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atomic force microscopy investigation of a chlorella virus, pbcv-1.a virus pbcv-1, which infects certain fresh water algae and has been shown by transmission and cryo-electron microscopy to exist as a triskaidecahedron, was imaged using atomic force microscopy (afm). from afm the particles have diameters of about 190nm and the overall structure is in all important respects consistent with existing models. the surface lattice of the virion is composed of trimeric capsid proteins distributed according to p3 symmetry to create a honeycomb arrangement of raised edg ...200515721579
a novel potassium channel encoded by ectocarpus siliculosus virus.kcv, the first identified viral potassium channel encoded by the green algae paramecium bursaria chlorella virus (pbcv-1), conducted k(+) selective currents when expressed in heterologous systems. this k(+) channel was proposed to be important for pbcv-1 infection and replication. in the present study, we identified and functionally characterized a novel k(+) channel kesv, encoded by ectocarpus siliculosus virus that infects filamentous marine brown algae. kesv encodes a protein of 124 amino aci ...200515607752
chlorella viruses.chlorella viruses or chloroviruses are large, icosahedral, plaque-forming, double-stranded-dna-containing viruses that replicate in certain strains of the unicellular green alga chlorella. dna sequence analysis of the 330-kbp genome of paramecium bursaria chlorella virus 1 (pbcv-1), the prototype of this virus family (phycodnaviridae), predict approximately 366 protein-encoding genes and 11 trna genes. the predicted gene products of approximately 50% of these genes resemble proteins of known fun ...200616877063
virion-associated restriction endonucleases of chloroviruses.chloroviruses are large, double-stranded-dna, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae. the prototype of the genus is paramecium bursaria chlorella virus 1 (pbcv-1). chlorovirus genomes contain various amounts of methylated nucleotides due to virus-encoded dna methyltransferases (mtases); about 25% of the mtases are associated with companion dna site-specific (restriction) endonucleases (reases). these enzymes constitute virally encoded restriction-modific ...200616873267
catalytic mechanism and structure of viral flavin-dependent thymidylate synthase thyx.by using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase thyx from paramecium bursaria chlorella virus-1 (pbcv-1). site-directed mutagenesis experiments have identified several residues implicated in either nadph oxidation or deprotonation activity of pbcv-1 thyx. chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (his53) crucial for nadph ...200616707489
structural insights of the specificity and catalysis of a viral histone h3 lysine 27 methyltransferase.set domain lysine methyltransferases are known to catalyze site and state-specific methylation of lysine residues in histones that is fundamental in epigenetic regulation of gene activation and silencing in eukaryotic organisms. here we report the three-dimensional solution structure of the set domain histone lysine methyltransferase (vset) from paramecium bursaria chlorella virus 1 bound to cofactor s-adenosyl-l-homocysteine and a histone h3 peptide containing mono-methylated lysine 27. the dim ...200616603186
structure of a197 from sulfolobus turreted icosahedral virus: a crenarchaeal viral glycosyltransferase exhibiting the gt-a fold.sulfolobus turreted icosahedral virus (stiv) was the first icosahedral virus characterized from an archaeal host. it infects sulfolobus species that thrive in the acidic hot springs (ph 2.9 to 3.9 and 72 to 92 degrees c) of yellowstone national park. the overall capsid architecture and the structure of its major capsid protein are very similar to those of the bacteriophage prd1 and eukaryotic viruses paramecium bursaria chlorella virus 1 and adenovirus, suggesting a viral lineage that predates t ...200616840342
ability of viral topoisomerase ii to discern the handedness of supercoiled dna: bimodal recognition of dna geometry by type ii enzymes.previous studies with human and bacterial topoisomerases suggest that the type ii enzyme utilizes two distinct mechanisms to recognize the handedness of dna supercoils. it has been proposed that the ability of some type ii enzymes, such as human topoisomerase iialpha and escherichia coli topoisomerase iv, to distinguish supercoil geometry during dna relaxation is mediated by elements in the variable c-terminal domain of the protein. in contrast, the ability of human topoisomerase iialpha and top ...200616981727
