The Experts below are selected from a list of 150 Experts worldwide ranked by ideXlab platform
C. Cheng Kao - One of the best experts on this subject based on the ideXlab platform.
-
Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection
Journal of virology, 2016Co-Authors: Haley Hoover, Stefani Middleton, Joseph Che-yen Wang, Adam Zlotnick, Robert C. Vaughan, C. Cheng KaoAbstract:ABSTRACT The four Brome Mosaic Virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection. IMPORTANCE How icosahedral Viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome Mosaic Virus (BMV) is an RNA Virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.
-
Brome Mosaic Virus Infection of Rice Results in Decreased Accumulation of RNA1
Molecular plant-microbe interactions : MPMI, 2015Co-Authors: Masahiko Kitayama, Haley Hoover, Stefani Middleton, C. Cheng KaoAbstract:Brome Mosaic Virus (BMV) (the Russian strain) infects monocot plants and has been studied extensively in barley and wheat. Here, we report BMV can systemically infect rice (Oryza sativa var. japonica), including cultivars in which the genomes have been determined. The BMV capsid protein can be found throughout the inoculated plants. However, infection in rice exhibits delayed symptom expression or no symptoms when compared with wheat (Triticum aestivum). The sequences of BMV RNAs isolated from rice did not reveal any nucleotide changes in RNA1 or RNA2, while RNA3 had only one synonymous nucleotide change from the inoculum sequence. Preparations of purified BMV virions contained RNA1 at a significantly reduced level relative to the other two RNAs. Analysis of BMV RNA replication in rice revealed that minus-strand RNA1 was replicated at a reduced rate when compared with RNA2. Thus, rice appears to either inhibit RNA1 replication or lacks a sufficient amount of a factor needed to support efficient RNA1 repli...
-
Self-Assembly of Brome Mosaic Virus Capsids: Insights from Shorter Time-Scale Experiments†
The journal of physical chemistry. A, 2008Co-Authors: Chao Chen, C. Cheng Kao, Bogdan DragneaAbstract:An amended kinetic model for the self-assembly of empty capsids of Brome Mosaic Virus is proposed. The model has been modified to account for a new feature in the assembly kinetics revealed by time-course light scattering experiments at higher temporal resolution than previously attempted. To be able to simulate the sharp takeoff from the initial lag phase to the growth phase in the kinetic curves, a monomer activation step was proposed.
-
Selective repression of translation by the Brome Mosaic Virus 1a RNA replication protein.
Journal of virology, 2006Co-Authors: K. Gopinath, C. Cheng KaoAbstract:Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the Brome Mosaic Virus (BMV) 1a protein can repress viral RNA replication in a dosage-dependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5′-untranslated region (5′ UTR); BMV RNA3 that lacks a B box in its 5′ UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.
-
Brome Mosaic Virus RNA Syntheses In Vitro and in Barley Protoplasts
Journal of virology, 2003Co-Authors: K. Sivakumaran, M. Hema, C. Cheng KaoAbstract:The RNA replicase extracted from Brome Mosaic Virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber Mosaic Virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.
Jozef J. Bujarski - One of the best experts on this subject based on the ideXlab platform.
-
next generation sequencing reveals packaging of host rnas by Brome Mosaic Virus
Virus Research, 2018Co-Authors: N Shrestha, Jozef J. Bujarski, Philipp H Weber, Sean V Burke, William P Wysocki, Melvin R DuvallAbstract:Abstract Although RNA Viruses evolved the mechanisms of specific encapsidation, miss-packaging of cellular RNAs has been reported in such RNA Virus systems as flock house Virus or cucumber necrosis Virus. To find out if Brome Mosaic Virus (BMV), a tripartite RNA Virus, can package cellular RNAs, BMV was propagated in barley and in Nicotiana benthamiana hosts, purified by cesium chloride (CsCl) gradient ultracentrifugation followed by nuclease treatment to remove any contaminating cellular (host) RNAs. The extracted virion RNA was then sequenced by using next-generation sequencing (NGS RNA-Seq) with the Illumina protocol. Bioinformatic analysis revealed the content of host RNAs ranging from 0.07% for BMV extracted from barley to 0.10% for the Virus extracted from N. benthamiana. The Viruses from two sources appeared to co-encapsidate different patterns of host-RNAs, including ribosomal RNAs (rRNAs), messenger RNAs (mRNAs) but also mitochondrial and plastid RNAs and, interestingly, transposable elements, both transposons and retrotransposons. Our data reveal that BMV virions can carry host RNAs, having a potential to mediate horizontal gene transfer (HGT) in plants.
