The Experts below are selected from a list of 1863 Experts worldwide ranked by ideXlab platform
James L. Van Etten - One of the best experts on this subject based on the ideXlab platform.
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Cryopreservation of Paramecium bursaria Chlorella Virus-1 during an active infection cycle of its host.
PloS one, 2019Co-Authors: Samantha R. Coy, James L. Van Etten, Alyssa N. Alsante, Steven W. WilhelmAbstract:Best practices in laboratory culture management often include cryopreservation of microbiota, but this can be challenging with some Virus particles. By preserving viral isolates researchers can mitigate genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting Virus, Paramecium bursaria Chlorella Virus 1 (PBCV-1). We explored cryotolerance of the infectivity of this Virus particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in its Chlorella variabilis NC64A host, and found that Virus survivorship was highest (69.5 ± 16.5%) when the infected host is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the cell as a vehicle for viral cryopreservation resulted in 24.9-30.1 fold increases in PBCV-1 survival based on 95% confidence intervals of frozen Virus particles and Virus cryopreserved at 240 minutes post-infection. Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that cryopreservation of infected hosts may be a reliable mechanism for Virus persistence in non-growth permitting circumstances in the environment, such as ancient permafrosts.
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Near-atomic structure of a giant Virus
Nature Publishing Group, 2019Co-Authors: Qianglin Fang, Thomas Klose, Dongjie Zhu, Irina Agarkova, Jagat Adhikari, Yue Liu, Zhenguo Chen, Yingyuan Sun, Michael L. Gross, James L. Van EttenAbstract:Nucleocytoplasmic large DNA Viruses are a group of Viruses that infect many eukaryotic hosts. Here, the authors provide a 3.5 Å resolution icosahedrally-averaged capsid structure of Paramecium bursaria Chlorella Virus 1 and show how 1800 minor capsid proteins form a hexagonal network below the outer capsid shell
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structural studies demonstrating a bacteriophage like replication cycle of the eukaryote infecting paramecium bursaria Chlorella Virus 1
PLOS Pathogens, 2017Co-Authors: Elad Milrot, James L. Van Etten, Eyal Shimoni, Katya Rechav, Tali Dadosh, Tamar Unger, Abraham MinskyAbstract:A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria Chlorella Virus-1 (PBCV-1). Our studies reveal that early infection stages of this eukaryotic-infecting Virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the Virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA Viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle.
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Deep RNA Sequencing Reveals Hidden Features and Dynamics of Early Gene Transcription in Paramecium bursaria Chlorella Virus 1
2016Co-Authors: Guillaume Blanc, David D Dunigan, James R. Gurnon, Giane M. Yanai-balser, Irina V. Agarkova, Michael Mozar, Janet M. Rowe, Yuannan Xia, Jean-jack Riethoven, James L. Van EttenAbstract:Paramecium bursaria Chlorella Virus 1 (PBCV-1) is the prototype of the genus ChloroVirus (family Phycodnaviridae) that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i.), transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41 % or the poly (A+)-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i.) was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most o
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response of mammalian macrophages to challenge with the chloroVirus acanthocystis turfacea Chlorella Virus 1
