in the presence of P2 suggest that this interaction involves the dynamic C-terminal tail of P7, more specifically an acidic cluster therein. Patterns of chemical shift changes induced on P2 by the P7 C-terminus resemble those seen in the presence of single-stranded RNA suggesting similarities in binding. This association between P2 and P7 reduces the affinity of the former toward template RNA and results in its decreased activity both in de novo RNA synthesis and in extending a short primer. Given the presence of C-terminal acidic tracts on all cystoviral P7 proteins, the electrostatic nature of the P2/P7 interaction is likely conserved within the family and could constitute a mechanism through which P7 regulates transcription in Cystoviruses.
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structure of the rna directed rna polymerase from the Cystovirus φ12
Proteins, 2013Co-Authors: Zhen Ren, Ranajeet Ghose, M C FranklinAbstract:We have determined the structure of P2, the self-priming RdRp from Cystovirus φ12 in two crystal forms (A, B) at resolutions of 1.7 A and 2.1 A. Form A contains Mg(2+) bound at a site that deviates from the canonical noncatalytic position seen in form B. These structures provide insight into the temperature sensitivity of a ts-mutant. However, the tunnel through which template ssRNA accesses the active site is partially occluded by a flexible loop; this feature, along with suboptimal positioning of other structural elements that prevent the formation of a stable initiation complex, indicate an inactive conformation in crystallo.
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Dynamics on multiple timescales in the RNA-directed RNA polymerase from the Cystovirus ϕ6
Nucleic acids research, 2010Co-Authors: Zhen Ren, Haiyan Wang, Ranajeet GhoseAbstract:The de novo initiating RNA-directed RNA polymerase (RdRP), P2, forms the central machinery in the infection cycle of the bacteriophage ϕ6 by performing the dual tasks of replication and transcription of the double-stranded RNA genome in the host cell. By measurement and quantitative analysis of multiple-quantum spin-relaxation data for the δ1 positions of Ile residues that are distributed over the 3D-fold of P2, we find that the enzyme is dynamic both on the fast (ps–ns) and slow (µs–ms) timescales. The characteristics of several motional modes including those that coincide with the catalytic timescale (500–800/s) are altered in the presence of substrate analogs and single-stranded RNA templates. These studies reveal the plasticity of this finely tuned molecular machine and represent a first step towards linking structural information available from a host of crystal structures to catalytic mechanisms and timescales obtained from the measurements of kinetics for homologous systems in solution.
Minna M. Poranen - One of the best experts on this subject based on the ideXlab platform.
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Microbial production of lipid-protein vesicles using enveloped bacteriophage phi6
Microbial Cell Factories, 2019Co-Authors: Outi L. Lyytinen, Daria Starkova, Minna M. PoranenAbstract:Background Cystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The Cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli . Results Using transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13 g/cm^3 in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles. Conclusions Our results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.
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Recognition of six additional Cystoviruses: Pseudomonas virus phi6 is no longer the sole species of the family Cystoviridae
Archives of Virology, 2018Co-Authors: Sari Mäntynen, Lotta-riina Sundberg, Minna M. PoranenAbstract:Cystoviridae is a family of bacterial viruses (bacteriophages) with a tri-segmented dsRNA genome. It includes a single genus Cystovirus , which has presently only one recognised virus species, Pseudomonas virus phi6 . However, a large number of additional dsRNA phages have been isolated from various environmental samples, indicating that such viruses are more widespread and abundant than previously recognised. Six of the additional dsRNA phage isolates (Pseudomonas phages phi8, phi12, phi13, phi2954, phiNN and phiYY) have been fully sequenced. They all infect Pseudomonas species, primarily plant pathogenic Pseudomonas syringae strains. Due to the notable genetic and structural similarities with Pseudomonas phage phi6, we propose that these viruses should be included into the Cystovirus genus (and consequently into the Cystoviridae family). Here, we present an updated taxonomy of the family Cystoviridae and give a short overview of the properties of the type member phi6 as well as the putative new members of the family.
Meng Xiaolin - One of the best experts on this subject based on the ideXlab platform.
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amplification of dendrolimus puctatus whenshanesis cytovirus dpcpv w in vitro in sf21 cell
Virologica Sinica, 2002Co-Authors: Meng XiaolinAbstract:We have studied amphification of DpCPV-W in vitro in Sf21 cell in this paper.The results showed that DpCPV-W could amplify in vitro in Sf21 cell and form normal polyhedra.Accessional rev combinant enhancin in medium could synergist the infective rate of DpCPV-W in Sf21 cell in vitro .Bioassay indicated that the toxicity of DpCPV-W polyhedra amplified in Sf21 cell was as violence as the polyhedra amplified in pine caterpillar.By using the plaque purfication technique in Sf21 cell,DpCPV-W could be purified in a single virion clone level.