chloroviruses encode a bifunctional dcmp-dctp deaminase that produces two key intermediates in dttp formation.the chlorovirus pbcv-1, like many large double-stranded dna-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis. this report describes the characterization of the pbcv-1 dcmp deaminase, which produces dump, a key intermediate in the synthesis of dttp. as predicted, the recombinant protein has dcmp deaminase activity that is activated by dctp and inhibited by dttp. unexpectedly, however, the viral enzyme also has dctp deaminase activity, pr ...200717475641
structure and function of a chlorella virus-encoded glycosyltransferase.paramecium bursaria chlorella virus-1 encodes at least five putative glycosyltransferases that are probably involved in the synthesis of the glycan components of the viral major capsid protein. the 1.6 a crystal structure of one of these glycosyltransferases (a64r) has a mixed alpha/beta fold containing a central, six-stranded beta sheet flanked by alpha helices. crystal structures of a64r, complexed with udp, cmp, or gdp, established that only udp bound to a64r in the presence of mn(2+), consis ...200717850743
sequence and annotation of the 288-kb atcv-1 virus that infects an endosymbiotic chlorella strain of the heliozoon acanthocystis turfacea.acanthocystis turfacea chlorella virus (atcv-1), a prospective member of the family phycodnaviridae, genus chlorovirus, infects a unicellular, eukaryotic, chlorella-like green alga, chlorella sag 3.83, that is a symbiont in the heliozoon a. turfacea. the 288,047-bp atcv-1 genome is the first virus to be sequenced that infects chlorella sag 3.83. atcv-1 contains 329 putative protein-encoding and 11 trna-encoding genes. the protein-encoding genes are almost evenly distributed on both strands and i ...200717276475
chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway.two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (aih) and n-carbamoylputrescine amidohydrolase (cpa) were cloned from the chloroviruses pbcv-1, ny-2a and mt325. they were expressed in escherichia coli to form c-terminal (his)6-tagged proteins and the recombinant proteins were purified by ni2+-binding affinity chromatography. the biochemical properties of the two enzymes are similar to aih and cpa enzymes from arabidopsis thaliana and pseudomonas aeruginosa. ...200717101165
sequence and annotation of the 369-kb ny-2a and the 345-kb ar158 viruses that infect chlorella nc64a.viruses ny-2a and ar158, members of the family phycodnaviridae, genus chlorovirus, infect the fresh water, unicellular, eukaryotic, chlorella-like green alga, chlorella nc64a. the 368,683-bp genome of ny-2a and the 344,690-bp genome of ar158 are the two largest chlorella virus genomes sequenced to date; ny-2a contains 404 putative protein-encoding and 7 trna-encoding genes and ar158 contains 360 putative protein-encoding and 6 trna-encoding genes. the protein-encoding genes are almost evenly dis ...200717027058
sequence and annotation of the 314-kb mt325 and the 321-kb fr483 viruses that infect chlorella pbi.viruses mt325 and fr483, members of the family phycodnaviridae, genus chlorovirus, infect the fresh water, unicellular, eukaryotic, chlorella-like green alga, chlorella pbi. the 314,335-bp genome of mt325 and the 321,240-bp genome of fr483 are the first viruses that infect chlorella pbi to have their genomes sequenced and annotated. furthermore, these genomes are the two smallest chlorella virus genomes sequenced to date, mt325 has 331 putative protein-encoding and 10 trna-encoding genes and fr4 ...200717023017
kinetic and thermodynamic characterization of the rna guanylyltransferase reaction.an rna guanylyltransferase activity is involved in the synthesis of the cap structure found at the 5' end of eukaryotic mrnas. the rna guanylyltransferase activity is a two-step ping-pong reaction in which the enzyme first reacts with gtp to produce the enzyme-gmp covalent intermediate with the concomitant release of pyrophosphate. in the second step of the reaction, the gmp moiety is then transferred to a diphosphorylated rna. both reactions were previously shown to be reversible. in this study ...200818298088