Journal of Virology, 2015Co-Authors: Robert H. Yolken, James L. Van Etten, Irina V. Agarkova, Thomas M Petro, You Zhou, David D DuniganAbstract:ABSTRACT It was recently reported that 44% of the oropharyngeal samples from the healthy humans in a study cohort had DNA sequences similar to that of the chloroVirus ATCV-1 (Acanthocystisturfacea Chlorella Virus 1, family Phycodnaviridae) and that these study subjects had decreases in visual processing and visual motor speed compared with individuals in whom no Virus was detected. Moreover, mice inoculated orally with ATCV-1 developed immune responses to ATCV-1 proteins and had decreases in certain cognitive domains. Because heightened interleukin-6 (IL-6), nitric oxide (NO), and ERK mitogen-activated protein (MAP) kinase activation from macrophages are linked to cognitive impairments, we evaluated cellular responses and viral PFU counts in murine RAW264.7 cells and primary macrophages after exposure to ATCV-1 in vitro for up to 72 h after a Virus challenge. Approximately 8% of the ATCV-1 inoculum was associated with macrophages after 1 h, and the percentage increased 2- to 3-fold over 72 h. Immunoblot assays with rabbit anti-ATCV-1 antibody detected a 55-kDa protein consistent with the viral capsid protein from 1 to 72 h and increasing de novo synthesis of a previously unidentified 17-kDa protein beginning at 24 h. Emergence of the 17-kDa protein did not occur and persistence of the 55-kDa protein declined over time when cells were exposed to heat-inactivated ATCV-1. Moreover, starting at 24 h, RAW264.7 cells exhibited cytopathic effects, annexin V staining, and cleaved caspase 3. Activation of ERK MAP kinases occurred in these cells by 30 min postchallenge, which preceded the expression of IL-6 and NO. Therefore, ATCV-1 persistence in and induction of inflammatory factors by these macrophages may contribute to declines in the cognitive abilities of mice and humans. IMPORTANCE Virus infections that persist in and stimulate inflammatory factors in macrophages contribute to pathologies in humans. A previous study showed that DNA sequences homologous to the chloroVirus ATCV-1 were found in a significant fraction of oropharyngeal samples from a healthy human cohort. We show here that ATCV-1, whose only known host is a eukaryotic green alga (Chlorellaheliozoae) that is an endosymbiont of the heliozoon Acanthocystisturfacea, can unexpectedly persist within murine macrophages and trigger inflammatory responses including factors that contribute to immunopathologies. The inflammatory factors that are produced in response to ATCV-1 include IL-6 and NO, whose induction is preceded by the activation of ERK MAP kinases. Other responses of ATCV-1-challenged macrophages include an apoptotic cytopathic effect, an innate antiviral response, and a metabolic shift toward aerobic glycolysis. Therefore, mammalian encounters with chloroViruses may contribute to chronic inflammatory responses from macrophages.
Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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kinetic analysis of dna strand joining by Chlorella Virus dna ligase and the role of nucleotidyltransferase motif vi in ligase adenylylation
Journal of Biological Chemistry, 2012Co-Authors: Poulami Samai, Stewart ShumanAbstract:Chlorella Virus DNA ligase (ChVLig) is an instructive model for mechanistic studies of the ATP-dependent DNA ligase family. ChVLig seals 3′-OH and 5′-PO4 termini via three chemical steps: 1) ligase attacks the ATP α phosphorus to release PPi and form a covalent ligase-adenylate intermediate; 2) AMP is transferred to the nick 5′-phosphate to form DNA-adenylate; 3) the 3′-OH of the nick attacks DNA-adenylate to join the polynucleotides and release AMP. Each chemical step requires Mg2+. Kinetic analysis of nick sealing by ChVLig-AMP revealed that the rate constant for phosphodiester synthesis (kstep3 = 25 s−1) exceeds that for DNA adenylylation (kstep2 = 2.4 s−1) and that Mg2+ binds with similar affinity during step 2 (Kd = 0.77 mm) and step 3 (Kd = 0.87 mm). The rates of DNA adenylylation and phosphodiester synthesis respond differently to pH, such that step 3 becomes rate-limiting at pH ≤ 6.5. The pH profiles suggest involvement of one and two protonation-sensitive functional groups in catalysis of steps 2 and 3, respectively. We suggest that the 5′-phosphate of the nick is the relevant protonation-sensitive moiety and that a dianionic 5′-phosphate is necessary for productive step 2 catalysis. Motif VI, located at the C terminus of the OB-fold domain of ChVLig, is a conserved feature of ATP-dependent DNA ligases and GTP-dependent mRNA capping enzymes. Presteady state and burst kinetic analysis of the effects of deletion and missense mutations highlight the catalytic contributions of ChVLig motif VI, especially the Asp-297 carboxylate, exclusively during the ligase adenylylation step.
