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At least 145 records · Page 8Linked to original sources
PubMed1997

Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1.

We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step. These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.

Adenosine Monophosphate↗
PubMed2000

A hypothesis for DNA viruses as the origin of eukaryotic replication proteins.

The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.

Amino Acid Sequence↗
PubMed2001

RNA triphosphatase component of the mRNA capping apparatus of Paramecium bursaria Chlorella virus 1.

Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5' diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the gamma-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.

Acid Anhydride Hydrolases↗
PubMed2005

The marine algal virus PpV01 has an icosahedral capsid with T=219 quasisymmetry.

Phaeocystis pouchetii virus (PpV01) infects and lyses the haptophyte Phaeocystis pouchetii (Hariot) Lagerheim and was first isolated from Norwegian coastal waters. We have used electron cryomicroscopy and three-dimensional image reconstruction methods to examine the native morphology of PpV01 at a resolution of 3 nm. The icosahedral capsid of PpV01 has a maximum diameter of 220 nm and is composed of 2,192 capsomers arranged with T=219 quasisymmetry. One specific capsomer in each asymmetric unit contains a fiber-like protrusion. Density attributed to the presence of a lipid membrane appears just below (inside) the capsid. PpV01 is the largest icosahedral virus whose capsid structure has been determined in three dimensions from images of vitrified samples. Striking similarities in the structures of PpV01 and a number of other large double-stranded DNA viruses are consistent with a growing body of evidence that they share a common evolutionary origin.

Capsid↗
PubMed2005

Chlorella virus-encoded deoxyuridine triphosphatases exhibit different temperature optima.

A putative deoxyuridine triphosphatase (dUTPase) gene from chlorella virus PBCV-1 was cloned, and the recombinant protein was expressed in Escherichia coli. The recombinant protein has dUTPase activity and requires Mg(2+) for optimal activity, while it retains some activity in the presence of other divalent cations. Kinetic studies of the enzyme revealed a K(m) of 11.7 microM, a turnover k(cat) of 6.8 s(-1), and a catalytic efficiency of k(cat)/K(m) = 5.8 x 10(5) M(-1) s(-1). dUTPase genes were cloned and expressed from two other chlorella viruses IL-3A and SH-6A. The two dUTPases have similar properties to PBCV-1 dUTPase except that IL-3A dUTPase has a lower temperature optimum (37 degrees C) than PBCV-1 dUTPase (50 degrees C). The IL-3A dUTPase differs from the PBCV-1 enzyme by nine amino acids, including two amino acid substitutions, Glu81-->Ser81 and Thr84-->Arg84, in the highly conserved motif III of the proteins. To investigate the difference in temperature optima between the two enzymes, homology modeling and docking simulations were conducted. The results of the simulation and comparisons of amino acid sequence suggest that adjacent amino acids are important in the temperature optima. To confirm this suggestion, three site-directed amino acid substitutions were made in the IL-3A enzyme: Thr84-->Arg84, Glu81-->Ser81, and Glu81-->Ser81 plus Thr84-->Arg84. The single substitutions affected the optimal temperature for enzyme activity. The temperature optimum increased from 37 to 55 degrees C for the enzyme containing the two amino acid substitutions. We postulate that the change in temperature optimum is due to reduction in charge and balkiness in the active cavity that allows more movement of the ligand and protein before the enzyme and substrate complex is formed.

Amino Acid Motifs↗
PubMed2006

Potassium ion channels of Chlorella viruses cause rapid depolarization of host cells during infection.

Previous studies have established that chlorella viruses encode K(+) channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs(+) to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of Chlorella NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [t(1/2)], about 9 min) and slowest with virus NY-2A (t(1/2), about 12 min). Cs(+) inhibited membrane depolarization only in viruses that encode a Cs(+)-sensitive K(+) channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of Chlorella cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K(+) conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host.

Cell Membrane↗
PubMed2003

Unusual life style of giant chlorella viruses.