-
Infection and RNA recombination of Brome Mosaic Virus in Arabidopsis thaliana.
Virology, 2004Co-Authors: Aleksandra Dzianott, Jozef J. BujarskiAbstract:Ecotypes of Arabidopsis thaliana supported the replication and systemic spread of Brome Mosaic Virus (BMV) RNAs. Infection was induced either by manual inoculation with viral RNA or by BMV virions, demonstrating that Virus disassembly did not prevent infection. When in vitro-transcribed BMV RNAs 1-3 were used, production of subgenomic RNA4 was observed, showing that BMV RNA replication and transcription had occurred. Furthermore, inoculations of the transgenic Arabidopsis line that expressed a suppressor of RNA interference (RNAi) pathway markedly increased the BMV RNA concentrations. Inoculations with designed BMV RNA3 recombination vectors generated both homologous and nonhomologous BMV RNA-RNA recombinants. Thus, all cellular factors essential for BMV RNA replication, transcription, and RNA recombination were shown to be present in Arabidopsis. The current scope of understanding of the model Arabidopsis plant system should facilitate the identification of these factors governing the BMV life cycle.
-
Structure-based rationale for the rescue of systemic movement of Brome Mosaic Virus by spontaneous second-site mutations in the coat protein gene.
Journal of virology, 1997Co-Authors: S. Flasinski, Aleksandra Dzianott, Jeffrey A. Speir, John E. Johnson, Jozef J. BujarskiAbstract:We describe spontaneous second-site reversions within the coat protein open reading frame that rescue the systemic-spread phenotype and increase virion stability of a mutant of Brome Mosaic Virus. Based on the crystal structure of the related cowpea chlorotic mottle Virus, we show that the modified residues are spatially clustered to affect the formation of hexamers and pentamers and therefore virion stability.
-
Molecular Studies of Genetic RNA–RNA Recombination in Brome Mosaic Virus
Advances in virus research, 1994Co-Authors: Jozef J. Bujarski, Peter D. Nagy, S. FlasinskiAbstract:Publisher Summary It is well known that DNA-based organisms rearrange and repair their genomic DNA through recombination processes, and that these rearrangements serve as a powerful source of variability and adaptation for these organisms. In RNA Viruses' genetic recombination is defined as any process leading to the exchange of information between viral RNAs. There are two types of recombination events: legitimate and illegitimate. While legitimate (homologous) recombination occurs between closely related sequences at corresponding positions, illegitimate (nonhomologous) recombination could happen at any position among the unrelated RNA molecules. In order to differentiate between the symmetrical and asymmetrical homologous crosses, Lai defined the former as homologous recombination and the latter as aberrant homologous recombination. This chapter uses Brome Mosaic Virus (BMV), a multicomponent plant RNA Virus, as an example to discuss the progress in studying the mechanism of genetic recombination in positive-stranded RNA Viruses. Studies described in this chapter summarize the molecular approaches used to increase the frequency of recombination among BMV RNA segments and, more importantly, to target the sites of crossovers to specific BMV RNA regions. It demonstrates that the latter can be accomplished by introducing local complementarities to the recombining substrates.
-
molecular studies of genetic rna rna recombination in Brome Mosaic Virus
Advances in Virus Research, 1994Co-Authors: Jozef J. Bujarski, Peter D. Nagy, S. FlasinskiAbstract:Publisher Summary It is well known that DNA-based organisms rearrange and repair their genomic DNA through recombination processes, and that these rearrangements serve as a powerful source of variability and adaptation for these organisms. In RNA Viruses' genetic recombination is defined as any process leading to the exchange of information between viral RNAs. There are two types of recombination events: legitimate and illegitimate. While legitimate (homologous) recombination occurs between closely related sequences at corresponding positions, illegitimate (nonhomologous) recombination could happen at any position among the unrelated RNA molecules. In order to differentiate between the symmetrical and asymmetrical homologous crosses, Lai defined the former as homologous recombination and the latter as aberrant homologous recombination. This chapter uses Brome Mosaic Virus (BMV), a multicomponent plant RNA Virus, as an example to discuss the progress in studying the mechanism of genetic recombination in positive-stranded RNA Viruses. Studies described in this chapter summarize the molecular approaches used to increase the frequency of recombination among BMV RNA segments and, more importantly, to target the sites of crossovers to specific BMV RNA regions. It demonstrates that the latter can be accomplished by introducing local complementarities to the recombining substrates.
Tetsuro Okuno - One of the best experts on this subject based on the ideXlab platform.
-
Brome Mosaic Virus replicase proteins localize with the movement protein at infection-specific cytoplasmic inclusions in infected barley leaf cells.