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structure function analysis of the ob and latch domains of Chlorella Virus dna ligase
Journal of Biological Chemistry, 2011Co-Authors: Poulami Samai, Stewart ShumanAbstract:Chlorella Virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5′-phosphate nucleotide and the 3′-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.
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functional dissection of the dna interface of the nucleotidyltransferase domain of Chlorella Virus dna ligase
Journal of Biological Chemistry, 2011Co-Authors: Poulami Samai, Stewart ShumanAbstract:Chlorella Virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3′-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3′-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3′-OH nucleoside in the catalysis of DNA 5′-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig.
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solution nmr studies of Chlorella Virus dna ligase adenylate
Journal of Molecular Biology, 2010Co-Authors: Andrea Piserchio, Stewart Shuman, Pravin A Nair, Ranajeet GhoseAbstract:Abstract DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella Virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone 15 N spin relaxation and 15 N, 1 H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate.
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functional dynamics in Chlorella Virus dna ligase
Biophysical Journal, 2009Co-Authors: Andrea Piserchio, Stewart Shuman, Pravin A Nair, Ranajeet GhoseAbstract:DNA ligases specifically recognize and seal double stranded nicked DNA by catalyzing the formation of a phospho-diester bond between the 3′OH and 5′ phosphate termini. In physiological conditions these ubiquitous enzymes are essential for DNA replication, repair and recombination, while in tumor cells they can play a critical role in apoptosis resistance.Chlorella Virus ligase is a pluripotent ATP-dependent ligase composed by two domains, a N-terminal nucleotidyltransferase domain, hosting the catalytic site, and a C-terminal OB-domain, both of which participate to DNA binding. A number of crystallographic studies have elucidated important structural details of the nick-sealing process. DNA binding, in particular, appears to require a large reorientation of the two domains, as well as relevant structural rearrangements localized mainly in the N-terminal region (1). Indeed this protein appears to be a highly dynamic system whose internal motions are closely linked to both the DNA recognition and to the catalytic process. So far, however, the actual nature of these motions is still largely unknown, not only for Chlorella Virus ligase, but also for the entire protein family.We therefore tried to close this gap by undertaking the analysis of the dynamic properties of Chlorella Virus liagase by solution NMR spectroscopy.Reference:(1) Nair P.A., Nandakumar J., Smith P., Odell M., Lima C.D., Shuman S. (2008) Nat Struct Mol Biol. 14, 770-8.
James R. Gurnon - One of the best experts on this subject based on the ideXlab platform.
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Deep RNA Sequencing Reveals Hidden Features and Dynamics of Early Gene Transcription in Paramecium bursaria Chlorella Virus 1
2016Co-Authors: Guillaume Blanc, David D Dunigan, James R. Gurnon, Giane M. Yanai-balser, Irina V. Agarkova, Michael Mozar, Janet M. Rowe, Yuannan Xia, Jean-jack Riethoven, James L. Van EttenAbstract:Paramecium bursaria Chlorella Virus 1 (PBCV-1) is the prototype of the genus ChloroVirus (family Phycodnaviridae) that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i.), transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41 % or the poly (A+)-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i.) was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most o
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Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria Chlorella Virus.
Cellular microbiology, 2015Co-Authors: Elad Milrot, James L. Van Etten, James R. Gurnon, Yael Mutsafi, Yael Fridmann-sirkis, Eyal Shimoni, Katya Rechav, Abraham MinskyAbstract:The increasing interest in cytoplasmic factories generated by eukaryotic-infecting Viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA Virus Paramecium bursaria Chlorella Virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and MimiVirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by MimiVirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway.
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deep rna sequencing reveals hidden features and dynamics of early gene transcription in paramecium bursaria Chlorella Virus 1
PLOS ONE, 2014Co-Authors: Guillaume Blanc, James R. Gurnon, Irina V. Agarkova, Michael Mozar, Janet M. Rowe, Yuannan Xia, Jean-jack Riethoven, Giane M Yanaibalser, David D DuniganAbstract:Paramecium bursaria Chlorella Virus 1 (PBCV-1) is the prototype of the genus ChloroVirus (family Phycodnaviridae) that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i.), transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+)-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i.) was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.
