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Kristin K Wobbe - One of the best experts on this subject based on the ideXlab platform.
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Mutational analysis of Turnip crinkle virus movement Protein P8.
Molecular plant pathology, 2001Co-Authors: Muslum Akgoz, Que N. Nguyen, Ann E. Talmadge, Katherine E. Drainville, Kristin K WobbeAbstract:Summary Turnip crinkle virus encodes two Proteins, P8 and p9, that are both required for cell-to-cell movement. The P8 movement Protein has been demonstrated to bind RNA in a cooperative manner, although, similar to many other plant virus movement Proteins, it contains no canonical RNA binding domain(s). However, three positively charged regions of P8 may potentially form ionic interactions with the RNA backbone. To identify functional regions of P8, a series of alanine and deletion scanning mutations were produced. The effects of these mutations were analysed using both in vitro RNA binding assays and in vivo infections of susceptible (Di-3) and resistant (Di-17) Arabidopsis thaliana plants. Several mutants that have reduced RNA binding ability were also demonstrated to be movement deficient and replication competent. Based on these results, there appear to be two regions, located between amino acids 18 and 31, and 50 and 72, that are required for RNA binding. Furthermore, additional regions (amino acids 12-15, and 34-37) appear to play a role in vivo unrelated to in vitro RNA binding activity.
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a single amino acid change in turnip crinkle virus movement Protein P8 affects rna binding and virulence on arabidopsis thaliana
Journal of Virology, 1998Co-Authors: Kristin K Wobbe, Dmaris Amick Dempsey, Muslum Akgoz, Daniel F KlessigAbstract:Comparison of the symptoms caused by turnip crinkle virus strain M (TCV-M) and TCV-B infection of a resistant Arabidopsis thaliana line termed Di-17 demonstrates that TCV-B has a greater ability to spread in planta. This ability is due to a single amino acid change in the viral movement Protein P8 and inversely correlates with P8 RNA binding affinity.
Dennis H. Bamford - One of the best experts on this subject based on the ideXlab platform.
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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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a novel virus host cell membrane interaction membrane voltage dependent endocytic like entry of bacteriophage φ6 nucleocapsid
Journal of Cell Biology, 1999Co-Authors: Minna M. Poranen, Rimantas Daugelavicius, Paivi M Ojala, Michael W Hess, Dennis H. BamfordAbstract:Studies on the virus-cell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virus-host membrane interac- tions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P.J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1467-1473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1821-1829). f 6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The vi- ral entry is initiated by fusion between the virus mem- brane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a Protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the inter- action of the nucleocapsid with the host plasma mem- brane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma mem- brane is independent of membrane voltage ( DC ) and the K 1 and H 1 gradients. However, the following inter- nalization is dependent on plasma membrane voltage ( DC ), but does not require a high ATP level or K 1 and H 1 gradients. Moreover, the nucleocapsid shell Protein, P8, is the viral component mediating the membrane- nucleocapsid interaction.
