The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Dennis H. Bamford - One of the best experts on this subject based on the ideXlab platform.
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eLS - Membrane-Containing Bacteriophages
eLS, 2015Co-Authors: Minna M. Poranen, Dennis H. Bamford, Hanna M. OksanenAbstract:Viruses exhibit vast diversity in their form and function and are by far the most numerous (estimates 1030–1032) organisms on earth. The largest group among viruses is bacteriophages (phages), the viruses that infect bacteria, with over 6000 identified members. Vast majority of the phages are composed of protein and nucleic acid with a head–tail morphology. Polyhedral, filamentous or pleomorphic phages comprise only less than 4% of the described bacteriophages, and a minority of these has lipid constituents in addition to nucleic acid and protein. These membrane-containing bacteriophages form a diverse group of viruses and the major virus morphotypes are represented by the members of the virus families Corticoviridae, Tectiviridae, Cystoviridae and Plasmaviridae. The lipids as a bilayer can either form the outermost layer of the virion or be enclosed within the virus capsid. In both cases, the viral membranes are involved in cell entry processes. Key Concepts Membrane-containing bacteriophages are a diverse group of bacterial viruses. Bacteriophages with a membrane are usually sensitive to organic solvents and detergents. Lipids in the viral membrane have a bilayer structure. The virion proteins are all virus specific, but lipids are derived from host cytoplasmic membrane. During virus morphogenesis, the virus-specific membrane proteins exclude host proteins during formation of the viral membrane. The viral protein-rich membranes have an essential role during entry mediating the translocation of the genome across the bacterial cell envelopes. Keywords: bacterial virus; lipid-containing bacteriophage; membrane-containing bacteriophage; virus evolution; life cycle
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Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution
Nucleic acids research, 2013Co-Authors: Kamel El Omari, Dennis H. Bamford, Minna M. Poranen, Denis E. Kainov, Roman Tuma, Jonathan M. Grimes, David I. Stuart, Christoph Meier, Geoff Sutton, Erika J. ManciniAbstract:Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, 12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from 6, 8 and 13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in 8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in 12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the 12 enzyme.
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Assembly of Large Icosahedral Double-Stranded RNA Viruses
Advances in experimental medicine and biology, 2011Co-Authors: Minna M. Poranen, Dennis H. BamfordAbstract:Double-stranded RNA (dsRNA) viruses are a diverse group of viruses infecting hosts from bacteria to higher eukaryotes. Among the hosts are humans, domestic animals, and economically important plant species. Fine details of high-resolution virion structures have revealed common structural characteristics unique to these viruses including an internal icosahedral capsid built from 60 asymmetric dimers (120 monomers!) of the major coat protein. Here we focus mainly on the structures and assembly principles of large icosahedral dsRNA viruses belonging to the families of Cystoviridae and Reoviridae. It is obvious that there are a variety of assembly pathways utilized by different viruses starting from similar building blocks and reaching in all cases a similar capsid architecture. This is true even with closely related viruses indicating that the assembly pathway per se is not an indicator of relatedness and is achieved with minor changes in the interacting components.
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Phospholipids act as secondary receptor during the entry of the enveloped, double-stranded RNA bacteriophage φ6.
Journal of General Virology, 2010Co-Authors: Virginija Cvirkaitė-krupovič, Minna M. Poranen, Dennis H. BamfordAbstract:Bacteriophage phi6 is the type member of the family Cystoviridae and infects Gram-negative Pseudomonas syringae cells. The virion consists of a protein-rich lipid envelope enclosing a nucleocapsid. The nucleocapsid covers the icosahedral polymerase complex that encloses the double-stranded RNA genome. Here, we demonstrate that nucleocapsid surface protein P8 is the single nucleocapsid component interacting with the cytoplasmic membrane. This interaction takes place between P8 and phospholipid. Based on this and previous studies, we propose a model where the periplasmic nucleocapsid interacts with the phospholipid head groups and, when the membrane voltage exceeds the threshold of 110 mV, this interaction drives the nucleocapsid through the cytoplasmic membrane, resulting in an intracellular vesicle containing the nucleocapsid.