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Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus
Journal of virology, 2012Co-Authors: Ronald L. Cerny, James R. Gurnon, Giane M. Yanai-balser, Irina V. Agarkova, Andrew T. Bauman, Jared C. Roach, Leslie C. Lane, Kurt Wulser, Jason C. VitekAbstract:The 331-kbp chloroVirus Paramecium bursaria Chlorella Virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique Virus-encoded proteins associated with the virion (about 35% of the coding capacity of the Virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroViruses, phycodnaViruses, and nuclear-cytoplasmic large DNA Viruses. The genes encoding these proteins are dispersed throughout the Virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.
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Paramecium bursaria Chlorella Virus 1 Encodes a Polyamine Acetyltransferase
The Journal of biological chemistry, 2012Co-Authors: Zachary Charlop-powers, James L. Van Etten, James R. Gurnon, Jean Jakoncic, Ming-ming ZhouAbstract:Paramecium bursaria Chlorella Virus 1 (PBCV-1), a large DNA Virus that infects green algae, encodes a histone H3 lysine 27-specific methyltransferase that functions in global transcriptional silencing of the host. PBCV-1 has another gene a654l that encodes a protein with sequence similarity to the GCN5 family histone acetyltransferases. In this study, we report a 1.5 Å crystal structure of PBCV-1 A654L in a complex with coenzyme A. The structure reveals a unique feature of A654L that precludes its acetylation of histone peptide substrates. We demonstrate that A654L, hence named viral polyamine acetyltransferase (vPAT), acetylates polyamines such as putrescine, spermidine, cadaverine, and homospermidine present in both PBCV-1 and its host through a reaction dependent upon a conserved glutamate 27. Our study suggests that as the first virally encoded polyamine acetyltransferase, vPAT plays a possible key role in the regulation of polyamine catabolism in the host during viral replication.
Neil Osheroff - One of the best experts on this subject based on the ideXlab platform.
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dna methylation impacts the cleavage activity of Chlorella Virus topoisomerase ii
Biochemistry, 2005Co-Authors: Jennifer S Dickey, James L. Van Etten, Neil OsheroffAbstract:Topoisomerase II from Paramecium bursaria Chlorella Virus-1 (PBCV-1) and Chlorella Virus Marburg-1 (CVM-1) displays an extraordinarily high in vitro DNA cleavage activity that is 30−50 times higher than that of human topoisomerase IIα. This remarkable scission activity may reflect a unique role played by the type II enzyme during the viral life cycle that extends beyond the normal control of DNA topology. Alternatively, but not mutually exclusively, it may reflect an adaptation to some aspect of the viral environment that differs from the in vitro conditions. To this point, the genomes of many Chlorella Viruses contain high levels of N6-methyladenine (6mA) and 5-methylcytosine (5mC), but the DNA employed in vitro is unmodified. Therefore, to determine whether methylation impacts the ability of Chlorella Virus topoisomerase II to cleave DNA, the effects of 6mA and 5mC on the PBCV-1 and CVM-1 enzymes were examined. Results indicate that 6mA strongly inhibits DNA scission mediated by both enzymes, while 5mC ...
