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PubMed2006

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 FR483 has 335 putative protein-encoding and 9 tRNA-encoding genes. The protein-encoding genes are almost evenly distributed on both strands, and intergenic space is minimal. Approximately 40% of the viral gene products resemble entries in public databases, including some that are the first of their kind to be detected in a virus. For example, these unique gene products include an aquaglyceroporin in MT325, a potassium ion transporter protein and an alkyl sulfatase in FR483, and a dTDP-glucose pyrophosphorylase in both viruses. Comparison of MT325 and FR483 protein-encoding genes with the prototype chlorella virus PBCV-1 indicates that approximately 82% of the genes are present in all three viruses.

Aquaglyceroporins↗
PubMed2006

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 function, including many that are completely unexpected for a virus. In addition, the chlorella viruses have several features and encode many gene products that distinguish them from most viruses. These products include: (1) multiple DNA methyltransferases and DNA site-specific endonucleases, (2) the enzymes required to glycosylate their proteins and synthesize polysaccharides such as hyaluronan and chitin, (3) a virus-encoded K(+) channel (called Kcv) located in the internal membrane of the virions, (4) a SET domain containing protein (referred to as vSET) that dimethylates Lys27 in histone 3, and (5) PBCV-1 has three types of introns; a self-splicing intron, a spliceosomal processed intron, and a small tRNA intron. Accumulating evidence indicates that the chlorella viruses have a very long evolutionary history. This review mainly deals with research on the virion structure, genome rearrangements, gene expression, cell wall degradation, polysaccharide synthesis, and evolution of PBCV-1 as well as other related viruses.

Base Sequence↗
PubMed1997

Large-scale production and plaque titration of European Chlorella viruses.

Viruses of the exsymbiotic green freshwater algae Chlorella, family Phycodnaviridae, appear to be distributed worldwide but those found in North American algae have been characterized in detail. The distinct European Chlorella viruses were studied and it was necessary to adapt both large scale purification and the plaque titration assay to the host organisms' different physiology and to our specific laboratory needs. In the virus purification scheme, a precipitation step with polyethylene glycol was introduced which allows high yield recovery of infective particles from large volumes by rapid low-speed centrifugation. In the plaque assay, a standardized algal culture was introduced. The influence of other factors, e.g. circadian rhythm, on plaque growth is also described.

Centrifugation, Density Gradient↗
PubMed2005

Isolation and characterization of a new type of chlorovirus that infects an endosymbiotic Chlorella strain of the heliozoon Acanthocystis turfacea.

A novel virus, named Acanthocystis turfacea Chlorella virus (ATCV), that infects endosymbiotic Chlorella algae of the heliozoon Acanthocystis turfacea was isolated from freshwater samples. Electron microscopic analysis of ATCV revealed that the viral capsid has a distinct icosahedral shape with a diameter of 140-190 nm. Filamentous structures extending from some of the virus vertices, which may aid attachment of the virus to host cells, were also observed. The capsid is made up of one major coat protein of about 50 kDa and contains a large dsDNA genome. ATCV is a member of the genus Chlorovirus, which belongs to the family Phycodnaviridae, a group of large, icosahedral, dsDNA-containing viruses that infect algae and are ubiquitous in natural environments. However, ATCV is clearly distinct from the prototype Chlorovirus, Paramecium bursaria Chlorella virus (PBCV-1), in some aspects of its genome structure and gene content and therefore must be regarded as a member of a new group of Chlorella viruses.

Biological Evolution↗
PubMed2004

Isolation and phylogenetic analysis of novel viruses infecting the phytoplankton Phaeocystis globosa (Prymnesiophyceae).

Viruses infecting the harmful bloom-causing alga Phaeocystis globosa (Prymnesiophyceae) were readily isolated from Dutch coastal waters (southern North Sea) in 2000 and 2001. Our data show a large increase in the abundance of putative P. globosa viruses during blooms of P. globosa, suggesting that viruses are an important source of mortality for this alga. In order to examine genetic relatedness among viruses infecting P. globosa and other phytoplankton, DNA polymerase gene (pol) fragments were amplified and the inferred amino acid sequences were phylogenetically analyzed. The results demonstrated that viruses infecting P. globosa formed a closely related monophyletic group within the family Phycodnaviridae, with at least 96.9% similarity to each other. The sequences grouped most closely with others from viruses that infect the prymnesiophyte algae Chrysochromulina brevifilum and Chrysochromulina strobilus. Whether the P. globosa viruses belong to the genus Prymnesiovirus or form a separate group needs further study. Our data suggest that, like their phytoplankton hosts, the Chrysochromulina and Phaeocystis viruses share a common ancestor and that these prymnesioviruses and their algal host have coevolved.