chlorella viruses evoke a rapid release of k+ from host cells during the early phase of infection.infection of chlorella nc64a cells by pbcv-1 produces a rapid depolarization of the host probably by incorporation of a viral-encoded k(+) channel (kcv) into the host membrane. to examine the effect of an elevated conductance, we monitored the virus-stimulated efflux of k(+) from the chlorella cells. the results indicate that all 8 chlorella viruses tested evoked a host specific k(+) efflux with a concomitant decrease in the intracellular k(+). this k(+) efflux is partially reduced by blockers o ...200818045641
chlorovirus-mediated membrane depolarization of chlorella alters secondary active transport of solutes.paramecium bursaria chlorella virus 1 (pbcv-1) is the prototype of a family of large, double-stranded dna, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus chlorovirus. pbcv-1 infection results in rapid host membrane depolarization and potassium ion release. one interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (kcv) that are considered responsible for the host membrane depolarizat ...200818842725
epigenetic transcriptional repression of cellular genes by a viral set protein.viruses recruit host proteins to secure viral genome maintenance and replication. however, whether they modify host histones directly to interfere with chromatin-based transcription is unknown. here we report that paramecium bursaria chlorella virus 1 (pbcv-1) encodes a functional set domain histone lys methyltransferase (hkmtase) termed vset, which is linked to rapid inhibition of host transcription after viral infection. we show that vset is packaged in the pbcv-1 virion, and that it contains ...200819160493
differential role of nadp+ and nadph in the activity and structure of gdp-d-mannose 4,6-dehydratase from two chlorella viruses.gdp-d-mannose 4,6-dehydratase (gmd) is a key enzyme involved in the synthesis of 6-deoxyhexoses in prokaryotes and eukaryotes. paramecium bursaria chlorella virus-1 (pbcv-1) encodes a functional gmd, which is unique among characterized gmds because it also has a strong stereospecific nadph-dependent reductase activity leading to gdp-d-rhamnose formation (tonetti, m., zanardi, d., gurnon, j., fruscione, f., armirotti, a., damonte, g., sturla, l., de flora, a., and van etten, j.l. (2003) j. biol. ...200817974560
cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage l5.the genomes of mycobacteriophages of the l5 family, which includes the lytic phage d29, contain several genes putatively linked to nucleotide-metabolizing functions. two such genes, 48 and 50, encoding thymidylate synthase and ribonucleotide reductase (rnr), respectively, were overexpressed in escherichia coli and the recombinant proteins were biochemically characterized. it was established that gp50 was a class ii rnr having properties similar to that of the corresponding enzyme from lactobacil ...200818248423
phylogenetic analysis of members of the phycodnaviridae virus family, using amplified fragments of the major capsid protein gene.algal viruses are considered ecologically important by affecting host population dynamics and nutrient flow in aquatic food webs. members of the family phycodnaviridae are also interesting due to their extraordinary genome size. few algal viruses in the phycodnaviridae family have been sequenced, and those that have been have few genes in common and low gene homology. it has hence been difficult to design general pcr primers that allow further studies of their ecology and diversity. in this stud ...200818359826
[identification of chlorella viruses in paramecium bursaria clones by pulse-field electrophoresis].the ciliates paramecium bursaria contain endosymbiotic green algae chlorella spp. in their cytoplasm. the algae isolated from p. bursaria are sensitive to large dna-containing viruses of the family phycodnaviridae. the type virus of this family is pbcv-1 (paramecium bursaria chlorella virus). investigation of the total dna of p. bursaria clones by pulse-field electrophoresis (pep) revealed a pronounced band on pep profiles of some p. bursaria clones; the band was formed by dna molecules of appro ...200819004349
the capsid proteins of a large, icosahedral dsdna virus.chilo iridescent virus (civ) is a large (approximately 1850 a diameter) insect virus with an icosahedral, t=147 capsid, a double-stranded dna (dsdna) genome, and an internal lipid membrane. the structure of civ was determined to 13 a resolution by means of cryoelectron microscopy (cryoem) and three-dimensional image reconstruction. a homology model of p50, the civ major capsid protein (mcp), was built based on its amino acid sequence and the structure of the homologous paramecium bursaria chlore ...200919027752
