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James L. Van Etten - One of the best experts on this subject based on the ideXlab platform.
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catalysis of Chlorovirus production by the foraging of bursaria truncatella on paramecia bursaria containing endosymbiotic algae
Microorganisms, 2021Co-Authors: Zeina Alameeli, David D Dunigan, Maitham M Alsammak, John P Delong, James L. Van EttenAbstract:Chloroviruses are large viruses that replicate in chlorella-like green algae and normally exist as mutualistic endosymbionts (referred to as zoochlorellae) in protists such as Paramecium bursaria. Chlorovirus populations rise and fall in indigenous waters through time; however, the factors involved in these virus fluctuations are still under investigation. Chloroviruses attach to the surface of P. bursaria but cannot infect their zoochlorellae hosts because the viruses cannot reach the zoochlorellae as long as they are in the symbiotic phase. Predators of P. bursaria, such as copepods and didinia, can bring Chloroviruses into contact with zoochlorellae by disrupting the paramecia, which results in an increase in virus titers in microcosm experiments. Here, we report that another predator of P. bursaria, Bursaria truncatella, can also increase Chlorovirus titers. After two days of foraging on P. bursaria, B. truncatella increased infectious Chlorovirus abundance about 20 times above the controls. Shorter term foraging (3 h) resulted in a small increase of Chlorovirus titers over the controls and more foraging generated more Chloroviruses. Considering that B. truncatella does not release viable zoochlorellae either during foraging or through fecal pellets, where zoochlorellae could be infected by Chlorovirus, we suggest a third pathway of predator virus catalysis. By engulfing the entire protist and digesting it slowly, virus replication can occur within the predator and some of the virus is passed out through a waste vacuole. These results provide additional support for the hypothesis that predators of P. bursaria are important drivers of Chlorovirus population sizes and dynamics.
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Chlorovirus pbcv 1 multidomain protein a111 114r has three glycosyltransferase functions involved in the synthesis of atypical n glycans
Viruses, 2021Co-Authors: Eric A. Noel, Garry A Duncan, Cristina De Castro, Immacolata Speciale, Anna Notaro, James L. Van EttenAbstract:The structures of the four N-linked glycans from the prototype Chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known Chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all Chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the Chloroviruses do not use the canonical host endoplasmic reticulum–Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes.
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pursuit of Chlorovirus genetic transformation and crispr cas9 mediated gene editing
PLOS ONE, 2021Co-Authors: Eric A. Noel, Donald P Weeks, James L. Van EttenAbstract:Genetic and molecular modifications of the large dsDNA Chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of Chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific Chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit Chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the Chlorovirus genome, and that further experimentation is warranted and worthwhile.
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smrt sequencing of paramecium bursaria chlorella virus 1 reveals diverse methylation stability in adenines targeted by restriction modification systems
Frontiers in Microbiology, 2020Co-Authors: James L. Van Etten, Samantha R. Coy, Eric R Gann, Spiridon E Papoulis, Michael Holder, Nadim J Ajami, Joseph F Petrosino, Erik R Zinser, Steven W. WilhelmAbstract:Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the Chlorovirus model system; where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype Chlorovirus, PBCV-1, using single-molecule real time (SMRT) sequencing (aka PacBio). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of Chloroviruses and their giant virus relatives.
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Recovery of infectious PBCV-1 frozen at various times after PBCV-1 infection of the C. variabilis NC64A host.
2019Co-Authors: Samantha R. Coy, James L. Van Etten, Alyssa N. Alsante, Steven W. WilhelmAbstract:Viability of Chlorovirus PBCV-1 was assayed by monitoring plaque formation of cell-associated viruses that were collected at different times during an active infection cycle of the NC64A host. Open circles denote replicate plaque titers, with the average represented by the solid line.
James L. Van Etten - One of the best experts on this subject based on the ideXlab platform.