Animals↗
PubMed2005

Algal viruses with distinct intraspecies host specificities include identical intein elements.

Heterosigma akashiwo virus (HaV) is a large double-stranded DNA virus infecting the single-cell bloom-forming raphidophyte (golden brown alga) H. akashiwo. A molecular phylogenetic sequence analysis of HaV DNA polymerase showed that it forms a sister group with Phycodnaviridae algal viruses. All 10 examined HaV strains, which had distinct intraspecies host specificities, included an intein (protein intron) in their DNA polymerase genes. The 232-amino-acid inteins differed from each other by no more than a single nucleotide change. All inteins were present at the same conserved position, coding for an active-site motif, which also includes inteins in mimivirus (a very large double-stranded DNA virus of amoebae) and in several archaeal DNA polymerase genes. The HaV intein is closely related to the mimivirus intein, and both are apparently monophyletic to the archaeal inteins. These observations suggest the occurrence of horizontal transfers of inteins between viruses of different families and between archaea and viruses and reveal that viruses might be reservoirs and intermediates in horizontal transmissions of inteins. The homing endonuclease domain of the HaV intein alleles is mostly deleted. The mechanism keeping their sequences basically identical in HaV strains specific for different hosts is yet unknown. One possibility is that rapid and local changes in the HaV genome change its host specificity. This is the first report of inteins found in viruses infecting eukaryotic algae.

Amino Acid Sequence↗
PubMed2006

Locus-specific gene expression pattern suggests a unique propagation strategy for a giant algal virus.

Emiliania huxleyi virus strain 86 is the largest algal virus sequenced to date and is unique among the Phycodnaviridae since its genome is predicted to contain six RNA polymerase subunit genes. We have used a virus microarray to profile the temporal transcription strategy of this unusual virus during infection. There are two distinct transcription phases to the infection process. The primary phase is dominated by a group of coding sequences (CDSs) expressed by 1 h postinfection that are localized to a subregion of the genome. The CDS of the primary group have no database homologues, and each is associated with a unique promoter element. The remainder of the CDSs are expressed in a secondary phase between 2 and 4 hours postinfection. Compartmentalized transcription of the two distinctive phases is discussed. We hypothesize that immediately after infection the nucleic acid of the virus targets the host nucleus, where primary-phase genes are transcribed by host RNA polymerase which recognizes the viral promoter. Secondary-phase transcription may then be conducted in the cytoplasm.

Cell Nucleus↗
PubMed1993

Ultrastructural studies on a chlorella virus from Germany.

This is the first report on the morphology and fine structure of chlorella virus Göttingen-1 (CVG-1), an European member of the newly approved family Phycodnaviridae, which infects certain unicellular, eukaryotic, exsymbiotic, green algae. CVG-1 are polyhedral particles 145-160 nm in diameter. The capsid consists of two shells, apparently composed of ca. 7 nm subunits. Whereas the outer shell of the capsid appears to be icosahedral, the inner shell appears to be irregular underneath one vertex of the virion. In this vertex the inner shell is separated from the outer shell leaving a distinct space ("empty vertex") of unknown function.

Capsid↗
PubMed2000

Flow cytometric detection of viruses.

Representatives from several different virus families (Baculoviridae, Herpesviridae, Myoviridae, Phycodnaviridae, Picornaviridae, Podoviridae, Retroviridae, and Siphoviridae) were stained using a variety of highly fluorescent nucleic acid specific dyes (SYBR Green I, SYBR Green II, OliGreen, PicoGreen) and examined using a standard flow cytometer equipped with a standard 15 mW argon-ion laser. The highest green fluorescence intensities were obtained using SYBR Green I. DNA viruses with genome sizes between 48.5 and 300 kb could easily be detected. The fluorescence signals of the small genome-sized RNA viruses (7.4-14.5 kb) were found at the limit of detection. No significant linear relationship could be found between genome size and the green fluorescence intensity of the SYBR Green I stained virus preparations. To our knowledge, this is the first report of detecting and discriminating between a wide range of different viruses directly using flow cytometry. This rapid and precise assay represents a new and promising tool in the field of virology.

Benzothiazoles↗
PubMed2002

Viral ion channels: structure and function.