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Mutational Analysis of the Role of Nucleoside Triphosphatase P4 in the Assembly of the RNA Polymerase Complex of Bacteriophage φ6
Journal of virology, 1998Co-Authors: Anja O. Paatero, Leonard Mindich, Dennis H. BamfordAbstract:The complex double-stranded RNA (dsRNA) viruses share a number of characteristics. The genomes are segmented, and the innermost particle in the virion comprises RNA-dependent RNA polymerase activity. The outer Protein (or lipid) layers are designed to recognize the host cell and to deliver the polymerase particle into it. The double-stranded genome is contained within the polymerase particle throughout the infection; only the positive-sense transcripts of the genome segments are translocated to the cell interior for Protein production and particle assembly. Newly formed polymerase particles express plus-strand synthesis, thus multiplying the number of transcribing particles and allowing the production of large amounts of the structural Proteins. Complex dsRNA viruses that infect bacterial, plant, and animal hosts have been found. Bacteriophage φ6 infects Pseudomonas syringae cells. It is surrounded by a lipid envelope that encloses the nucleocapsid (33). The nucleocapsid is composed of a core, the polymerase complex particle, which is surrounded by a shell of Protein P8. The icosahedral core is composed of four Protein species and three dsRNA genome segments (15, 32). Protein P1 forms the particle skeleton, and the rest of the core Proteins are associated with it (12, 21). Protein P2 contains the RNA polymerase active site, P7 is needed for stable genome packaging (9, 10), and Protein P4 is a nonspecific nucleoside triphosphatase (NTPase) that plays a role in providing the energy for the RNA translocation reaction (packaging) (7, 20, 27). The proposed numbers of these Proteins in the polymerase complex are 120 for P1, 12 for P2, 120 for P4, and 60 for P7 (10, 11, 14). The genes encoding the polymerase complex Proteins are all located in genome segment L (17). A cDNA copy of the L segment expressed in Escherichia coli produces empty polymerase complex particles, procapsids (6). These package plus-sense transcripts and synthesize the corresponding minus strands inside the particle in vitro (8). Protein P8 assembly onto these particles leads to the formation of nucleocapsids that are infectious to spheroplasts of the host cell. This infection process produces infectious enveloped virions (23, 24). Protein P4 is a nonspecific NTPase cleaving ribo-, deoxyribo-, and dideoxyribonucleoside triphosphates to the corresponding diphosphates (27). The Protein is 331 amino acids long (ca. 35 kDa) and forms doughnut-shaped homomultimers in the presence of divalent cations and ATP or ADP (11). The enzymatic activity is associated only with the multimeric form of the Protein. The activity is enhanced by calcium and zinc ions as well as single-stranded RNA and is down-regulated by magnesium ions (11, 27). The P4 NTPase is the only one detected in the polymerase particle and, since the RNA packaging reaction is dependent on the presence of nucleotides that can be cleaved by P4, it is considered to be the energy source for the RNA translocation reaction into the procapsid (7, 27). P4 is shown to contain about 30% of both α-helix and β-strand, suggesting an α/β fold with significant amounts of loops and turns (11). It has previously been shown that Proteins P2 and P7 associate with a particle containing Proteins P1 and P4 (3, 6, 10). However, due to the difficulties based on the insolubility of P4-deficient particles, it has not been possible to determine the assembly requirements of Protein P4. In this investigation, we used mutational analysis to investigate Protein P4 assembly behavior. Both random and targeted mutations are produced in gene 4. The corresponding mutant Proteins are assayed for solubility, enzymatic activity, multimer formation, and assembly on particles lacking P4. We propose an assembly pathway in which the multimer is formed first and then is assembled on the P1 particle without the aid of any other phage Proteins.
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Protein p7 of phage phi6 rna polymerase complex acquiring of rna packaging activity by in vitro assembly of the purified Protein onto deficient particles
Journal of Molecular Biology, 1997Co-Authors: Jarmo T Juuti, Dennis H. BamfordAbstract:Abstract The RNA polymerase complex of double-stranded RNA bacteriophage φ6 is composed of four Proteins, P1, P2, P4 and P7. These four Proteins are capable of performing all the functions required for the replication of the double-stranded RNAs of the φ6 genome. The polymerase complex containing the three genomic dsRNA segments is the core particle of the φ6 virion. In this study purified Protein P7 was found to form highly asymmetric dimers. Using polyclonal anti-P7 antibody, P7 was shown to be accessible on the surface of the nucleocapsid. Treatment of nucleocapsids with polyclonal anti-P7 antibody released coat Protein P8 with ensuing activation of the plus strand RNA synthesis from the resulting core particles. Purified P7 could be assembled onto particles lacking P7 and particles lacking both P2 (RNA polymerase) and P7. In both cases RNA packaging activity was acquired. Assembly of P7 onto deficient particles took place also in the absence of host Proteins. Protein P7 is known to be necessary for stable packaging of the three genomic φ6 plus strand RNAs into preformed polymerase complex particles. Additionally, Protein P7 seems to be involved in the regulation of plus strand synthesis (i.e. transcription) as a fidelity factor. Particles lacking Protein P7 produce anomalous size transcripts. Analysis of the polymerase complex stability revealed that Proteins P2, P4 and P7 are independently associated with the major structural Protein P1. The number of P7 molecules in one virion was estimated to be 60 and a location at the 5-fold symmetry position is proposed.