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Size-dependent Catalysis of Chlorovirus Population Growth by A Messy Feeding Predator
Microbial Ecology, 2018Co-Authors: John P Delong, James L. Van Etten, Zeina Al-ameeli, Shelby Lyon, David D DuniganAbstract:Many Chloroviruses replicate in endosymbiotic zoochlorellae that are protected from infection by their symbiotic host. To reach the high virus concentrations that often occur in natural systems, a mechanism is needed to release zoochlorellae from their hosts. We demonstrate that the ciliate predator Didinium nasutum foraging on zoochlorellae-bearing Paramecium bursaria can release live zoochlorellae from the ruptured prey cell that can then be infected by Chloroviruses. The catalysis process is very effective, yielding roughly 95% of the theoretical infectious virus yield as determined by sonication of P. bursaria . Chlorovirus activation is more effective with smaller Didinia , as larger Didinia typically consume entire P. bursaria cells without rupturing them, precluding the release of zoochlorellae. We also show that the timing of Chlorovirus growth is tightly linked to the predator-prey cycle between Didinium and Paramecium , with the most rapid increase in Chloroviruses temporally linked to the peak foraging rate of Didinium , supporting the idea that predator-prey cycles can drive cycles of Chlorovirus abundance.
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Structure of the N-glycans from the Chlorovirus NE-JV-1
Antonie van Leeuwenhoek, 2017Co-Authors: Immacolata Speciale, Garry A Duncan, James L. Van Etten, Irina Agarkova, Cristina De CastroAbstract:Results from recent studies are breaking the paradigm that all viruses depend on their host machinery to glycosylate their proteins. Chloroviruses encode several genes involved in glycan biosynthesis and some of their capsid proteins are decorated with N-linked oligosaccharides with unique features. Here we describe the elucidation of the N-glycan structure of an unusual Chlorovirus, NE-JV-1, that belongs to the Pbi group. The host for NE-JV-1 is the zoochlorella Micractinium conductrix . Spectroscopic analyses established that this N-glycan consists of a core region that is conserved in all of the Chloroviruses. The one difference is that the residue 3OMe- l -rhamnose is acetylated at the O-2 position in a non-stoichiometric fashion.
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Three-year survey of abundance, prevalence and genetic diversity of Chlorovirus populations in a small urban lake
Archives of Virology, 2016Co-Authors: Cristian F. Quispe, David D Dunigan, Olivia Sonderman, Anya Seng, Brenna Rasmussen, Garrett Weber, Claire Mueller, James L. Van EttenAbstract:Inland water environments cover about 2.5 percent of our planet and harbor huge numbers of known and still unknown microorganisms. In this report, we examined water samples for the abundance, prevalence, and genetic diversity of a group of infectious viruses (Chloroviruses) that infect symbiotic chlorella-like green algae. Samples were collected on a weekly basis for a period of 24 to 36 months from a recreational freshwater lake in Lincoln, Nebraska, and assayed for infectious viruses by plaque assay. The numbers of infectious virus particles were both host- and site-dependent. The consistent fluctuations in numbers of viruses suggest their impact as key factors in shaping microbial community structures in the water surface. Even in low-viral-abundance months, infectious Chlorovirus populations were maintained, suggesting either that the viruses are very stable or that there is ongoing viral production in natural hosts.