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Chlorella Virus marburg topoisomerase ii high dna cleavage activity as a characteristic of Chlorella Virus type ii enzymes
Biochemistry, 2005Co-Authors: Jennifer S Dickey, Tae-jin Choi, James L. Van Etten, Neil OsheroffAbstract:Although the formation of a covalent enzyme-cleaved DNA complex is a prerequisite for the essential functions of topoisomerase II, this reaction intermediate has the potential to destabilize the genome. Consequently, all known eukaryotic type II enzymes maintain this complex at a low steady-state level. Recently, however, a novel topoisomerase II was discovered in Paramecium bursaria Chlorella Virus-1 (PBCV-1) that has an exceptionally high DNA cleavage activity [Fortune et al. (2001) J. Biol. Chem. 276, 24401-24408]. If robust DNA cleavage is critical to the physiological functions of Chlorella Virus topoisomerase II, then this remarkable characteristic should be conserved throughout the viral family. Therefore, topoisomerase II from Chlorella Virus Marburg-1 (CVM-1), a distant family member, was expressed in yeast, isolated, and characterized. CVM-1 topoisomerase II is 1058 amino acids in length, making it the smallest known type II enzyme. The viral topoisomerase II displayed a high DNA strand passage activity and a DNA cleavage activity that was approximately 50-fold greater than that of human topoisomerase IIalpha. High DNA cleavage appeared to result from a greater rate of scission rather than promiscuous DNA site utilization, inordinately tight DNA binding, or diminished religation rates. Despite the fact that CVM-1 and PBCV-1 topoisomerase II share approximately 67% amino acid sequence identity, the two enzymes displayed clear differences in their DNA cleavage specificity/site utilization. These findings suggest that robust DNA cleavage is intrinsic to the viral enzyme and imply that Chlorella Virus topoisomerase II plays a physiological role beyond the control of DNA topology.
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Site-specific DNA cleavage by Chlorella Virus topoisomerase II
Biochemistry, 2002Co-Authors: John M. Fortune, R. Stephen Lloyd, Oleg V. Lavrukhin, James L. Van Etten, Jennifer S Dickey, Neil OsheroffAbstract:The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella Virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.
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topoisomerase ii from Chlorella Virus pbcv 1 has an exceptionally high dna cleavage activity
Journal of Biological Chemistry, 2001Co-Authors: John M. Fortune, Stephen R Lloyd, Oleg V. Lavrukhin, James L. Van Etten, James R. Gurnon, Neil OsheroffAbstract:Abstract Chlorella Virus PBCV-1 topoisomerase II is the only functional type II enzyme known to be encoded by a Virus that infects eukaryotic cells. However, it has not been established whether the protein is expressed following viral infection or whether the enzyme has any catalytic features that distinguish it from cellular type II topoisomerases. Therefore, the present study characterized the physiological expression of PBCV-1 topoisomerase II and individual reaction steps catalyzed by the enzyme. Results indicate that the topoisomerase II gene is widely distributed among ChlorellaViruses and that the protein is expressed 60–90 min after viral infection of algal cells. Furthermore, the enzyme has an extremely high DNA cleavage activity that sets it apart from all known eukaryotic type II topoisomerases. Levels of DNA scission generated by the viral enzyme are ∼30 times greater than those observed with human topoisomerase IIα. The high levels of cleavage are not due to inordinately tight enzyme-DNA binding or to impaired DNA religation. Thus, they most likely reflect an elevated forward rate of scission. The robust DNA cleavage activity of PBCV-1 topoisomerase II provides a unique tool for studying the catalytic functions of type II topoisomerases.
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Topoisomerase II from Chlorella Virus PBCV-1. Characterization of the smallest known type II topoisomerase.
The Journal of biological chemistry, 2000Co-Authors: Oleg V. Lavrukhin, Neil Osheroff, John M. Fortune, Thomas G. Wood, Dwight E. Burbank, James L. Van Etten, R. Stephen LloydAbstract:Abstract Type II topoisomerases, a family of enzymes that govern topological DNA interconversions, are essential to many cellular processes in eukaryotic organisms. Because no data are available about the functions of these enzymes in the replication of Viruses that infect eukaryotic hosts, this led us to express and characterize the first topoisomerase II encoded by one of such Viruses. Paramecium bursaria Chlorella Virus 1 (PBCV-1) infects certain Chlorella-like green algae and encodes a 120-kDa protein with a similarity to type II topoisomerases. This protein was expressed inSaccharomyces cerevisiae and was highly active in relaxation of both negatively and positively supercoiled plasmid DNA, catenation of plasmid DNA, and decatenation of kinetoplast DNA networks. Its optimal activity was determined, and the omission of Mg2+ or its replacement with other divalent cations abolished DNA relaxation. All activities of the recombinant enzyme were ATP dependent. Increasing salt concentrations shifted DNA relaxation from a normally processive mechanism to a distributive mode. Thus, even though the PBCV-1 enzyme is considerably smaller than other eukaryotic topoisomerase II enzymes (whose molecular masses are typically 160–180 kDa), it displays all the catalytic properties expected for a type II topoisomerase.