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double stranded rna bacteriophage phi 6 Protein p4 is an unspecific nucleoside triphosphatase activated by calcium ions
Journal of Virology, 1995Co-Authors: Anja O. Paatero, J E Syvaoja, Dennis H. BamfordAbstract:Double-stranded RNA bacteriophage phi 6 has an envelope surrounding the nucleocapsid (NC). The NC is composed of a surface Protein, P8, and Proteins P1, P2, P4, and P7, which form a dodecahedral polymerase complex enclosing the segmented viral genome. Empty polymerase complex particles (procapsids) package positive-sense viral single-stranded RNAs provided that energy is available in the form of nucleoside triphosphates (NTPs). Photoaffinity labelling of both the NC and the procapsid has earlier been used to show that ATP binds to Protein P4 and that the NC hydrolyzes NTPs. Using the NC and the NC core particles (NCs lacking surface Protein P8) and purified Protein P4, we demonstrate here that multimeric P4 is the active NTPase. Isolation of multimeric P4 is successful only in the presence of NTPs. The activity of P4 is the same in association with the viral particles as it is in pure form. P4 is an unspecific NTPase hydrolyzing ribo-NTPs, deoxy NTPs, and dideoxy NTPs to the corresponding nucleoside diphosphates. The Km of the reaction for ATP, GTP, and UTP is around 0.2 to 0.3 mM. The NTP hydrolysis by P4 absolutely requires residual amounts of Mg2+ ions and is greatly activated when the Ca2+ concentration reaches 0.5 mM. Competition experiments indicate that Mg2+ and Ca2+ ions have approximately equal binding affinities for P4. They might compete for a common binding site. The nucleotide specificity and enzymatic properties of the P4 NTPase are similar to the NTP hydrolysis reaction conditions needed to translocate and condense the viral positive-sense RNAs to the procapsid particle.
Yongwang Zhong - One of the best experts on this subject based on the ideXlab platform.
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rice black streaked dwarf virus minor core Protein P8 is a nuclear dimeric Protein and represses transcription in tobacco protoplasts
FEBS Letters, 2007Co-Authors: Huijun Liu, Chunhong Wei, Yongwang ZhongAbstract:Virus-encoding nuclear transcriptional regulators play important roles in the viral life cycle. Most of these Proteins exhibit intrinsic transcriptional activation or repression activity, and are involved in the regulation of the expression of virus genome itself or important cellular genes to facilitate viral replication and inhibit antiviral responses. Here, we report that the minor core Protein P8 of Rice black-streaked dwarf virus, a dsRNA virus infecting host plants and insects, is targeted to the nucleus of insect and plant cells via its N-terminal 1–40 amino acids and possesses potent active transcriptional repression activity in Bright Yellow-2 tobacco suspension cells. Moreover, P8, like many transcriptional regulatory Proteins, is capable of forming homo-dimers within insect cells and in vitro. All these data suggest that P8 is likely to enter the nucleus of host cell and play an important role as a negative transcriptional regulator of host gene expression during the process of virus–host interaction.
Toshihiro Omura - One of the best experts on this subject based on the ideXlab platform.