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eLS - Bacteriophages: Lipid‐containing
Encyclopedia of Life Sciences, 2010Co-Authors: Hanna M. Oksanen, Minna M. Poranen, Dennis H. BamfordAbstract:Viruses exhibit vast diversity in their form and function and are by far the most numerous (estimates 1030–1032) organisms on earth. The largest group among viruses is bacteriophages, the viruses that infect bacteria, with over 5000 identified members. Majority of the phages are composed of protein and nucleic acid with a head–tail morphology. Polyhedral, filamentous or pleomorphic phages comprise only less than 4% of the described bacteriophages, and a minority of these have lipid constituents in addition to nucleic acid and protein. These lipid-containing bacteriophages form a diverse group of viruses and they are classified in four virus families Corticoviridae, Cystoviridae, Plasmaviridae and Tectiviridae. The lipids can be either as the outermost layer of the virion or they can locate internally enclosed within the virus capsid. In both cases, the viral membranes are involved in cell entry processes. Key Concepts: The lipid-containing bacteriophages are a diverse group of bacterial viruses. The lipid-containing bacteriophages are sensitive to organic solvents and detergents. Lipids in the viral membrane have a bilayer structure. The virion proteins are all virus specific, but lipids are derived from host cytoplasmic membrane. During virus morphogenesis, the virus-specific proteins exclude host proteins during formation of the viral membrane. The viral protein-rich membranes have an essential role during entry mediating the translocation of the genome across the host cell envelopes. Keywords: bacterial viruses; lipid-containing bacteriophages; virus evolution; life cycles
Paul Gottlieb - One of the best experts on this subject based on the ideXlab platform.
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Enveloped Icosahedral Phages – dsRNA (φ6)
Reference Module in Life Sciences, 2019Co-Authors: Paul Gottlieb, Aleksandra AlimovaAbstract:Abstract The virulent lipid-containing bacteriophage φ6, containing a genome of three double – stranded RNA segments, was the first isolated member of the Cystoviridae family. Discovered in 1973 it remains the type species. Beginning in 1999, additional species have been isolated, for the most part, from pseudomonad bacterial plant pathogens. All share similar architecture comprising the genome segments enclosed in a core surrounded by a lipid envelope that carries the host attachment proteins. The structural and replicative features are similar to the Reoviridae family and establishment of in-vitro assembly and rescue assays made the bacteriophage family the subject of considerable investigation.
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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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Bacteriophage φ6--structure investigated by fluorescence Stokes shift spectroscopy.
Photochemistry and photobiology, 2011Co-Authors: Alvin Katz, Hui Wei, Alexandra Alimova, Garrett E. Katz, Elina Futerman, Paul GottliebAbstract:The Stokes shift of tryptophan (Trp) fluorescence from layers of the lipid-containing bacteriophage ϕ6 are compared to determine the relative effect of the layers on virus hydrophobicity. In the inner most layer, the empty procapsid (PC) which contains 80% – 90% of the virion Trp residues, λmax = 339.8 nm. The PC emission is substantially more red-shifted than the other ϕ6 layers and nearer to that of the Pseudomonad host cell than the other ϕ6 layers. The Trp emission from the nucleocapsid (NC) with λmax = 337.4 nm, is blue shifted by 2.4 nm relative to the PC although the number of Trp in the NC is identical to the PC. This shift represents an increase in Trp hydrophobicity, likely a requirement for the maintenance of A-form dsRNA. Fluorescence from the completely assembled virion indicates it is in a considerably more hydrophobic environment with λmax = 330.9 nm. Density measurements show that the water content in the NC does not changed during envelope assembly, therefore the blue-shifted ϕ6 emission suggests that the envelope changes the PC environment, probably via the P8 layer. This change in hydrophobicity likely arises from charge redistribution or envelope-induced structural changes in the PC