Archives of virology, 2001Co-Authors: Koji Dohi, Kazuyuki Mise, Iwao Furusawa, Masashi Mori, Tetsuro OkunoAbstract:Subcellular localization of the Brome Mosaic Virus replicase-related 1a and 2a proteins, and the 3a movement protein in infected barley leaves was examined by immunogold electron microscopy. The 1a and 2a proteins colocalized at infection-specific electron-dense cytoplasmic inclusions. The 3a protein was also detected in these inclusions. The inclusions were oval or amorphous, and contained electron-lucent regions. Electron microscopic autoradiography showed that the inclusions were sites of [3H]uridine incorporation, suggesting that they are sites of viral RNA synthesis.
-
Brome Mosaic Virus defective rnas generated during infection of barley plants
Journal of General Virology, 1999Co-Authors: Tri Asmira Damayanti, Kazuyuki Mise, Iwao Furusawa, Hideaki Nagano, Tetsuro OkunoAbstract:Brome Mosaic Virus (BMV) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective RNAs (D-RNAs). The D-RNAs were detected in total and virion RNAs extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking D-RNA. The D-RNAs were derived from genomic RNA3 by double or mainly single deletions in the 3a protein ORF, and formed a heterogeneous population. By using in vitro transcripts of D-RNA synthesized from full-length cDNA clones, the D-RNAs were shown to replicate in a helper Virus-dependent manner and to be packaged into virions in barley protoplasts. Subgenomic RNA4 was produced from the D-RNA and the coat protein was also expressed. Existence of the D-RNAs together with BMV genomic RNAs in inoculated protoplasts decreased the accumulation of 3a protein but it had no apparent effect on the accumulation of BMV genomic RNA3 or the coat protein. This is the first report of naturally occurring D-RNAs generated during prolonged infection with BMV.
-
Expression of Brome Mosaic Virus-encoded replicase genes in transgenic tobacco plants.
Journal of General Virology, 1992Co-Authors: Masashi Mori, Kazuyuki Mise, Tetsuro Okuno, Iwao FurusawaAbstract:We introduced replicase genes of Brome Mosaic Virus (BMV) to Nicotiana tabacum cv. Petit Habana (SR1) using two different types of transformation vectors containing cDNAs of BMV RNA 1 and RNA 2. One type (V type) contains cDNA from which complete viral RNAs are transcribed. These RNAs can function as templates for viral replicase. The other type (M type) contains cDNA from which viral RNAs without their 3′ non-coding regions are transcribed; these RNAs can only function as mRNA. Viral replicase expressed from the integrated cDNAs in both V and M type transgenic plants can complement an infection by BMV RNA 3.
-
infectivity of plasmids containing Brome Mosaic Virus cdna linked to the cauliflower Mosaic Virus 35s rna promoter
Journal of General Virology, 1991Co-Authors: Masashi Mori, Kazuyuki Mise, Tetsuro Okuno, Kappei Kobayashi, Iwao FurusawaAbstract:Full-length biologically active cDNAs of Brome Mosaic Virus genomic RNAs 1, 2 and 3 were constructed by joining cDNA fragments. The cDNAs were constructed so that, at the 5′ ends, unique SnaBI sites were present at the site of initiation of transcription. The cDNAs were inserted between a modified cauliflower Mosaic Virus (CaMV) 35S RNA promoter and terminator regions derived from CaMV DNA, and cloned into pUC18. When a mixture of the plasmid DNAs was inoculated onto Chenopodium hybridum leaves, local lesions appeared 5 to 6 days later. However, no symptoms appeared in similarly inoculated barley plants. Plasmid cDNAs with extra sequences at the 5′ end were infectious but RNAs transcribed from cDNAs with similar sequences were not.
Iwao Furusawa - One of the best experts on this subject based on the ideXlab platform.
-
Brome Mosaic Virus replicase proteins localize with the movement protein at infection-specific cytoplasmic inclusions in infected barley leaf cells.
Archives of virology, 2001Co-Authors: Koji Dohi, Kazuyuki Mise, Iwao Furusawa, Masashi Mori, Tetsuro OkunoAbstract:Subcellular localization of the Brome Mosaic Virus replicase-related 1a and 2a proteins, and the 3a movement protein in infected barley leaves was examined by immunogold electron microscopy. The 1a and 2a proteins colocalized at infection-specific electron-dense cytoplasmic inclusions. The 3a protein was also detected in these inclusions. The inclusions were oval or amorphous, and contained electron-lucent regions. Electron microscopic autoradiography showed that the inclusions were sites of [3H]uridine incorporation, suggesting that they are sites of viral RNA synthesis.