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Expression of Chlorovirus MT325 aquaglyceroporin (aqpv1) in tobacco and its role in mitigating drought stress
Planta, 2014Co-Authors: Saadia Bihmidine, Mingxia Cao, Tala Awada, David D Dunigan, James L. Van Etten, Ming Kang, Tom E. ClementeAbstract:Main conclusionsA Chlorovirusaquaglyceroporin expressed in tobacco is localized to the plastid and plasma membranes. Transgenic events display improved response to water deficit. Necrosis in adult stage plants is observed. Aquaglyceroporins are a subclass of the water channel aquaporin proteins (AQPs) that transport glycerol along with other small molecules transcellular in addition to water. In the studies communicated herein, we analyzed the expression of the aquaglyceroporin gene designated, aqpv1, from Chlorovirus MT325, in tobacco (Nicotiana tabacum), along with phenotypic changes induced by aqpv1 expression in planta. Interestingly, aqpv1 expression under control of either a constitutive or a root-preferred promoter, triggered local lesion formation in older leaves, which progressed significantly after induction of flowering. Fusion of aqpv1 with GFP suggests that the protein localized to the plasmalemma, and potentially with plastid and endoplasmic reticulum membranes. Physiological characterizations of transgenic plants during juvenile stage growth were monitored for potential mitigation to water dry-down (i.e., drought) and recovery. Phenotypic analyses on drought mimic/recovery of juvenile transgenic plants that expressed a functional aqpv1 transgene had higher photosynthetic rates, stomatal conductance, and water use efficiency, along with maximum carboxylation and electron transport rates when compared to control plants. These physiological attributes permitted the juvenile aqpv1 transgenic plants to perform better under drought-mimicked conditions and hastened recovery following re-watering. This drought mitigation effect is linked to the ability of the transgenic plants to maintain cell turgor.
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Chloroviruses: Not Your Everyday Plant Virus
Trends in Plant Science, 2011Co-Authors: James L. Van Etten, David D DuniganAbstract: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 genes and 11 tRNA genes. About 40% of the PBCV-1 encoded proteins resemble proteins of known function including many that are completely unexpected for a virus. In many respects, Chlorovirus infection resembles bacterial infection by tailed bacteriophages.
Van Etten James L. - One of the best experts on this subject based on the ideXlab platform.
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Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans
DigitalCommons@University of Nebraska - Lincoln, 2021Co-Authors: Noel Eric, De Castro Cristina, Speciale Immacolata, Duncan, Garry A, Notaro Anna, Van Etten James L.Abstract:The structures of the four N-linked glycans from the prototype Chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known Chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of ge- netic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all Chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the Chloroviruses do not use the canonical host endoplasmic reticulum–Golgi glycosyla- tion pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes
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Identification of a Chlorovirus PBCV-1 Protein Involved in Degrading the Host Cell Wall during Virus Infection
DigitalCommons@University of Nebraska - Lincoln, 2021Co-Authors: Agarkova Irina, Dunigan, David D., Ghosh, Jayadri S., Quispe, Cristian F, Lane, Leslie C, Duncan, Garry A, Milrot Elad, Minsky Abraham, Esmael Ahmed, Van Etten James L.Abstract:Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561LD4, with cell wall degrading activity. An A561LD4 homolog was present in all 52 genomically sequenced Chloroviruses, infecting four different algal hosts. A561LD4 degraded the cell walls of all four Chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561LD4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561LD4 increased the specific infectivity of PBCV-1 from about 25–30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates
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Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae
DigitalCommons@University of Nebraska - Lincoln, 2020Co-Authors: Murry Carter, Agarkova Irina, Thiel Gerhard, Hertel Brigitte, Ghosh, Jayadri S., Fitzgerald, Fiona C., Carlson, Roger M., Kukovetz Kerri, Rauh Oliver, Van Etten James L.Abstract:Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of Chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG Chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane -helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG Chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels
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Diversity of tRNA Clusters in the Chloroviruses