proteins. Introduction The bacteriophage ϕ6 is a member of Cystoviridae, a family of viruses that are related to reoviruses. The cystoviruses contain a genome composed of three segments of doubled-stranded RNA (dsRNA) and are structurally analogous to reoviruses in that they have a multiple layered architecture. ϕ6 has a three-layer structure, shown schematically in Fig. 1. The inner core of the virus consists of four proteins designated P1, P2, P4 and P7, which together form the procapsid (PC). The PC has the capability of packaging three segments of viral message RNA that is subsequently replicated to dsRNA. The three dsRNA segments are named according to their size: small (S), medium (M) and large (L). Of the four proteins that comprise the PC, P1 is the major structural protein that is believed to alter its conformation during the RNA packaging reactions (1). The atomic structure of P1 has not been established, however studies of the isolated P1 from ϕ8 suggest that it is tetrameric. P1 from ϕ6 has only been purified in small amounts as a monomer but in vitro assembly assays show a fourth-order concentration dependence, further suggesting it is tetrameric (2, 3). The remaining three procapsid proteins, P2, P4, and P7 together constitute the RNA packaging/replication portal and the atomic structure of each has been determined (4–6). P2 is the RNA-directed RNA polymerase (RdRP) a monomeric protein that replicates and transcribes the dsRNA viral genome (7). P4 is a hexameric nucleotide-triphosphorylase that packages each viral mRNA segment. P4 is located at the five-fold axis of symmetry of the PC creating a symmetry mismatch by imposing its six-fold symmetry (8). The position of the P7 dimer within the assembled PC has not been confirmed but data from single particle reconstructions of the ϕ12 virus particle tentatively identified electron densities at the five-fold axis and surrounding the P4 hexamer as being P7. Fig. 1 A) Schematic of ϕ6 and inner layers: B) ϕ6−P3; C) NC; and D) PC. The cystovirus replicative mechanism starts with assembly of the empty PC. This is followed by the specific selection and packaging of three segments of viral mRNA within the PC (7, 9–11). The dodecahedral-shaped PC contains the RNA packaging and replication apparatus at each of 12 portals constituting multiple polymerase elements (PX). The PX consists of P2, P4 and, based on cryo-electron tomography of the related ϕ12 virus, P7 (12). The dsRNA segments are packaged in a specific size order and replicated to the double-stranded format utilizing RdRP only after packaging is complete. The RdRPs are located at each of the 12 portals and appear to undergo a position shift from the three-fold axis to the five-fold axis of the PX after PC expansion (12, 13). The PC undergoes a specific and sequential expansion during RNA packaging that selectively exposes RNA binding sites for each of the three RNA segments (1, 4, 14). The genomic RNA interacts with the internal structure of the PC and may modify P1, the main structural component. Mutations in the genome RNA positive stranded segments that alter packaging order have resulted in selection of P1 mutations that alter the overall charge distribution of the PC (15, 16). After packaging and RNA replication, a matrix composed of 200 trimeric copies of protein P8, is assembled around the filled PC. The P8 matrix is an open structure which can allow passage of water, cations and other smaller molecules (1). The PC, P8 and genome together form the nucleocapsid (NC). A lipid-bilayer-membrane envelope, derived from the Pseudomonas host-cell, is assembled around the NC. The mechanism of viral envelope assembly is not well understood. It involves the non-structural P12 protein which is not present in the completely assembled virion. The envelope contains four membrane proteins, P6, P9, P10 and P13, within a lipid bilayer. Based on cryo-electron microscopy of the related ϕ12 virus, protein P5 is likely positioned between the NC and the envelope (12, 17). P3 proteins form external spikes on the envelope whose function are to bind to the host cell receptor. In ϕ6, the three discrete layers can be selectively removed to reveal the subviral elements (17, 18). Alternatively, recombinant RNA packaging-competent procapsid structures can be assembled (7, 19). In the work presented here, we employ fluorescence Stokes shift