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PubMed1998

Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC.

A novel gene encoding a cytosine-5-DNA methyltransferase recognizing the dinucleotide GpC was cloned from Chlorella virus NYs-1 and expressed in both Escherichia coli and Saccharomyces cerevisiae . The gene was sequenced and a predicted polypeptide of 362 amino acids with a molecular weight of 41.903 kDa was identified. The protein contains several amino acid motifs with high similarity to those of other known 5-methylcytosine-forming methyltransferases. In addition, this enzyme, named M. Cvi PI, shares 66% identity and 76% similarity with M. Cvi JI, the only other cytosine-5-DNA methyltransferase cloned from a Chlorella virus. The short, frequently occurring recognition sequence of the new methyltransferase will be very useful for in vivo chromatin structure studies in both yeast and higher organisms.

Amino Acid Sequence↗
PubMed2004

Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification.

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.

Amino Acid Sequence↗
PubMed2001

A variable region on the chlorovirus CVK2 genome contains genes possibly involved in the host range determination.

A 22.6-kbp variable region near the left end of the chlorovirus CVK2 genome which was characterized. This region contained a tandem array of 5 gene copies for Vp260-like protein, a viral surface glycoprotein. The authentic 104-kDa Vp260 was encoded at another site on the genome and contained 13 internally located, tandem repeats of 61-65 amino acids like the prominent Rickettsia surface antigen. By Northern and Western blot analyses, these genes were demonstrated to be expressed late in infection and the proteins were incorporated into virions. These results implied that the extra copies of Vp260-like proteins may be involved in host range in the natural environment.

Gene Dosage↗
PubMed2001

Regulatory mechanism of the gene expression during chlorovirus infection cycle.

In chlorovirus infection, immediate-early gene expression occurs within 5-20 min post infection (p.i.). We identified 16 major immediate-early transcripts. On Northern blot analysis, these transcripts showed unusual behaviors: (I) after 40 min p.i., their sizes reduced by approximately 100 nt, most likely caused by loss of a poly(A) tail and (II) after 40 min p.i., often appeared a few larger additional transcripts that may be formed by read-through from an upstream gene or into a downstream gene. These results indicate that the transcription of the viral genes changes drastically between before and after 40 min p.i.

Chlorella↗
PubMed2000

Characterization of immediate early genes expressed in chlorovirus infection.

By Southern blot analysis of restriction fragments of a chlorovirus CVK2 genomic contig with probes of RNA expressed immediate early in infection, sixteen genes were specifically found to be expressed in the host cells. These genes include those for aminoacyl-tRNA synthetase, nucleolin, ribosomal protein S5, hyaluronan synthase, TFIID etc. All of these transcripts were polyadenylated and most likely expressed in the host nucleus. The structural characteristics of these genes are discussed in connection with their expression mechanism. The biological importance of the gene products in viral infection are also considered.

Chlorella↗
PubMed1995

Infectivity of algal viruses studied by chlorophyll fluorescence.

Algal virus infection proceeds via the specific recognition of the host cell wall, penetration of the cell wall and transfer of genetic material into the cytoplasm of the host cell. This process is similar to that which occurs when bacteriophage infect bacteria so that techniques and concepts developed to study bacteriophage are applicable to algal virus studies. By measuring virus-induced changes in chlorophyll fluorescence we have redefined classical studies on the distribution of infectivity. We show that infectivity does not follow a Poisson distribution with a fixed mean, n. By analysing the infectivity of algal viruses over a broad range of virus:cell ratios we have obtained a corrected Poisson distribution that reflects the probability of multiple virus particles attached per cell and is equally applicable to algal viruses and bacteriophage.

Chlorella↗
PubMed1996

Comparison of two DNA viruses infecting the marine brown algae Ectocarpus siliculosus and E. fasciculatus.

The marine brown algal genus Ectocarpus contains two species, E. siliculosus and E. fasciculatus. Field populations of both species include plants with infection symptoms caused by DNA viruses. We have established clonal cultures from infected and normal host plants and investigated the properties of the endogenous viruses. Both host species contain virus particles with a hexagonal cross-section and a diameter of ca. 150 nm. The genomes of both virus types consist of double-stranded DNA, approximately 320 kb in size. Restriction digestion with Sfil revealed differences between the two virus genomes. However, PCR experiments suggest that at least one gene, which encodes a major capsid protein, is quite similar in both virus species. In cross-infection experiments the E. siliculosus virus did not initiate an infection cycle in E. fasciculatus. In contrast, the E. fasciculatus virus infected E. siliculosus zoospores. The resulting plants showed aberrant symptoms and produced virus particles which were not infectious. We conclude that the two Ectocarpus species are hosts for different, but closely related viruses.