chlorella virus atcv-1 encodes a functional potassium channel of 82 amino acids.chlorella virus pbcv-1 (paramecium bursaria chlorella virus-1) encodes the smallest protein (94 amino acids, named kcv) previously known to form a functional k+ channel in heterologous systems. in this paper, we characterize another chlorella virus encoded k+ channel protein (82 amino acids, named atcv-1 kcv) that forms a functional channel in xenopus oocytes and rescues saccharomyces cerevisiae mutants that lack endogenous k+ uptake systems. compared with the larger pbcv-1 kcv, atcv-1 kcv lacks ...200919267691
characterization of a monothiol glutaredoxin encoded by chlorella virus pbcv-1.annotation of the 330-kb chlorella virus pbcv-1 genome identified a 237 nucleotide gene (a438l) that codes for a protein with approximately 35% amino acid identity to glutaredoxins (grx) found in other organisms. the pbcv-1 protein resembles classical grxs in both size (9 kda) and location of the active site (n-terminus). however, the pbcv-1 grx is unusual because it contains a monothiol active site (cpys) rather than the typical dithiol active site (cpyc). to examine this unique active site, fo ...200919697117
an icosahedral algal virus has a complex unique vertex decorated by a spike.paramecium bursaria chlorella virus-1 is an icosahedrally shaped, 1,900-a-diameter virus that infects unicellular eukaryotic green algae. a 5-fold symmetric, 3d reconstruction using cryoelectron microscopy images has now shown that the quasiicosahedral virus has a unique vertex, with a pocket on the inside and a spike structure on the outside of the capsid. the pocket might contain enzymes for use in the initial stages of infection. the unique vertex consists of virally coded proteins, some of w ...200919541619
the a312l 5'-utr of chlorella virus pbcv-1 is a translational enhancer in arabidopsis thaliana.pbcv-1 (paramecium bursaria chlorella virus) is a large double stranded dna virus that replicates in certain eukaryotic chlorella like green algae. the pbcv-1 a312l gene encodes a 33-kda protein whose function currently is unknown. the 5'-utr of the a312l mrna is 153 nucleotides, longer than the 5'-utr in any other pbcv-1 gene. the sequence 5'-aaac was repeated 17 times within 156bp 5' to the a312l gene start codon and this sequence was repeated 13 times continuously in the 5'-utr of the mrna. r ...200919118587
fast and slow gating are inherent properties of the pore module of the k+ channel kcv.kcv from the chlorella virus pbcv-1 is a viral protein that forms a tetrameric, functional k+ channel in heterologous systems. kcv can serve as a model system to study and manipulate basic properties of the k+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. we present a characterization of kcv properties at the single-channel level. in symmetric 100 mm k+, single-channel conductanc ...200919720961
sequence-specific 1h n, 13c, and 15n backbone resonance assignments of the 34 kda paramecium bursaria chlorella virus 1 (pbcv1) dna ligase.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 beta-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) resonances of this 34.2 kda protein, the first for a dna ligase in ...200919636951
revised mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain.acanthamoebae polyphaga mimivirus (apm) is the largest known dsdna virus. the viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. a capsid protein sequence, d13l, was deduced from the apm l425 coding gene and was shown to be the most abundant protein found within the viral particle. however this protein remained poorly characterised until now. a revised protein sequence deposited in a database suggested an additional n-termina ...200919432951
chlorella viruses prevent multiple infections by depolarizing the host membrane.previous experiments established that when the unicellular green alga chlorella nc64a is inoculated with two viruses, usually only one virus replicates in a single cell. that is, the viruses mutually exclude one another. in the current study, we explore the possibility that virus-induced host membrane depolarization, at least partially caused by a virus-encoded k(+) channel (kcv), is involved in this mutual exclusion. two chlorella viruses, pbcv-1 and ny-2a, were chosen for the study because (i) ...200919386783