DigitalCommons@University of Nebraska - Lincoln, 2020Co-Authors: Duncan, Garry A, Dunigan, David D, Ph. D., Van Etten James L.Abstract:Viruses rely on their host’s translation machinery for the synthesis of their own proteins. Problems belie viral translation when the host has a codon usage bias (CUB) that is different from an infecting virus due to differences in the GC content between the host and virus genomes. Here, we examine the hypothesis that Chloroviruses adapted to host CUB by acquisition and selection of tRNAs that at least partially favor their own CUB. The genomes of 41 Chloroviruses comprising three clades, each infecting a different algal host, have been sequenced, assembled and annotated. All 41 viruses not only encode tRNAs, but their tRNA genes are located in clusters. While differences were observed between clades and even within clades, seven tRNA genes were common to all three clades of Chloroviruses, including the tRNAArg gene, which was found in all 41 Chloroviruses. By comparing the codon usage of one Chlorovirus algal host, in which the genome has been sequenced and annotated (67% GC content), to that of two of its viruses (40% GC content), we found that the viruses were able to at least partially overcome the host’s CUB by encoding tRNAs that recognize AU-rich codons. Evidence presented herein supports the hypothesis that a Chlorovirus tRNA cluster was present in the most recent common ancestor (MRCA) prior to divergence into three clades. In addition, the MRCA encoded a putative isoleucine lysidine synthase (TilS) that remains in 39/41 Chloroviruses examined herein, suggesting a strong evolutionary pressure to retain the gene. TilS alters the anticodon of tRNA that normally recognizes AUG to then recognize AUA, a codon for isoleucine. This is advantageous to the Chloroviruses because the AUA codon is 12–13 times more common in the Chloroviruses than their host, further helping the Chloroviruses to overcome CUB. Among large DNA viruses infecting eukaryotes, the presence of tRNA genes and tRNA clusters appear to be most common in the Phycodnaviridae and, to a lesser extent, in the Mimiviridae
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Chloroviruses lure hosts through long-distance chemical signaling
DigitalCommons@University of Nebraska - Lincoln, 2019Co-Authors: Dunigan David, Agarkova Irina, Al-sammak, Maitham Ahmed, Al-ameeli Zeina, Delong John, Van Etten James L.Abstract:Chloroviruses exist in aquatic systems around the planet where they infect certain eukaryotic green algae that are mutualistic endosymbionts in a variety of protists and metazoans. Natural Chlorovirus populations are seasonally dynamic but the precise temporal changes in these populations and the mechanisms that underlie them have, heretofore, been unclear. We recently reported the novel concept that predator/prey-mediated virus activation regulates Chlorovirus population dynamics, and in the current manuscript demonstrate virus packaged chemotactic modulation of prey behavior. Viruses have not previously been reported to act as chemotactic/chemo-attractive agents. Rather, viruses as extracellular entities are generally viewed as non-metabolically active spore-like agents that await further infection events upon collisions with appropriate host cells. That a virus might actively contribute to its fate via chemotaxis and change the behavior of an organism independent of infection is unprecedented
David D Dunigan - One of the best experts on this subject based on the ideXlab platform.
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catalysis of Chlorovirus production by the foraging of bursaria truncatella on paramecia bursaria containing endosymbiotic algae
Microorganisms, 2021Co-Authors: Zeina Alameeli, David D Dunigan, Maitham M Alsammak, John P Delong, James L. Van EttenAbstract:Chloroviruses are large viruses that replicate in chlorella-like green algae and normally exist as mutualistic endosymbionts (referred to as zoochlorellae) in protists such as Paramecium bursaria. Chlorovirus populations rise and fall in indigenous waters through time; however, the factors involved in these virus fluctuations are still under investigation. Chloroviruses attach to the surface of P. bursaria but cannot infect their zoochlorellae hosts because the viruses cannot reach the zoochlorellae as long as they are in the symbiotic phase. Predators of P. bursaria, such as copepods and didinia, can bring Chloroviruses into contact with zoochlorellae by disrupting the paramecia, which results in an increase in virus titers in microcosm experiments. Here, we report that another predator of P. bursaria, Bursaria truncatella, can also increase Chlorovirus titers. After two days of foraging on P. bursaria, B. truncatella increased infectious Chlorovirus abundance about 20 times above the controls. Shorter term foraging (3 h) resulted in a small increase of Chlorovirus titers over the controls and more foraging generated more Chloroviruses. Considering that B. truncatella does not release viable zoochlorellae either during foraging or through fecal pellets, where zoochlorellae could be infected by Chlorovirus, we suggest a third pathway of predator virus catalysis. By engulfing the entire protist and digesting it slowly, virus replication can occur within the predator and some of the virus is passed out through a waste vacuole. These results provide additional support for the hypothesis that predators of P. bursaria are important drivers of Chlorovirus population sizes and dynamics.