Viral ion channels are short auxiliary membrane proteins with a length of ca. 100 amino acids. They are found in enveloped viruses from influenza A, influenza B and influenza C (Orthomyxoviridae), and the human immunodeficiency virus type 1 (HIV-1, Retroviridae). The channels are called M2 (influenza A), NB (influenza B), CM2 (influenza C) and Vpu (HIV-1). Recently, in Paramecium bursaria chlorella virus (PBCV-1, Phycodnaviridae), a K+ selective ion channel has been discovered. The viral channels form homo oligomers to allow an ion flux and represent miniaturised systems. Proton conductivity of M2 is established; NB, Vpu and the potassium channel from PBC-1 conduct ions; for CM2 ion conductivity is still under proof. This review summarises the current knowledge of these short viral membrane proteins. Their discovery is outlined and experimental evidence for their structure and function is discussed. Studies using computational methods are presented as well as investigations of drug-protein interactions.

Amino Acid Sequence↗
PubMed2003

Evidence for the evolution of ascoviruses from iridoviruses.

Ascoviruses (family Ascoviridae) are large, enveloped, double-stranded (ds)DNA viruses that attack lepidopteran larvae and pupae, and are unusual in that they are transmitted by parasitic wasps during oviposition. Previous comparisons of DNA polymerase sequences from vertebrate and invertebrate viruses suggested that ascoviruses are closely related to iridoviruses. This relationship was unexpected because these viruses differ markedly in virion symmetry, genome configuration and cellular pathology. Here we present evidence based on sequence comparisons and phylogenetic analyses of a greater range of ascovirus proteins and their homologues in other large dsDNA viruses that ascoviruses evolved from iridoviruses. Consensus trees for the major capsid protein, DNA polymerase, thymidine kinase and ATPase III from representative ascoviruses, algal viruses (family Phycodnaviridae), vertebrate and invertebrate iridoviruses (family Iridoviridae) and African swine fever virus (ASFV; family Asfarviridae) showed that ascovirus proteins clustered most closely with those of the lepidopteran iridovirus Chilo iridescent virus (CIV) (Invertebrate iridescent virus 6). Moreover, analysis of the presence or absence of homologues of an additional 50 proteins encoded in the genome of Spodoptera frugiperda ascovirus (SfAV-1a) showed that about 40 % occurred in CIV, with lower percentages encoded by the genomes of, respectively, vertebrate iridoviruses, phycodnaviruses and ASFV. The occurrence of three of these genes in SfAV-1a but not CIV was indicative of the evolutionary differentiation of ascoviruses from invertebrate iridoviruses.

Adenosine Triphosphatases↗
PubMed1993

Expression of the gene encoding a translational elongation factor 3 homolog of Chlorella virus CVK2.

A gene encoding a putative translational elongation factor 3 (EF-3) on the genome of Chlorella virus CVK2 has been cloned and sequenced. It encodes for a predicted polypeptide of 1120 amino acids (aa) with a molecular mass of 127 kDa. The overall amino acid sequence of CVK2 EF-3 (vEF-3) showed 36.1% identity and 83.6% similarity to that of Saccharomyces cerevisiae EF-3. Functional domains including two sets of ATP-binding motifs were extremely well conserved between vEF-3 and yeast EF-3; 63.6% identity and 92.4% similarity in total 330-aa portions. Northern blot analysis indicated that the vEF-3 gene was transcribed in the host cells early, at 20 min postinfection (p.i.), as well as late, 3-4 hr p.i. Western blot analyses with anti-vEF-3 antibody detected the 120 kDa vEF-3 protein product after 40 min p.i. It was present until the final stages of infection but absent in the virion. The vEF-3 gene was highly conserved among all Chlorella viruses isolated in Japan.

Amino Acid Sequence↗
PubMed1994

Hairpin loop structure at the termini of the chlorella virus PBCV-1 genome.

The termini of the chlorella virus PBCV-1 330-kb dsDNA genome consist of 35-nucleotide-long, incompletely base-paired, covalently closed hairpin loops that exist in one of two forms. The two forms are complementary when the 35 nucleotide sequences are inverted with respect to one another (flip and flop). Each hairpin loop structure is followed by an identical 2221-bp inverted repeat sequence after which the DNA sequence diverges. The strategy for cloning the PBCV-1 DNA hairpin ends may be useful for cloning other hairpin termini.

Base Sequence↗
PubMed1994

Protein glycosylation and myristylation in Chlorella virus PBCV-1 and its antigenic variants.

Chlorella virus PBCV-1 particles contain three glycoproteins, the major capsid protein Vp54 and two minor proteins Vp280 and Vp260. The major capsid protein is myristylated as well as glycosylated. Both modifications are in the carboxyl-terminal portion of the protein. A gene which is modified in a PBCV-1 antiserum-resistant mutant was cloned and sequenced. This gene has an open reading frame of 3099 bases and encodes one of the two large virion glycoproteins (Vp260). Vp260 contains 13 tandem repeats of 61 to 65 amino acids. The mutation deletes the equivalent of four of the amino acid repeat sequences and duplicates one of these sequences.