initial events associated with virus pbcv-1 infection of chlorella nc64a.chlorella viruses (or chloroviruses) are very large, plaque-forming viruses. the viruses are multilayered structures containing a large double-stranded dna genome, a lipid bilayered membrane, and an outer icosahedral capsid shell. the viruses replicate in certain isolates of the coccal green alga, chlorella. sequence analysis of the 330-kbp genome of paramecium bursaria chlorella virus 1 (pbcv-1), the prototype of the virus family phycodnaviridae, reveals <365 protein-encoding genes and 11 trna ...201021152366
isolation of the phycodnavirus pbcv-1 by biological laser printing.the phycodnaviridae family of viruses is diverse genetically but similar morphologically. these viruses infect eukaryotic algal hosts from both fresh and marine waters, and are an important component of aqueous environments. they play important roles in the dynamics of algal blooms, nutrient cycling, algal community structure, and possibly gene transfer between organisms. as such, it is important to identify new viruses within the phycodnaviridae family. biological laser printing (biolp) was use ...201020399229
microarray analysis of paramecium bursaria chlorella virus 1 transcription.paramecium bursaria chlorella virus 1 (pbcv-1), a member of the family phycodnaviridae, is a large double-stranded dna, plaque-forming virus that infects the unicellular green alga chlorella sp. strain nc64a. the 330-kb pbcv-1 genome is predicted to encode 365 proteins and 11 trnas. to monitor global transcription during pbcv-1 replication, a microarray containing 50-mer probes to the pbcv-1 365 protein-encoding genes (cdss) was constructed. competitive hybridization experiments were conducted b ...201019828609
characterization of viruses infecting a eukaryotic chlorella-like green alga.nineteen plaque-forming viruses of the unicellular, eukaryotic chlorella-like green alga, strain nc64a, were isolated from various geographic regions in the united states and characterized. like the previously described virus, pbcv-1, all of the new viruses were large polyhedrons, sensitive to chloroform, and contained large dsdna genomes of ca. 300 kbp. all of the viral dnas contained 5-methyldeoxycytidine which varied from 0.1 to 47% of the deoxycytidine. in addition, 10 of the viral dnas cont ...20103006334
viral-encoded enzymes that target host chromatin functions.ever since their existence, there has been an everlasting arms race between viruses and their host cells. host cells have developed numerous strategies to silence viral gene expression whereas viruses always find their ways to overcome these obstacles. recent studies show that viruses have also evolved to take full advantage of existing cellular chromatin components to activate or repress its own genes when needed. while in most cases viruses encode certain proteins to recruit or inhibit cellula ...201019716451
tat-mediated delivery of a dna repair enzyme to skin cells rapidly initiates repair of uv-induced dna damage.uv light causes dna damage in skin cells, leading to more than one million cases of non-melanoma skin cancer diagnosed annually in the united states. although human cells possess a mechanism (nucleotide excision repair) to repair uv-induced dna damage, mutagenesis still occurs when dna is replicated before repair of these photoproducts. although human cells have all the enzymes necessary to complete an alternate repair pathway, base excision repair (ber), they lack a dna glycosylase that can ini ...201020927123
modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase.the repair of cis-syn cyclobutane pyrimidine dimers (cpds) can be initiated via the base excision repair (ber) pathway, utilizing pyrimidine dimer-specific dna glycosylase/lyase enzymes (pdgs). however, prior to incision at lesion sites, these enzymes bind to non-damaged dnas through charge-charge interactions. following initial binding to dna containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. if a subset of these lesions are in close proximity, cluster ...201121889915
viral proteins function as ion channels.viral ion channels are short membrane proteins with 50-120 amino acids and play an important role either in regulating virus replication, such as virus entry, assembly and release or modulating the electrochemical balance in the subcellular compartments of host cells. this review summarizes the recent advances in viral encoded ion channel proteins (or viroporins), including pbcv-1 kcv, influenza m2, hiv-1 vpu, hcv p7, picornavirus 2b, and coronavirus e and 3a. we focus on their function and mech ...201120478263