Capsid↗
PubMed1999

Persistent virus integration into the genome of its algal host, Ectocarpus siliculosus (Phaeophyceae).

The brown alga Ectocarpus siliculosus frequently carries an endogenous virus, E. siliculosus virus (EsV-1), the genome of which is a circular, double-stranded DNA molecule of about 320 kbp. After infection, which occurs in the unicellular spores or gametes, the virus is present latently in all somatic cells of the host. Virus multiplication is restricted to cells of the reproductive organs. It has been an open question whether the latent viral DNA occurs as a free episome or becomes integrated into the host genome. PCR studies showed that viral DNA co-migrates with high molecular mass DNA in pulsed-field gel electrophoresis, which confirms that latent viral DNA is integrated into the host genome.

DNA, Plant↗
PubMed2004

Glycosyltransferases encoded by viruses.

Studies of cellular biology in recent decades have highlighted the crucial roles of glycans in numerous important biological processes, raising the concept of glycomics that is now considered as important as genomics, transcriptomics and proteomics. For millions of years, viruses have been co-evolving with their hosts. Consequently, during this co-evolution process, viruses have acquired mechanisms to mimic, hijack or sabotage host processes that favour their replication, including mechanisms to modify the glycome. The importance of the glycome in the regulation of host-virus interactions has recently led to a new concept called 'glycovirology'. One fascinating aspect of glycovirology is the study of how viruses affect the glycome. Viruses reach that goal either by regulating expression of host glycosyltransferases or by expressing their own glycosyltransferases. This review describes all virally encoded glycosyltransferases and discusses their established or putative functions. The description of these enzymes illustrates several intriguing aspects of virology and provides further support for the importance of glycomics in biological processes.

Bacteriophages↗
PubMed2003

Detection of four oxidation sites in viral prolyl-4-hydroxylase by top-down mass spectrometry.

Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl-4-hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 +/- 3, and <2%, respectively; "top-down" MS/MS of these ions show oxidation at the sites R(28)-V(31), E(95)-F(107), and K(216) of 22%, 28%, and 34%, respectively, but with a possible (approximately 4%) fourth site at V(125)-D(150). However, for the doubly oxidized molecular ions (increasing the precursor oxygen content from 0.94 to 2), MS/MS showed an easily observable approximately 13% oxygen at the V(125)-D(150) site. For the "bottom-up" approach, detection of the approximately 4% oxidation at the V(125)-D(150) site by MS analysis of a proteolysis mixture would have been very difficult. The unmodified peptide containing this site would represent a few percent of the proteolysis mixture; the oxidized peptide not only would be just approximately 4% of this, but the uniqueness of its mass value (approximately 1-2 kD) would be far less than the 11,933 Dalton value used here. Using different molecular ion precursors for top-down MS/MS also provides kinetic data from a single sample, that is, from molecular ions with 0.94 and 2 oxygens. Little oxidation occurs at V(125)-D(150) until K(216) is oxidized, suggesting that these are competitively catalyzed by the iron center; among several prolyl-4-hydroxylases the K(216), H(137), and D(139) are conserved residues.

Amino Acid Sequence↗
PubMedDate not supplied

Viral control of phytoplankton populations--a review.

Phytoplankton population dynamics are the result of imbalances between reproduction and losses. Losses include grazing, sinking, and natural mortality. As the importance of microbes in aquatic ecology has been recognized, so has the potential significance of viruses as mortality agents for phytoplankton. The field of algal virus ecology is steadily changing and advancing as new viruses are isolated and new methods are developed for quantifying the impact of viruses on phytoplankton dynamics and diversity. With this development, evidence is accumulating that viruses can control phytoplankton dynamics through reduction of host populations, or by preventing algal host populations from reaching high levels. The identification of highly specific host ranges of viruses is changing our understanding of population dynamics. Viral-mediated mortality may not only affect algal species succession, but may also affect intraspecies succession. Through cellular lysis, viruses indirectly affect the fluxes of energy, nutrients, and organic matter, especially during algal bloom events when biomass is high. Although the importance of viruses is presently recognized, it is apparent that many aspects of viral-mediated mortality of phytoplankton are still poorly understood. It is imperative that future research addresses the mechanisms that regulate virus infectivity, host resistance, genotype richness, abundance, and the fate of viruses over time and space.

Biodiversity↗
PubMed2005

Complete genome sequence and lytic phase transcription profile of a Coccolithovirus.

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Among the 472 predicted genes of the 407,339-base pair genome are a variety of unexpected genes, most notably those involved in biosynthesis of ceramide, a sphingolipid known to induce apoptosis. Uniquely for algal viruses, it also contains six RNA polymerase subunits and a novel promoter, suggesting this virus encodes its own transcription machinery. Microarray transcriptomic analysis reveals that 65% of the predicted virus-encoded genes are expressed during lytic infection of E. huxleyi.