Paramecium bursaria chlorella virus (PBCV-1) is the prototype of a family of large, icosahedral, plaque-forming, dsDNA viruses that replicate in certain unicellular, eukaryotic chlorella-like green algae. Its 330-kb genome contains approximately 373 protein-encoding genes and 11 tRNA genes. The predicted gene products of approximately 50% of these genes resemble proteins of known function, including many that are unexpected for a virus, e.g., ornithine decarboxylase, hyaluronan synthase, GDP-D-mannose 4,6 dehydratase, and a potassium ion channel protein. In addition to their large genome size, the chlorella viruses have other features that distinguish them from most viruses. These features include: (a) The viruses encode multiple DNA methyltransferases and DNA site-specific endonucleases. (b) The viruses encode at least some, if not all, of the enzymes required to glycosylate their proteins. (c) PBCV-1 has at least three types of introns, a self-splicing intron in a transcription factor-like gene, a spliceosomal processed intron in its DNA polymerase gene, and a small intron in one of its tRNA genes. (d) Many chlorella virus-encoded proteins are either the smallest or among the smallest proteins of their class. (e) Accumulating evidence indicates that the chlorella viruses have a very long evolutionary history.

Chlorella↗
PubMed1999

Giant viruses infecting algae.

Paramecium bursaria chlorella virus (PBCV-1) is the prototype of a family of large, icosahedral, plaque-forming, double-stranded-DNA-containing viruses that replicate in certain unicellular, eukaryotic chlorella-like green algae. DNA sequence analysis of its 330, 742-bp genome leads to the prediction that this phycodnavirus has 376 protein-encoding genes and 10 transfer RNA genes. The predicted gene products of approximately 40% of these genes resemble proteins of known function. The chlorella viruses have other features that distinguish them from most viruses, in addition to their large genome size. These features include the following: (a) The viruses encode multiple DNA methyltransferases and DNA site-specific endonucleases; (b) PBCV-1 encodes at least part, if not the entire machinery to glycosylate its proteins; (c) PBCV-1 has at least two types of introns--a self-splicing intron in a transcription factor-like gene and a splicesomal processed type of intron in its DNA polymerase gene. Unlike the chlorella viruses, large double-stranded-DNA-containing viruses that infect marine, filamentous brown algae have a circular genome and a lysogenic phase in their life cycle.

Biological Evolution↗
PubMed2005

Giant viruses in the oceans: the 4th Algal Virus Workshop.

Giant double-stranded DNA viruses (such as record breaking Acanthamoeba polyphaga Mimivirus), with particle sizes of 0.2 to 0.6 microm, genomes of 300 kbp to 1.200 kbp, and commensurate complex gene contents, constitute an evolutionary mystery. They challenge the common vision of viruses, traditionally seen as highly streamlined genomes optimally fitted to the smallest possible--filterable--package. Such giant viruses are now discovered in increasing numbers through the systematic sampling of ocean waters as well as freshwater aquatic environments, where they play a significant role in controlling phyto- and bacterio- plankton populations. The 4th Algal Virus Workshop showed that the study of these ecologically important viruses is now massively entering the genomic era, promising a better understanding of their diversity and, hopefully, some insights on their origin and the evolutionary forces that shaped their genomes.

DNA Viruses↗
PubMed2006

Genome comparison of two Coccolithoviruses.

BACKGROUND: The Coccolithoviridae is a recently discovered family of viruses that infect the marine coccolithophorid Emiliania huxleyi. Following on from the sequencing of the type strain EhV-86, we have sequenced a second strain, EhV-163. RESULTS: We have sequenced approximately 80% of the EhV-163 genome, equating to more than 200 full length CDSs. Conserved and variable CDSs and a gene replacement have been identified in the EhV-86 and EhV-163 genomes. CONCLUSION: The sequencing of EhV-163 has provided a wealth of information which will aid the re-annotating of the EhV-86 genome and identified a gene insertion in EhV-163.

Base Sequence↗
PubMedDate not supplied

Chlorella viruses encode multiple DNA methyltransferases.

The >320 kb dsDNA genomes of 16 viruses which infect Chlorella strain NC64A and 5 viruses infecting Chlorella strain Pbi were tested for their sensitivity/resistance to more than 80 DNA restriction endonucleases. From the known methylation sensitivities of these enzymes to site-specific 5-methylcytosine and N6-methyladenine DNA modifications, we deduce that the 16 NC64A viruses encode at least 13 different sequence-specific DNA methyltransferases and the 5 Pbi viruses encode at least 7 sequence-specific DNA methyltransferases. Each DNA methyltransferase has a 2 to 4 base pair DNA recognition sequence. Some individual viruses encode as many as ten different DNA methyltransferases, making these chlorella virus genomes among the most concentrated sources of DNA methyltransferase genes known.