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Size-dependent Catalysis of Chlorovirus Population Growth by A Messy Feeding Predator
Microbial Ecology, 2018Co-Authors: John P Delong, James L. Van Etten, Zeina Al-ameeli, Shelby Lyon, David D DuniganAbstract:Many Chloroviruses replicate in endosymbiotic zoochlorellae that are protected from infection by their symbiotic host. To reach the high virus concentrations that often occur in natural systems, a mechanism is needed to release zoochlorellae from their hosts. We demonstrate that the ciliate predator Didinium nasutum foraging on zoochlorellae-bearing Paramecium bursaria can release live zoochlorellae from the ruptured prey cell that can then be infected by Chloroviruses. The catalysis process is very effective, yielding roughly 95% of the theoretical infectious virus yield as determined by sonication of P. bursaria . Chlorovirus activation is more effective with smaller Didinia , as larger Didinia typically consume entire P. bursaria cells without rupturing them, precluding the release of zoochlorellae. We also show that the timing of Chlorovirus growth is tightly linked to the predator-prey cycle between Didinium and Paramecium , with the most rapid increase in Chloroviruses temporally linked to the peak foraging rate of Didinium , supporting the idea that predator-prey cycles can drive cycles of Chlorovirus abundance.
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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, James R. Gurnon, David D Dunigan, Irina V Agarkova, Michael Mozar, Giane Yanai-balser, 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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Three-year survey of abundance, prevalence and genetic diversity of Chlorovirus populations in a small urban lake
Archives of Virology, 2016Co-Authors: Cristian F. Quispe, David D Dunigan, Olivia Sonderman, Anya Seng, Brenna Rasmussen, Garrett Weber, Claire Mueller, James L. Van EttenAbstract:Inland water environments cover about 2.5 percent of our planet and harbor huge numbers of known and still unknown microorganisms. In this report, we examined water samples for the abundance, prevalence, and genetic diversity of a group of infectious viruses (Chloroviruses) that infect symbiotic chlorella-like green algae. Samples were collected on a weekly basis for a period of 24 to 36 months from a recreational freshwater lake in Lincoln, Nebraska, and assayed for infectious viruses by plaque assay. The numbers of infectious virus particles were both host- and site-dependent. The consistent fluctuations in numbers of viruses suggest their impact as key factors in shaping microbial community structures in the water surface. Even in low-viral-abundance months, infectious Chlorovirus populations were maintained, suggesting either that the viruses are very stable or that there is ongoing viral production in natural hosts.
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Chlorella cells and Chlorovirus Paramecium bursaria chlorella virus (PBCV-1).
2016Co-Authors: James L. Van Etten, David D DuniganAbstract:(A) Paramecium bursaria and its symbiotic chlorella cells. (B) Plaques formed by PBCV-1 on a lawn of Chlorella variabilis. (C) Five-fold averaged cryo-electron micrograph of PBCV-1 reveals a long narrow cylindrical spike structure at one vertex and fibers extending from one unique capsomer per trisymmetron. (D) PBCV-1 attached to the cell wall as viewed by the quick-freeze, deep etch procedure. Note the virions attached to the wall by fibers. (E) Surface view of the PBCV-1 spike structure and fibers. (F) Initial attachment of PBCV-1 to a C. variabilis cell wall. (G) Attachment of PBCV-1 to the algal wall and digestion of the wall at the point of attachment. This occurs within 1–3 minutes postinfection (PI) (H) Virion particles assemble in defined areas in the cytoplasm named virus assembly centers at ~4 hour PI. Note that both DNA containing (dark centers) and empty capsids. (I) A model depicting PBCV-1 assembly into infectious particles including generation of nuclei-derived cisternae decorated with ribosomes (red spheres), which serve as precursors (dark blue) for single bilayer viral membranes (light blue) in the viral assembly centers. (J) Localized lysis of cell plasma membrane and cell wall and release of progeny viruses at ~8 hours PI. This figure is modified with permission: Fig 1A–H and J is modified from reference 2 with permission and Fig I is modified from reference 18 with permission.