Amino Acid Sequence↗
PubMed1995

Large deletions in the genome of Chlorella virus CVK1.

Large (30-45 kbp) deletions were induced in the Chlorella virus CVK1 genome by UV irradiation. Restriction endonuclease maps of the mutant genomes showed that these deletions occurred in a region located from 1.5 kbp to 47 kbp from the left DNA end. The nucleotide sequences determined around the deletion boundaries indicate that the deletion process took place by both homologous and nonhomologous recombinations. In one case, the recombination site was within a region of about 600 bp, consisting of 40 tandem repetitions of a 15-bp sequence element. The deleted region may contain several multigene families. Northern blot analyses with probes including the genes for translational elongation factor 3 and DNA polymerase showed no discernible aberrancy in the gene expression patterns in the mutants. However, at least two protein bands were missing from the mutant virions.

Base Sequence↗
PubMed1995

Large deletions in antigenic variants of the chlorella virus PBCV-1.

Four spontaneously derived, antigenic variants of chlorella virus PBCV-1 contained 27- to 37-kb deletions in the left end of the 330-kb genome. Two of the mutants, which were serologically identical, had deletions that began from map position 4.9 or 16 and ended at position 42.2 kb. In total, the two deleted regions encoded 28 putative functional open reading frames (ORFs); these deletions probably arose from homologous recombination. The other two mutants, which were serologically identical but distinct from the first two mutants, lacked the entire left terminal 37 kb of the PBCV-1 genome, including an identical 2.2-kb inverted terminal repeat region present at both ends of the wild-type genome. The deleted left end region was replaced by the transposition of an inverted 7.7- or 18.5-kb copy of the right end of the PBCV-1 genome. The region deleted in these two viruses encoded 26 single-copy ORFs, of which 23 were common to those deleted in the first two mutant viruses. The junctions of the deletions/transpositions probably arose from nonhomologous recombination. Taken together, the results indicate that 40.1 kb of single-copy DNA encoding 31 ORFs at the left end of the genome are unnecessary for PBCV-1 replication in Chlorella strain NC64A in the laboratory. The results also indicate that the size of the inverted terminal repeat region in this virus can be highly variable and that the PBCV-1 DNA packaging process tolerates large changes in genome size.

Antigenic Variation↗
PubMed1995

Characterization of a repetitive DNA element in a brown algal virus.

We describe a family of repetitive sequences found in viruses infecting the brown alga Feldmannia sp. Previously we have demonstrated that the dsDNA genomes of viruses infecting one Feldmannia sp. isolate exist as two size classes of 160 and 179 kb. Repetitive sequences within these genomes were first demonstrated based on the anomalous hybridization among five BamHI fragments from digested virus DNA. Sequence analysis of one of those fragments, B2.4, revealed the presence of 173-bp direct repeats. The restriction maps of the cross-hybridizing BamHI fragments in the two FsV (Feldmannia sp. virus) genome size classes show that these repeats are not widely dispersed, rather they are confined to a small region of each virus genome. We estimate the number of these repeats in the 179-kb genome to be about 109 and in the 169-kb genome to be about 41. In the 179-kb genome, the repeats are contained within a 22-kb region, about 12% of that virus genome, and in the 160-kb genome the repeats are contained within a 10-kb region, about 6% of the genome. The difference in repeat numbers can account for 62% of the size difference between the two size classes of the FsV genome.

Base Sequence↗
PubMed1996

Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182.

Analysis of 94 kb of DNA, located between map positions 88 and 182 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 195 open reading frames (ORFs) 65 codons or longer. One hundred and five of the 195 ORFs were considered major ORFs. Twenty-six of the 105 major ORFs resembled genes in the databases including three chitinases, a chitosanase, three serine/threonine protein kinases, two additional protein kinases, a tyrosine protein phosphatase, two ankyrins, an ornithine decarboxylase, a copper/zinc-superoxide dismutase, a proliferating cell nuclear antigen, a DNA polymerase, a fibronectin-binding protein, the yeast Ski2 protein, an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease, and an amidase. The genes for the 105 major ORFs were evenly distributed along the genome and, except for one noncoding 1788-nucleotide stretch, the genes were close together. Unexpectedly, a 900-bp region in the 1788-bp noncoding sequence resembled a CpG island.

Amino Acid Sequence↗