structures of giant icosahedral eukaryotic dsdna viruses.in the last twenty years, numerous giant, dsdna, icosahedral viruses have been discovered and assigned to the nucleocytoplasmic large dsdna virus (ncldv) clade. the major capsid proteins of these viruses consist of two consecutive jelly-roll domains, assembled into trimers, with pseudo 6-fold symmetry. the capsomers are assembled into arrays that have either p6 (as in paramecium bursaria chlorella virus-1) or p3 symmetry (as in mimivirus). most of the ncldv viruses have a membrane that separates ...201121909343
method for rapid isolation and analysis of algal virus dna.a quick method has been developed for isolating viral dna from small cultures of eukaryotic algae infected with pbcv-1 or similar viruses. the dna preparations are virtually free of contaminating host dna and are suitable substrates for restriction enzymes. 5 ml cultures yield 8-25 micrograms of viral dna. many plaque-purified isolates from environmental water samples can be analyzed in a day.20112836467
paramecium bursaria chlorella virus 1 proteome reveals novel architectural and regulatory features of a giant virus.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 chlo ...201222696644
phycodnavirus potassium ion channel proteins question the virus molecular piracy hypothesis.phycodnaviruses are large dsdna, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. among the viral gene products are the smallest proteins known to form functional k(+) channels. to determine if these viral k(+) channels are the product of molecular piracy from their hosts, we compared the sequences of the k(+) channel pore modules from seven phycodnaviruses to the k(+) channels from chlorella variabilis and ectocarpus siliculosus, whose genomes have rec ...201222685610
paramecium bursaria chlorella virus 1 encodes a polyamine acetyltransferase.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 i ...201222277659
chloroviruses: not your everyday plant virus.viruses infecting higher plants are among the smallest viruses known and typically have four to ten protein-encoding genes. by contrast, many viruses that infect algae (classified in the virus family phycodnaviridae) are among the largest viruses found to date and have up to 600 protein-encoding genes. this brief review focuses on one group of plaque-forming phycodnaviruses that infect unicellular chlorella-like green algae. the prototype chlorovirus pbcv-1 has more than 400 protein-encoding gen ...201222100667
towards defining the chloroviruses: a genomic journey through a genus of large dna viruses.giant viruses in the genus chlorovirus (family phycodnaviridae) infect eukaryotic green microalgae. the prototype member of the genus, paramecium bursaria chlorella virus 1, was sequenced more than 15 years ago, and to date there are only 6 fully sequenced chloroviruses in public databases. presented here are the draft genome sequences of 35 additional chloroviruses (287 - 348 kb/319 - 381 predicted protein encoding genes) collected across the globe; they infect one of three different green alga ...201323497343
prolonged synthesis of hyaluronan by chlorella cells infected with chloroviruses.hyaluronan (ha) synthesis by microalgal chlorella cells in combination with chloroviruses represents a unique eco-friendly process for converting solar energy and co2 into useful materials. however, at the final stage of viral infection, infected host cells are completely lysed, and thus ha should be harvested before cell lysis. in the current study, two methods were investigated to improve the yield of ha: (i) adopting slow-growing chlorovirus isolates and (ii) modification of the virus replica ...201323273909
enzymatic synthesis of rnas capped with nucleotide analogues reveals the molecular basis for substrate selectivity of rna capping enzyme: impacts on rna metabolism.rna cap binding proteins have evolved to specifically bind to the n7-methyl guanosine cap structure found at the 5' ends of eukaryotic mrnas. the specificity of rna capping enzymes towards gtp for the synthesis of this structure is therefore crucial for mrna metabolism. the fact that ribavirin triphosphate was described as a substrate of a viral rna capping enzyme, raised the possibility that rnas capped with nucleotide analogues could be generated in cellulo. owing to the fact that this prospec ...201324086504