-
Brome Mosaic Virus defective rnas generated during infection of barley plants
Journal of General Virology, 1999Co-Authors: Tri Asmira Damayanti, Kazuyuki Mise, Iwao Furusawa, Hideaki Nagano, Tetsuro OkunoAbstract:Brome Mosaic Virus (BMV) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective RNAs (D-RNAs). The D-RNAs were detected in total and virion RNAs extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking D-RNA. The D-RNAs were derived from genomic RNA3 by double or mainly single deletions in the 3a protein ORF, and formed a heterogeneous population. By using in vitro transcripts of D-RNA synthesized from full-length cDNA clones, the D-RNAs were shown to replicate in a helper Virus-dependent manner and to be packaged into virions in barley protoplasts. Subgenomic RNA4 was produced from the D-RNA and the coat protein was also expressed. Existence of the D-RNAs together with BMV genomic RNAs in inoculated protoplasts decreased the accumulation of 3a protein but it had no apparent effect on the accumulation of BMV genomic RNA3 or the coat protein. This is the first report of naturally occurring D-RNAs generated during prolonged infection with BMV.
-
Expression of Brome Mosaic Virus-encoded replicase genes in transgenic tobacco plants.
Journal of General Virology, 1992Co-Authors: Masashi Mori, Kazuyuki Mise, Tetsuro Okuno, Iwao FurusawaAbstract:We introduced replicase genes of Brome Mosaic Virus (BMV) to Nicotiana tabacum cv. Petit Habana (SR1) using two different types of transformation vectors containing cDNAs of BMV RNA 1 and RNA 2. One type (V type) contains cDNA from which complete viral RNAs are transcribed. These RNAs can function as templates for viral replicase. The other type (M type) contains cDNA from which viral RNAs without their 3′ non-coding regions are transcribed; these RNAs can only function as mRNA. Viral replicase expressed from the integrated cDNAs in both V and M type transgenic plants can complement an infection by BMV RNA 3.
-
infectivity of plasmids containing Brome Mosaic Virus cdna linked to the cauliflower Mosaic Virus 35s rna promoter
Journal of General Virology, 1991Co-Authors: Masashi Mori, Kazuyuki Mise, Tetsuro Okuno, Kappei Kobayashi, Iwao FurusawaAbstract:Full-length biologically active cDNAs of Brome Mosaic Virus genomic RNAs 1, 2 and 3 were constructed by joining cDNA fragments. The cDNAs were constructed so that, at the 5′ ends, unique SnaBI sites were present at the site of initiation of transcription. The cDNAs were inserted between a modified cauliflower Mosaic Virus (CaMV) 35S RNA promoter and terminator regions derived from CaMV DNA, and cloned into pUC18. When a mixture of the plasmid DNAs was inoculated onto Chenopodium hybridum leaves, local lesions appeared 5 to 6 days later. However, no symptoms appeared in similarly inoculated barley plants. Plasmid cDNAs with extra sequences at the 5′ end were infectious but RNAs transcribed from cDNAs with similar sequences were not.
Katalin A. Hudak - One of the best experts on this subject based on the ideXlab platform.
-
depurination of Brome Mosaic Virus rna3 inhibits its packaging into Virus particles
Nucleic Acids Research, 2011Co-Authors: Rajita A Karran, Katalin A. HudakAbstract:Packaging of the segmented RNA genome of Brome Mosaic Virus (BMV) into discrete particles is an essential step in the Virus life cycle; however, questions remain regarding the mechanism of RNA packaging and the degree to which the viral coat protein controls the process. In this study, we used a plant-derived glycosidase, Pokeweed antiviral protein, to remove 14 specific bases from BMV RNA3 to examine the effect of depurination on Virus assembly. Depurination of A771 within ORF3 and A1006 in the intergenic region inhibited coat protein binding and prevented RNA3 incorporation into particles. The disruption of interaction was not based on sequence identity, as mutation of these two purines to pyrimidines did not decrease coat protein-binding affinity. Rather, we suggest that base removal results in decreased thermodynamic stability of local RNA structures required for packaging, and that this instability is detected by coat protein. These results describe a new level of discrimination by coat protein, whereby it recognizes damage to specific viral RNA elements in the form of base removal and selects against incorporating the RNA into particles.
-
pokeweed antiviral protein inhibits Brome Mosaic Virus replication in plant cells
Journal of Biological Chemistry, 2005Co-Authors: Daniel Picard, Cheng C Kao, Katalin A. HudakAbstract:Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal Viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate Brome Mosaic Virus (BMV) RNA in vitro, resulting in reduced viral protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection.