spectroscopy of each subviral layer to investigate the hydrophobic and hydrophilic qualities of the layers. The extent of hydrophobicity has implications for the internal environment of the virus in regard to packaged viral genomic RNA and viral replication. Trp fluorescence has been employed extensively to study proteins (20). It is well known that the Stokes shift and quantum efficiency of tryptophan (Trp) fluorescence are dependent on the local environment and protein structure (21, 22) with the emission being more red-shifted in a hydrophilic environment. Of the aromatic amino acids, Trp is both the predominant fluorophore (20, 23) and strongest absorber (20) and Trp is the predominant fluorophore in viruses. However, there have been very few reports on the use of Stokes shift spectroscopy to study proteins in phage. Kitchell et al. investigated Trp accessibility in bacteriophage f2 and observed that the blue-shifted Trp fluorescence indicated a hydrophobic Trp environment (24). Kneale and Van Resandt studied the DNA binding protein in bacteriophage Pf1 using spectral shifts and time-resolved measurements of Trp fluorescence (25). Urbaneja et al. investigated nucleic acid interactions in phage ϕ29 by Trp fluorescence (26). More recently, Alimova et al. used Trp Stokes shift fluorescence to study the infection processes of cystoviruses ϕ6 and ϕ12 (27, 28). In Alimova 2004, it was observed that the ϕ6 Trp emission exhibited a decrease in hydrophobicity shortly after infection. In the work present here, we compare the Trp emission for the different ϕ6 layers to examine the differences in hydrophobicity of the layers and its implication for protein interactions during viral assembly. To the best of our knowledge, this is the first time Stokes shift fluorescence spectroscopy has been used to probe the multi-layers of any viral system.
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Toroidal surface complexes of bacteriophage ϕ12 are responsible for host-cell attachment
Virology, 2011Co-Authors: Alejandra Leo-macias, Hui Wei, William J. Rice, David L. Stokes, Alvin Katz, Alexandra Alimova, Garrett E. Katz, Ruben Diaz-avalos, Guo Bin Hu, Paul GottliebAbstract:Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage ϕ12, a member of the Cystoviridae family, contains surface complexes that are toroidal in shape, are composed of six globular domains with six-fold symmetry, and have a discrete density connecting them to the virus membrane-envelope surface. The lack of this kind of spike in a reassortant of ϕ12 demonstrates that the gene for the hexameric spike is located in ϕ12's medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this and on protein mass estimates derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity.
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Three-Dimensional Structure of the Enveloped Bacteriophage Φ12: An Incomplete T = 13 Lattice Is Superposed on an Enclosed T = 1 Shell
PloS one, 2009Co-Authors: Hui Wei, R. Holland Cheng, John Berriman, William J. Rice, David L. Stokes, Alvin Katz, David Gene Morgan, Paul GottliebAbstract:Author(s): Wei, Hui; Cheng, R Holland; Berriman, John; Rice, William J; Stokes, David L; Katz, A; Morgan, David Gene; Gottlieb, Paul | Abstract: BACKGROUND:Bacteriophage phi12 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 phi12 obtained using cryo-electron microscopy and image processing techniques. We have collected images of isolated phi12 virions and generated reconstructions of both the entire particles and the polymerase complex (PC). We find that in the nucleocapsid (NC), the phi12 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 phi6 NC's but the detailed structure of the entire phi12 P8 layer is distinct from that found in the best classified cystovirus species phi6. 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. We believe these novel features correspond to dimers of protein P7. CONCLUSIONS/SIGNIFICANCE:In conclusion, we have determined that the phi12 NC surface is composed of an incomplete T = 13 P8 layer forming a net-like configuration. The significance of this finding in regard to cystovirus assembly is that vacancies in the lattice could have the potential to accommodate additional viral proteins that are required for RNA packaging and synthesis.
Minna M. Poranen - One of the best experts on this subject based on the ideXlab platform.