a virus-encoded potassium ion channel is a structural protein in the chlorovirus paramecium bursaria chlorella virus 1 virion.most chloroviruses encode small k(+) channels, which are functional in electrophysiological assays. the experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral k(+) channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. this hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chl ...201323918407
structure of n-linked oligosaccharides attached to chlorovirus pbcv-1 major capsid protein reveals unusual class of complex n-glycans.the major capsid protein vp54 from the prototype chlorovirus paramecium bursaria chlorella virus 1 (pbcv-1) contains four asn-linked glycans. the structure of the four n-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, pbcv-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the ...201323918378
evaluation of higher plant virus resistance genes in the green alga, chlorella variabilis nc64a, during the early phase of infection with paramecium bursaria chlorella virus-1.with growing industrial interest in algae plus their critical roles in aquatic systems, the need to understand the effects of algal pathogens is increasing. we examined a model algal host-virus system, chlorella variabilis nc64a and virus, pbcv-1. c. variabilis encodes 375 homologs to genes involved in rna silencing and in response to virus infection in higher plants. illumina rna-seq data showed that 325 of these homologs were expressed in healthy and early pbcv-1 infected (≤60min) cells. for e ...201323701839
dynamic attachment of chlorovirus pbcv-1 to chlorella variabilis.chloroviruses infect their hosts by specifically binding to and degrading the cell wall of their algal hosts at the site of attachment, using an intrinsic digesting enzyme(s). chlorovirus pbcv-1 stored as a lysate survived longer than virus alone, suggesting virus attachment to cellular debris may be reversible. ghost cells (algal cells extracted with methanol) were used as a model to study reversibility of pbcv-1 attachment because ghost cells are as susceptible to attachment and wall digestion ...201425240455
global analysis of chlorella variabilis nc64a mrna profiles during the early phase of paramecium bursaria chlorella virus-1 infection.the pbcv-1/chlorella variabilis nc64a system is a model for studies on interactions between viruses and algae. here we present the first global analyses of algal host transcripts during the early stages of infection, prior to virus replication. during the course of the experiment stretching over 1 hour, about a third of the host genes displayed significant changes in normalized mrna abundance that either increased or decreased compared to uninfected levels. the population of genes with significa ...201424608695
chlorovirus pbcv-1 encodes an active copper-zinc superoxide dismutase.superoxide dismutases (sods) are metalloproteins that protect organisms from toxic reactive oxygen species by catalyzing the conversion of superoxide anion to hydrogen peroxide and molecular oxygen. chlorovirus pbcv-1 encodes a 187-amino-acid protein that resembles a cu-zn sod with all of the conserved amino acid residues for binding copper and zinc (named cvsod). cvsod has an internal met that results in a 165-amino-acid protein (named tcvsod). both cvsod and tcvsod recombinant proteins inhibit ...201425142578
substrate interaction dynamics and oxygen control in the active site of thymidylate synthase thyx.thymidylate synthase thyx, required for dna synthesis in many pathogenic bacteria, is considered a promising antimicrobial target. it binds fad and three substrates, producing dtmp (2'-deoxythymidine-5'-monophosphate) from dump (2'-deoxyuridine-5'-monophosphate). however, thyx proteins also act as nadph oxidase by reacting directly with o2. in the present study we investigated the dynamic interplay between the substrates and their role in competing with this wasteful and potentially harmful oxid ...201424422556
deep rna sequencing reveals hidden features and dynamics of early gene transcription in paramecium bursaria chlorella virus 1.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. signi ...201424608750