Ming Kang - One of the best experts on this subject based on the ideXlab platform.
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Chlorovirus pbcv 1 encodes an active copper zinc superoxide dismutase
Journal of Virology, 2014Co-Authors: Garry A Duncan, Ming Kang, George A. Oyler, Charles A Kuszynski, Jiayin Zheng, Donald F Becker, James L. Van EttenAbstract: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 inhibited nitroblue tetrazolium reduction of superoxide anion generated in a xanthine-xanthine oxidase system in solution. tcvSOD was chosen for further characterization because it was easier to produce. Recombinant tcvSOD also inhibited a riboflavin photochemical reduction system in a polyacrylamide gel assay, which was blocked by the Cu-Zn SOD inhibitor cyanide but not by azide, which inhibits Fe and Mn SODs. A kcat/Km value for cvSOD was determined by stop-flow spectrophotometry as 1.28 × 108 M−1 s−1, suggesting that cvSOD-catalyzed O2− dismutation was not a diffusion controlled encounter. The cvsod gene was expressed as a late gene, and cvSOD activity was detected in purified virions. Superoxide accumulated rapidly during virus infection, and circumstantial evidence indicates that cvSOD aids its decomposition to benefit virus replication. Cu-Zn SOD homologs have been described to occur in 3 other families of large DNA viruses, poxviruses, baculoviruses, and mimiviruses, which group as a clade. Interestingly, cvSOD does not group in the same clade as the other virus SODs but instead groups in an expanded clade that includes Cu-Zn SODs from many cellular organisms. IMPORTANCE Virus infection often leads to an increase in toxic reactive oxygen species in the host, which can be detrimental to virus replication. Viruses have developed various ways to overcome this barrier. As reported in this article, the Chloroviruses often encode and package a functional Cu-Zn superoxide dismutase in the virion that presumably lowers the concentration of reactive oxygen induced early during virus infection.
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Chlorovirus Skp1-Binding Ankyrin Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery
Journal of virology, 2014Co-Authors: Eric A. Noel, Ming Kang, James L. Van Etten, Jiric Adamec, George A. OylerAbstract: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 PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic Chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced Chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three Chlorovirus types. Collectively, our results indicate that Chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. IMPORTANCE Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in the manuscript, the large Chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how Chloroviruses manipulate their host algal ubiquitination system will provide insights toward viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also further understanding of the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as Chloroviruses.
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Expression of Chlorovirus MT325 aquaglyceroporin (aqpv1) in tobacco and its role in mitigating drought stress
Planta, 2014Co-Authors: Saadia Bihmidine, Mingxia Cao, Tala Awada, David D Dunigan, James L. Van Etten, Ming Kang, Tom E. ClementeAbstract:Main conclusionsA Chlorovirusaquaglyceroporin expressed in tobacco is localized to the plastid and plasma membranes. Transgenic events display improved response to water deficit. Necrosis in adult stage plants is observed. Aquaglyceroporins are a subclass of the water channel aquaporin proteins (AQPs) that transport glycerol along with other small molecules transcellular in addition to water. In the studies communicated herein, we analyzed the expression of the aquaglyceroporin gene designated, aqpv1, from Chlorovirus MT325, in tobacco (Nicotiana tabacum), along with phenotypic changes induced by aqpv1 expression in planta. Interestingly, aqpv1 expression under control of either a constitutive or a root-preferred promoter, triggered local lesion formation in older leaves, which progressed significantly after induction of flowering. Fusion of aqpv1 with GFP suggests that the protein localized to the plasmalemma, and potentially with plastid and endoplasmic reticulum membranes. Physiological characterizations of transgenic plants during juvenile stage growth were monitored for potential mitigation to water dry-down (i.e., drought) and recovery. Phenotypic analyses on drought mimic/recovery of juvenile transgenic plants that expressed a functional aqpv1 transgene had higher photosynthetic rates, stomatal conductance, and water use efficiency, along with maximum carboxylation and electron transport rates when compared to control plants. These physiological attributes permitted the juvenile aqpv1 transgenic plants to perform better under drought-mimicked conditions and hastened recovery following re-watering. This drought mitigation effect is linked to the ability of the transgenic plants to maintain cell turgor.