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Multiple liquid crystalline geometries of highly compacted nucleic acid in a dsRNA virus
Nature, 2019Co-Authors: Serban L. Ilca, Minna M. Poranen, Jonathan M. Grimes, David I. Stuart, X. Sun, Abhay Kotecha, F. De Haas, Frank Dimaio, Kamel El Omari, Juha T. HuiskonenAbstract:Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations^ 1 – 8 . Because the release of dsRNA into the cytoplasm triggers host defence mechanisms^ 9 , dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription^ 10 – 12 . The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10–12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases^ 11 – 14 . However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses. The cryo-electron microscopy structure of the bacteriophage ɸ6 dsRNA genome shows that the genome is packaged in a spooled manner that is more similar to dsDNA viruses than to other dsRNA viruses.
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Multiple liquid crystalline geometries of highly compacted nucleic acid in a dsRNA virus.
Nature, 2019Co-Authors: Serban L. Ilca, Minna M. Poranen, Jonathan M. Grimes, David I. Stuart, X. Sun, K. El Omari, Abhay Kotecha, F. De Haas, Frank Dimaio, Juha T. HuiskonenAbstract:Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations1-8. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms9, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription10-12. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10-12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases11-14. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.
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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.
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ICTV Virus Taxonomy Profile: Cystoviridae.
The Journal of general virology, 2017Co-Authors: Minna M. Poranen, Sari MäntynenAbstract:The family Cystoviridae includes enveloped viruses with a tri-segmented dsRNA genome and a double-layered protein capsid. The innermost protein shell is a polymerase complex responsible for genome packaging, replication and transcription. Cystoviruses infect Gram-negative bacteria, primarily plant-pathogenic Pseudomonas syringae strains. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Cystoviridae, which is available at http://www.ictv.global/report/Cystoviridae.
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eLS - Membrane-Containing Bacteriophages
eLS, 2015Co-Authors: Minna M. Poranen, Dennis H. Bamford, Hanna M. OksanenAbstract:Viruses exhibit vast diversity in their form and function and are by far the most numerous (estimates 1030–1032) organisms on earth. The largest group among viruses is bacteriophages (phages), the viruses that infect bacteria, with over 6000 identified members. Vast majority of the phages are composed of protein and nucleic acid with a head–tail morphology. Polyhedral, filamentous or pleomorphic phages comprise only less than 4% of the described bacteriophages, and a minority of these has lipid constituents in addition to nucleic acid and protein. These membrane-containing bacteriophages form a diverse group of viruses and the major virus morphotypes are represented by the members of the virus families Corticoviridae, Tectiviridae, Cystoviridae and Plasmaviridae. The lipids as a bilayer can either form the outermost layer of the virion or be enclosed within the virus capsid. In both cases, the viral membranes are involved in cell entry processes. Key Concepts Membrane-containing bacteriophages are a diverse group of bacterial viruses. Bacteriophages with a membrane are usually sensitive to organic solvents and detergents. Lipids in the viral membrane have a bilayer structure. The virion proteins are all virus specific, but lipids are derived from host cytoplasmic membrane. During virus morphogenesis, the virus-specific membrane proteins exclude host proteins during formation of the viral membrane. The viral protein-rich membranes have an essential role during entry mediating the translocation of the genome across the bacterial cell envelopes. Keywords: bacterial virus; lipid-containing bacteriophage; membrane-containing bacteriophage; virus evolution; life cycle
Roman Tuma - One of the best experts on this subject based on the ideXlab platform.
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Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution
Nucleic acids research, 2013Co-Authors: Kamel El Omari, Dennis H. Bamford, Minna M. Poranen, Denis E. Kainov, Roman Tuma, Jonathan M. Grimes, David I. Stuart, Christoph Meier, Geoff Sutton, Erika J. ManciniAbstract:Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, 12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from 6, 8 and 13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in 8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in 12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the 12 enzyme.
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Mechanism of RNA packaging motor.