Apoptosis↗
PubMed1963

Algal virus: isolation.

Freshwater blue-green algae of the genera Lyngbya, Plectonema, and Phormidium are susceptible to a virus recently isolated from a waste-stabilization pond. Electron micrographs of a partially purified preparation show that the viral particle has an icosahedral structure about 66 mmicro in diameter.

Cyanobacteria↗
PubMed1964

BLUE-GREEN ALGAL VIRUS LPP-1: PURIFICATION AND PARTIAL CHARACTERIZATION.

The blue-green algal virus LPP-1 was concentrated by ultrafiltration and purified by density-gradient and differential centrifugation. The virus contains DNA and has a sedimentation coefficient of 548S. Electron micrographs of purified viral preparations show that the polyhedral particles have short tails, which are approximately one-fourth as long as the diameter of the head. Data presented in this report indicate that the blue-green algal virus more closely resembles bacteriophages than viruses infecting higher plants.

Bacteriophages↗
PubMed1997

Hyaluronan synthase of chlorella virus PBCV-1.

Sequence analysis of the 330-kilobase genome of the virus PBCV-1 that infects a chlorella-like green algae revealed an open reading frame, A98R, with similarity to several hyaluronan synthases. Hyaluronan is an essential polysaccharide found in higher animals as well as in a few pathogenic bacteria. Expression of the A98R gene product in Escherichia coli indicated that the recombinant protein is an authentic hyaluronan synthase. A98R is expressed early in PBCV-1 infection and hyaluronan is produced in infected algae. These results demonstrate that a virus can encode an enzyme capable of synthesizing a carbohydrate polymer and that hyaluronan exists outside of animals and their pathogens.

Amino Acid Sequence↗
PubMed2000

A potassium channel protein encoded by chlorella virus PBCV-1.

The large chlorella virus PBCV-1, which contains double-stranded DNA (dsDNA), encodes a 94-codon open reading frame (ORF) that contains a motif resembling the signature sequence of the pore domain of potassium channel proteins. Phylogenetic analyses of the encoded protein, Kcv, indicate a previously unidentified type of potassium channel. The messenger RNA encoded by the ORF leads to functional expression of a potassium-selective conductance in Xenopus laevis oocytes. The channel blockers amantadine and barium, but not cesium, inhibit this conductance, in addition to virus plaque formation. Thus, PBCV-1 encodes the first known viral protein that functions as a potassium-selective channel and is essential in the virus life cycle.

Amantadine↗
PubMed1964

GROWTH CHARACTERISTICS OF THE BLUE-GREEN ALGAL VIRUS LPP-1.

Safferman, Robert S. (Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio), and Mary-Ellen Morris. Growth characteristics of the blue-green algal virus LPP- 1. J. Bacteriol. 88:771-775. 1964.-The blue-green algal (BGA) virus, strain LPP-1, formed two distinct plaque variants. During subsequent propagation, each of the isolated variants eventually reverted to a mixture of both plaque types. The large-plaque-former, multiplying at a somewhat faster rate, showed maximal virus production within 60 hr after infection; maximal titer of the small-plaque mutant was reached after a 76-hr incubation period. No differences were observed in either their host spectra or their pH and thermal stability. Evidence was presented which demonstrated that BGA virus samples could be assayed with reasonable accuracy from plaque counts. Variations resulting from this plating technique were within the range of experimental error that has been generally reported for such a system.

Cyanobacteria↗
PubMed2006

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-modification (R/M) systems. Although several of the chlorovirus R/M systems are characterized, their biological functions are unknown. The PBCV-1 proteome reveals that two virus-encoded REases, but not their companion MTases, are virion associated, suggesting that viral REases might help degrade the host DNA early in infection. To test this hypothesis, host chromosomal DNA from PBCV-1-infected cells was examined by pulsed-field gel electrophoresis. Initiation of host chromosomal DNA degradation occurred within 5 min postinfection (p.i.). The DNA degradation was insensitive to protein synthesis inhibitors or UV inactivation of virus particles, consistent with the agent being a small protein associated with the virion. Nuclease activities, including those of the two predicted REases and an uncharacterized general nuclease(s), were detected in disrupted PBCV-1 particles. The general nuclease(s) degraded both host and viral DNAs in vitro, although the viral DNA was not degraded in vivo, suggesting differential intracellular trafficking of the virion-associated nucleases. Infection with chloroviruses lacking an R/M system(s) resulted in either delayed host chromosomal DNA degradation or no detectable host chromatin changes. These immediate-early events associated with chlorovirus infections may facilitate rapid switching of the host transcriptional apparatus to viral transcription, which begins within 5 to 10 min p.i.

Animals↗