chlorovirus skp1-binding ankyrin repeat protein interplay and mimicry of cellular ubiquitin ligase machinery.the ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. members of the genus chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large dna viruses (ncldv). chloroviruses encode an skp1 homolog and ankyrin repeat (ank) proteins. several chlorovirus-encoded ank repeats contain c-terminal domains characteristic of cellular f-boxes or related ncldv chordopox pran ...201425253343
efficient dna ligation in dna-rna hybrid helices by chlorella virus dna ligase.single-stranded dna molecules (ssdna) annealed to an rna splint are notoriously poor substrates for dna ligases. herein we report the unexpectedly efficient ligation of rna-splinted dna by chlorella virus dna ligase (pbcv-1 dna ligase). pbcv-1 dna ligase ligated ssdna splinted by rna with kcat ≈ 8 x 10(-3) s(-1) and k(m) < 1 nm at 25 °c under conditions where t4 dna ligase produced only 5'-adenylylated dna with a 20-fold lower kcat and a k(m) ≈ 300 nm. the rate of ligation increased with additio ...201424203707
the autonomous glycosylation of large dna viruses.glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/golgi apparatus to add carbohydrates to their nascent glycoproteins. in recent years, a newly discovered group of eukaryotic viruses, belonging to the nucleo-cytoplasmic large dna virus (ncldv) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. starting from initial observati ...201526690138
noninvasive measurement of electrical events associated with a single chlorovirus infection of a microalgal cell.chlorovirus paramecium bursaria chlorella virus 1 (pbcv-1) contains a viral-encoded k(+) channel imbedded in its internal membrane, which triggers host plasma membrane depolarization during virus infection. this early stage of infection was monitored at high resolution by recording the cell membrane depolarization of a single chlorella cell during infection by a single pbcv-1 particle. the measurement was achieved by depositing the cells onto a network of one-dimensional necklaces of au nanopart ...201627139597
influence of iron-doped apatite nanoparticles on viral infection examined in bacterial versus algal systems.the centers for disease control and prevention have estimated that each year, two million people in the united states become infected with antibiotic-resistant bacteria, of which, approximately 23000 die as a direct result of these infections. phage therapy, or the treatment of bacterial infection by specific, antagonistic viruses, provides one alternative to traditional antibiotics. bacteriophages, or phages, are bacteria-specific viruses that possess biological traits that allow for not only t ...201627775532
thermal stability and binding energetics of thymidylate synthase thyx.the bacterial thymidylate synthase thyx is a multisubstrate flavoenzyme that takes part in the de novo synthesis of thymidylate in a variety of microorganisms. herein we study the effect of fad and dump binding on the thermal stability of wild type (wt) thyx from the mesophilic paramecium bursaria chlorella virus-1 (pbcv-1) and from the thermophilic bacterium thermotoga maritima (tmthyx), and from two variants of tmthyx, y91f and s88w, using differential scanning calorimetry. the energetics unde ...201627268384
virus-host interactions: insights from the replication cycle of the large paramecium bursaria chlorella virus.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 elect ...201626248343
efficient in situ detection of mrnas using the chlorella virus dna ligase for padlock probe ligation.padlock probes are single-stranded dna molecules that are circularized upon hybridization to their target sequence by a dna ligase. in the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. the hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mrnas due to ...201727879431
characterization of a new chlorovirus type with permissive and non-permissive features on phylogenetically related algal strains.a previous report indicated that prototype chlorovirus pbcv-1 replicated in two chlorella variabilis algal strains, nc64a and syngen 2-3, that are ex-endosymbionts isolated from the protozoan paramecium bursaria. surprisingly, plaque-forming viruses on syngen 2-3 lawns were often higher than on nc64a lawns from indigenous water samples. these differences led to the discovery of viruses that exclusively replicate in syngen 2-3 cells, named only syngen (osy) viruses. osy-ne5, the prototype virus f ...201727816636
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