Advances in experimental medicine and biology, 2011Co-Authors: Erika J. Mancini, Roman TumaAbstract:P4 proteins are hexameric RNA packaging ATPases of dsRNA bacteriophages of the Cystoviridae family. P4 hexamers are integral part of the inner polymerase core and play several essential roles in the virus replication cycle. P4 proteins are structurally related to the hexameric helicases and translocases of superfamily 4 (SF4) and other RecA-like ATPases. Recombinant P4 proteins retain their 5’ to 3’ helicase and translocase activity in vitro and thus serve as a model system for studying the mechanism of action of hexameric ring helicases and RNA translocation. This review summarizes the different roles that P4 proteins play during virus assembly, genome packaging, and transcription. Structural and mechanistic details of P4 action are laid out to and subsequently compared with those of the related hexameric helicases and other packaging motors.
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Icosahedral Enveloped dsRNA Bacterial Viruses
Encyclopedia of Virology, 2008Co-Authors: Roman TumaAbstract:The Cystoviridae family encompasses nine lipid-containing bacteriophages (ϕ6–14) that infect plant pathogenic pseudomonads. A cystovirus has three segments of the dsRNA genome encapsidated within an icosahedral polymerase complex (PC) which is composed of four proteins. PC contains the RNA-dependent RNA polymerase (RdRP) which performs viral RNA replication and transcription. PC structure and function are analogous to those of the cores of dsRNA viruses belonging to the Reoviridae family. Structure, assembly, and host cell interactions of ϕ6, the type virus of Cystoviridae, have been extensively studied. Assembly proceeds in several stages producing first an empty PC into which the three genomic precursors (ssRNA) are sequentially packaged by a viral packaging motor (ATPase). The RNA precursors are then replicated inside the PC to produce a mature PC. PC is further enclosed by a nucleocapsid shell and a lipid envelope to yield the complete virion. Upon infection the envelope fuses with the bacterial outer membrane and the released nucleocapsid enters the cell using a process that resembles endocytosis. In vitro assembly and packaging systems have been developed. High-resolution structures of RdRP and the packaging ATPase have been obtained. Reverse genetics and carrier-state systems are available to aid prospective biotechnological applications.
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RNA packaging motor: From structure to quantum mechanical modelling and sequential-stochastic mechanism
Computational and Mathematical Methods in Medicine, 2008Co-Authors: Jelena Telenius, Erika J. Mancini, Anders Wallin, Michal Straka, Hongbo Zhang, Roman TumaAbstract:The bacteriophages of the Cystoviridae family package their single stranded RNA genomic precursors into empty capsid (procapsids) using a hexameric packaging ATPase motor (P4). This molecular motor shares sequence and structural similarity with RecA-like hexameric helicases. A concerted structural, mutational and kinetic analysis helped to define the mechanical reaction coordinate, i.e. the conformational changes associated with RNA translocation. The results also allowed us to propose a possible scheme of coupling between ATP hydrolysis and translocation which requires the cooperative action of three consecutive subunits. Here, we first test this model by preparing hexamers with defined proportions of wild type and mutant subunits and measuring their activity. Then, we develop a stochastic kinetic model which accounts for the catalytic cooperativity of the P4 hexamer. Finally, we use the available structural information to construct a quantum-chemical model of the chemical reaction coordinate and obtain a detailed description of the electron density changes during ATP hydrolysis. The model explains the results of the mutational analyses and yields new insights into the role of several conserved residues within the ATP binding pocket. These hypotheses will guide future experimental work.
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Cooperative Mechanism of RNA Packaging Motor
The Journal of biological chemistry, 2005Co-Authors: Jiří Lísal, Roman TumaAbstract:P4 is a hexameric ATPase that serves as the RNA packaging motor in double-stranded RNA bacteriophages from the Cystoviridae family. P4 shares sequence and structural similarities with hexameric helicases. A structure-based mechanism for mechano-chemical coupling has recently been proposed for P4 from bacteriophage phi12. However, coordination of ATP hydrolysis among the subunits and coupling with RNA translocation remains elusive. Here we present detailed kinetic study of nucleotide binding, hydrolysis, and product release by phi12 P4 in the presence of different RNA and DNA substrates. Whereas binding affinities for ATP and ADP are not affected by RNA binding, the hydrolysis step is accelerated and the apparent cooperativity is increased. No nucleotide binding cooperativity is observed. We propose a stochastic-sequential cooperativity model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. The translocation step appears coupled to hydrolysis, which is coordinated among three neighboring subunits. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive.