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three dimensional analysis of the association of viral particles with mitochondria during the replication of rice gall dwarf virus
Journal of Molecular Biology, 2011Co-Authors: Taiyun Wei, Naoyuki Miyazaki, Tamaki Ueharaichiki, Hiroyuki Hibino, Takumi Shimizu, Osamu Netsu, Akira Kikuchi, Takahide Sasaya, Kenji Iwasaki, Toshihiro OmuraAbstract:Examination of cultured insect vector cells that had been infected with Rice gall dwarf virus (RGDV), using transmission electron microscopy and confocal microscopy, revealed the presence of clusters of virus-coated mitochondria around viroplasms in which replication and assembly of RGDV occurred, suggesting a role for mitochondria in supplying the energy required for viral morphogenetic processes. Electron tomography revealed that RGDV particles on the surface of mitochondria are arrayed in an orderly but loose manner, unlike tightly packaged particles in vesicular compartments, suggesting the presence of counterpart molecules on the surface of mitochondria. The viral particles in close proximity to mitochondria were aligned along intermediate filaments, which might serve as scaffolds for the anchorage of these particles. RGDV has a putative mitochondrion-targeting sequence on the outer surface of the outer-capsid Protein P8. The arrangement of RGDV particles around mitochondria suggests that the region of the P8 Protein containing the mitochondrion-targeting sequence might attach to a molecule like a receptor on the outer mitochondrial membrane. Our analysis demonstrates the three-dimensional arrangement and molecular basis for the mitochondrial proximity of RGDV particles during viral replication.
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The atomic structure of rice dwarf virus reveals the self-assembly mechanism of component Proteins.
Structure, 2003Co-Authors: Atsushi Nakagawa, Naoyuki Miyazaki, Hiroshi Mizuno, Yasuo Watanabe, Junichiro Taka, Hisashi Naitow, Akira Ogawa, Zui Fujimoto, Takahiko Higashi, Toshihiro OmuraAbstract:Rice dwarf virus (RDV), the causal agent of rice dwarf disease, is a member of the genus Phytoreovirus in the family Reoviridae. RDV is a double-shelled virus with a molecular mass of approximately 70 million Dalton. This virus is widely prevalent and is one of the viruses that cause the most economic damage in many Asian countries. The atomic structure of RDV was determined at 3.5 A resolution by X-ray crystallography. The double-shelled structure consists of two different Proteins, the core Protein P3 and the outer shell Protein P8. The atomic structure shows structural and electrostatic complementarities between both homologous (P3-P3 and P8-P8) and heterologous (P3-P8) interactions, as well as overall conformational changes found in P3-P3 dimer caused by the insertion of amino-terminal loop regions of one of the P3 Protein into the other. These interactions suggest how the 900 Protein components are built into a higher-ordered virus core structure.
Hengmu Zhang - One of the best experts on this subject based on the ideXlab platform.
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interaction between southern rice black streaked dwarf virus minor core Protein P8 and a rice zinc finger transcription factor
Archives of Virology, 2017Co-Authors: Nianjun Cai, Jin Xue, Jian Yang, Jianping Chen, Hengmu ZhangAbstract:The fijivirus southern rice black-streaked dwarf virus (SRBSDV) causes one of the most serious viral diseases of rice in China and Vietnam. To better understand the molecular basis of SRBSDV infection, a yeast two-hybrid screen of a rice cDNA library was carried out using P8, a minor core Protein of SRBSDV, as the bait. A rice Cys2His2-type zinc finger Protein (OsZFP) was found to interact with SRBSDV P8. A strong interaction between SRBSDV P8 and OsZFP was then confirmed by pull-down assays, and bimolecular fluorescence complementation assays showed that the in vivo interaction was specifically localized in the nucleus of plant cells. Using a series of deletion mutants, it was shown that both the NTP-binding region of P8 and the first two zinc fingers of OsZFP were crucial for their interaction in plant cells. The localization in the nucleus and activation of transcription in yeast supports the notion that OsZFP is a transcription factor. SRBSDV P8 may play an important role in fijiviral infection and symptom development by interfering with the host transcription activity of OsZFP.