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RESEARCH ARTICLE Open Access Towards defining the Chloroviruses: a genomic journey through a genus of large DNA viruses
2013Co-Authors: Adrien Jeanniard, James R. Gurnon, David D Dunigan, Ming Kang, Irina V Agarkova, Jason Vitek, Garry Duncan, William O Mcclung, Megan Larsen, Jean-michel ClaverieAbstract:Background: 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 algal species. These new data allowed us to analyze the genomic landscape of 41 Chloroviruses, which revealed some remarkable features about these viruses. Results: Genome colinearity, nucleotide conservation and phylogenetic affinity were limited to Chloroviruses infecting the same host, confirming the validity of the three previously known subgenera. Clues for the existence of a fourth new subgenus indicate that the boundaries of Chlorovirus diversity are not completely determined. Comparison of the Chlorovirus phylogeny with that of the algal hosts indicates that Chloroviruses have changed hosts in their evolutionary history. Reconstruction of the ancestral genome suggests that the last common Chlorovirus ancestor had a slightly more diverse protein repertoire than modern Chloroviruses. However, more than half of the defined Chlorovirus gene families have a potential recent origin (after Chlorovirus divergence), among which a portion shows compositional evidence for horizontal gene transfer. Only a few of the putative acquire
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towards defining the Chloroviruses a genomic journey through a genus of large dna viruses
BMC Genomics, 2013Co-Authors: Adrien Jeanniard, James R. Gurnon, Garry A Duncan, David D Dunigan, Ming Kang, Irina V Agarkova, Jason Vitek, William O Mcclung, Megan L LarsenAbstract: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 algal species. These new data allowed us to analyze the genomic landscape of 41 Chloroviruses, which revealed some remarkable features about these viruses. Genome colinearity, nucleotide conservation and phylogenetic affinity were limited to Chloroviruses infecting the same host, confirming the validity of the three previously known subgenera. Clues for the existence of a fourth new subgenus indicate that the boundaries of Chlorovirus diversity are not completely determined. Comparison of the Chlorovirus phylogeny with that of the algal hosts indicates that Chloroviruses have changed hosts in their evolutionary history. Reconstruction of the ancestral genome suggests that the last common Chlorovirus ancestor had a slightly more diverse protein repertoire than modern Chloroviruses. However, more than half of the defined Chlorovirus gene families have a potential recent origin (after Chlorovirus divergence), among which a portion shows compositional evidence for horizontal gene transfer. Only a few of the putative acquired proteins had close homologs in databases raising the question of the true donor organism(s). Phylogenomic analysis identified only seven proteins whose genes were potentially exchanged between the algal host and the Chloroviruses. The present evaluation of the genomic evolution pattern suggests that Chloroviruses differ from that described in the related Poxviridae and Mimiviridae. Our study shows that the fixation of algal host genes has been anecdotal in the evolutionary history of Chloroviruses. We finally discuss the incongruence between compositional evidence of horizontal gene transfer and lack of close relative sequences in the databases, which suggests that the recently acquired genes originate from a still largely un-sequenced reservoir of genomes, possibly other unknown viruses that infect the same hosts.