Paul E Turne - One of the best experts on this subject based on the ideXlab platform.
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genomic and gene expression comparisons among phage resistant type iv pilus mutants of pseudomonas syringae pathovar phaseolicola
PLOS ONE, 2015Co-Authors: Mark Sistrom, Derek S Park, Heath E Obrie, Zheng Wang, David S Guttma, Jeffrey P Townsend, Paul E TurneAbstract:Pseudomonas syringae pv. phaseolicola (Pph) is a significant bacterial pathogen of agricultural crops, and phage Φ6 and other members of the dsRNA virus family Cystoviridae undergo lytic (virulent) infection of Pph, using the type IV pilus as the initial site of cellular attachment. Despite the popularity of Pph/phage Φ6 as a model system in evolutionary biology, Pph resistance to phage Φ6 remains poorly characterized. To investigate differences between phage Φ6 resistant Pph strains, we examined genomic and gene expression variation among three bacterial genotypes that differ in the number of type IV pili expressed per cell: ordinary (wild-type), non-piliated, and super-piliated. Genome sequencing of non-piliated and super-piliated Pph identified few mutations that separate these genotypes from wild type Pph--and none present in genes known to be directly involved in type IV pilus expression. Expression analysis revealed that 81.1% of gene ontology (GO) terms up-regulated in the non-piliated strain were down-regulated in the super-piliated strain. This differential expression is particularly prevalent in genes associated with respiration--specifically genes in the tricarboxylic acid cycle (TCA) cycle, aerobic respiration, and acetyl-CoA metabolism. The expression patterns of the TCA pathway appear to be generally up and down-regulated, in non-piliated and super-piliated Pph respectively. As pilus retraction is mediated by an ATP motor, loss of retraction ability might lead to a lower energy draw on the bacterial cell, leading to a different energy balance than wild type. The lower metabolic rate of the super-piliated strain is potentially a result of its loss of ability to retract.
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genomic and gene expression comparisons among phage resistant type iv pilus mutants of pseudomonas syringae pathovar phaseolicola
bioRxiv, 2015Co-Authors: Mark Sistrom, Derek S Park, Heath E Obrie, Zheng Wang, David S Guttma, Jeffrey P Townsend, Paul E TurneAbstract:Pseudomonas syringae pv. phaseolicola (Pph) is a significant bacterial pathogen of agricultural crops, and phage φ6 and other members of the dsRNA virus family Cystoviridae undergo lytic (virulent) infection of Pph, using the type IV pilus as the initial site of cellular attachment. Despite the popularity of Pph/phage φ6 as a model system in evolutionary biology, Pph resistance to phage φ6 remains poorly characterized. To investigate differences between phage φ6 resistant Pph strains, we examined genomic and gene expression variation among three bacterial genotypes that differ in the number of type IV pili expressed per cell: ordinary (wild-type), non-piliated, and super-piliated. Genome sequencing of non-piliated and super-piliated Pph identified few mutations that separate these genotypes from wild type Pph – and none present in genes known to be directly involved in type IV pilus expression. Expression analysis revealed that 81.1% of GO terms up-regulated in the non-piliated strain were down-regulated in the super-piliated strain. This differential expression is particularly prevalent in genes associated with respiration — specifically genes in the tricarboxylic acid cycle (TCA) cycle, aerobic respiration, and acetyl-CoA metabolism. The expression patterns of the TCA pathway appear to be generally up and down-regulated, in non-piliated and super-piliated Pph respectively. As pilus retraction is mediated by an ATP motor, loss of retraction ability might lead to a lower energy draw on the bacterial cell, leading to a different energy balance than wild type. The lower metabolic rate of the super-piliated strain is potentially a result of its loss of ability to retract.