Chlorella↗
PubMed2005

[Molecular ecology of microalgal viruses].

A great amount of virus particles exist in natural waters. Each virion is considered to have its own ecological role, affecting the maintenance and fluctuation of aquatic ecosystems. We have been studying viruses infectious to micro-plankton, especially those infecting phytoplankton. Red tides are caused by drastic increase in abundance of plankton. We succeeded in elucidating that viral infection is one of the most important factors determining the dynamics and termination of algal blooms by means of field survey and molecular experiments. In addition, we demonstrated that the interrelationship between viruses and their hosts are highly complicated, and might be determined by the molecular-structural difference of viral capsids among distinct virus ecotypes. Furthermore, in the process of our investigation on various aquatic algal viruses, their importance as genetic sources has also been suggested. In order to deeply understand the mechanism of aquatic ecosystem, more intensive studies as for aquatic viruses are urgently required.

Animals↗
PubMed1998

[Chinese Chlorella viruses and their molecular biological properties].

An extensive survey for Chlorella viruses revealed that Chlorella viruses are widely distributed over China. Eleven isolates of Chlorella viruses have been isolated, which lysis the Chlorella viruses sp. (strain NC64A). These isolates were named BJ-1, BJ-2, BJ-3, BJ-4, FJ-1, FJ-2, NJ-1, CDT-1, HCJ-1, SCB-1 and SCC-1 respectively. They have several common properties, including polyhedral morphology, and linear dsDNA genomes. However, the restriction patterns of viral DNA, the concentration of m5dC and m6dA in viral DNA, and the composition of viral structure protein are different among these isolates. All of viral major capsid proteins are 54,000 except FJ-1, whose major capsid protein is smaller than 54,000. Western blot analysis showed strong immunological cross reaction among all of viral proteins with PBCV-1's antiserum except FJ-1. It indicates that the homology between these isolates and PBCV-1 is high. Among these isolates, FJ-1 has some special properties.

Base Composition↗
PubMed2002

[The cell wall-degrading activities of lysin encoded by chlorella virus PBCV-1].

Lysin was isolated from Chlorella NC64A lysate which was infected by Chlorella virus PBCV-1. The enzyme property detection of lysin shows that it contains at least three enzyme activities: chitinase, chitosanase and beta-1, 3-Glucanase. This is consistent with the compose of Chlolrella cell wall. Chitinase and chitosanase, especially chitinase plays a important role in the process of virus entry. One 52 kD chitinase, one 56 kD chitosanase and one 36 kD chitosanase were obtained after purified by FPLC.

Cell Wall↗
PubMedDate not supplied

[The resistance of the DNA of cyanophage LPP-3 to the action of different restriction endonucleases].

Data on the study of structure peculiarities of cyanophage LPP-3 DNA are presented in the work. The length of cyanophage DNA calculated by means of the enzymatic hydrolysis by restrictases is 40 +/- 3.5 thou. pairs of bases. Cyanophage LPP-3 DNA was hydrolysed by more than 50 different restrictases. As a result of screening it was found out that the great number of restrictases, which recognized hexanucleotide sequences did not hydrolyze DNA of cyanophage LPP-3. A considerable deviation of the number of the observed sites of restriction from their theoretically expected number for restrictases Hae III and Cfr 131 was established. Restrictases-isoschisomeres with different sensitivity to the methylation of the recognition sites--Msp I, Hpa II and Sau 3A, MboI and DpnI were used to check the availability of methylated bases in LPP-3 DNA. Absence of methylated adenine in the site GATC and methylated cytosine in the second position of the site CCGG were established. The results obtained permit supposing that the expressed counterselection by the sites of recognition of many restriction endonucleases takes place in cyanophage LPP-3 DNA. It is supposed that apparently, this method of protection of its genome in LPP-3 is one of most important but the inconsiderable percentage of site-specific methylation of the virus DNA cannot be completely excluded.

Bacteriophages↗