Ranajeet Ghose - One of the best experts on this subject based on the ideXlab platform.
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solution nmr studies of Chlorella Virus dna ligase adenylate
Journal of Molecular Biology, 2010Co-Authors: Andrea Piserchio, Stewart Shuman, Pravin A Nair, Ranajeet GhoseAbstract:Abstract DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella Virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone 15 N spin relaxation and 15 N, 1 H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate.
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functional dynamics in Chlorella Virus dna ligase
Biophysical Journal, 2009Co-Authors: Andrea Piserchio, Stewart Shuman, Pravin A Nair, Ranajeet GhoseAbstract:DNA ligases specifically recognize and seal double stranded nicked DNA by catalyzing the formation of a phospho-diester bond between the 3′OH and 5′ phosphate termini. In physiological conditions these ubiquitous enzymes are essential for DNA replication, repair and recombination, while in tumor cells they can play a critical role in apoptosis resistance.Chlorella Virus ligase is a pluripotent ATP-dependent ligase composed by two domains, a N-terminal nucleotidyltransferase domain, hosting the catalytic site, and a C-terminal OB-domain, both of which participate to DNA binding. A number of crystallographic studies have elucidated important structural details of the nick-sealing process. DNA binding, in particular, appears to require a large reorientation of the two domains, as well as relevant structural rearrangements localized mainly in the N-terminal region (1). Indeed this protein appears to be a highly dynamic system whose internal motions are closely linked to both the DNA recognition and to the catalytic process. So far, however, the actual nature of these motions is still largely unknown, not only for Chlorella Virus ligase, but also for the entire protein family.We therefore tried to close this gap by undertaking the analysis of the dynamic properties of Chlorella Virus liagase by solution NMR spectroscopy.Reference:(1) Nair P.A., Nandakumar J., Smith P., Odell M., Lima C.D., Shuman S. (2008) Nat Struct Mol Biol. 14, 770-8.
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sequence specific 1hn 13c and 15n backbone resonance assignments of the 34 kda paramecium bursaria Chlorella Virus 1 pbcv1 dna ligase
Biomolecular Nmr Assignments, 2009Co-Authors: Andrea Piserchio, Stewart Shuman, Pravin A Nair, Ranajeet GhoseAbstract:Chlorella Virus DNA ligase (ChVLig) is a minimal (298-amino acid) pluripotent ATP-dependent ligase composed of three structural modules—a nucleotidyltransferase domain, an OB domain, and a β-hairpin latch—that forms a circumferential clamp around nicked DNA. ChVLig provides an instructive model to understand the chemical and conformational steps of nick repair. Here we report the assignment of backbone 13C, 15N, 1HN resonances of this 34.2 kDa protein, the first for a DNA ligase in full-length form.
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assignments of the 34 kda paramecium bursaria Chlorella Virus 1 pbcv1 dna ligase
2009Co-Authors: Andrea Piserchio, Stewart Shuman, Pravin A Nair, Ranajeet GhoseAbstract:Chlorella Virus DNA ligase (ChVLig) is a minimal (298-amino acid) pluripotent ATP-dependent ligase composed of three structural modules—a nucleo- tidyltransferase domain, an OB domain, and a b-hairpin latch—that forms a circumferential clamp around nicked DNA. ChVLig provides an instructive model to understand the chemical and conformational steps of nick repair. Here we report the assignment of backbone 13 C, 15 N, 1 H N res- onances of this 34.2 kDa protein, the first for a DNA ligase in full-length form.