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PubMed2005

A model-based parallel origin and orientation refinement algorithm for cryoTEM and its application to the study of virus structures.

We present a model-based parallel algorithm for origin and orientation refinement for 3D reconstruction in cryoTEM. The algorithm is based upon the Projection Theorem of the Fourier Transform. Rather than projecting the current 3D model and searching for the best match between an experimental view and the calculated projections, the algorithm computes the Discrete Fourier Transform (DFT) of each projection and searches for the central section ("cut") of the 3D DFT that best matches the DFT of the projection. Factors that affect the efficiency of a parallel program are first reviewed and then the performance and limitations of the proposed algorithm are discussed. The parallel program that implements this algorithm, called PO(2)R, has been used for the refinement of several virus structures, including those of the 500 Angstroms diameter dengue virus (to 9.5 Angstroms resolution), the 850 Angstroms mammalian reovirus (to better than 7A), and the 1800 Angstroms paramecium bursaria chlorella virus (to 15 Angstroms).

Algorithms↗
PubMed2006

Ab initio random model method facilitates 3D reconstruction of icosahedral particles.

Model-based, three-dimensional (3D) image reconstruction procedures require a starting model to initiate data analysis. We have designed an ab initio method, which we call the random model (RM) method, that automatically generates models to initiate structural analysis of icosahedral viruses imaged by cryo-electron microscopy. The robustness of the RM procedure was demonstrated on experimental sets of images for five representative viruses. The RM method also provides a straightforward way to generate unbiased starting models to derive independent 3D reconstructions and obtain a more reliable assessment of resolution. The fundamental scheme embodied in the RM method should be relatively easy to integrate into other icosahedral software packages.

Algorithms↗
PubMed2004

Immediate early genes expressed in chlorovirus infections.

Twenty-three chlorovirus genes expressed in host cells as early as 5-10 min postinfection (p.i.), or immediate early, were isolated and characterized. Some showed significant homology with those for transcriptional factors and mRNA-processing proteins including TFIIB, helicases, mRNA capping enzyme, nucleolin, and bean transcription factor. Others code for (i) factors influencing translation such as aminoacyl tRNA synthetases and ribosomal protein, and (ii) unknown proteins. Enzymes involved in polysaccharide synthesis were also found. All transcripts of these genes had a poly(A) tail, which decreased in size after 20 min p.i., possibly caused by the shortening by an exonuclease. Often, due to readthrough either from an upstream ORF or into a downstream ORF, a few extra transcripts for each gene appeared after 40 min p.i., suggesting a change in promoter selection and termination accuracy at this point. A typical TATA-box and a common element 5'-ATGACAA were in the promoter region of almost all of the immediate early genes, which may be recognized by host RNA polymerase and transcription factors.

Animals↗
PubMed2004

Vp130, a chloroviral surface protein that interacts with the host Chlorella cell wall.

A protein, Vp130, that interacts with the host cell wall was isolated from Chlorovirus CVK2. From its peptide sequence, the gene for Vp130 was identified on the PBCV-1 genomic sequence as an ORF combining A140R and A145R. In Vp130, the N-terminus was somehow modified and the C-terminus was occupied by 23-26 tandem repeats of a PAPK motif. In the internal region, Vp130 contained seven repeats of 70-73 amino acids, each copy of which was separated by PAPK sequences. This protein was well conserved among NC64A viruses. A recombinant rVp130N protein formed in Escherichia coli was shown not only to bind directly to the host cell wall in vitro but also to specifically bind to the host cells, as demonstrated by fluorescence microscopy. Because externally added rVp130N competed with CVK2 to bind to host cells, Vp130 is most likely to be a host-recognizing protein on the virion.

Amino Acid Sequence↗
PubMed2004

Genetic diversity in chlorella viruses flanking kcv, a gene that encodes a potassium ion channel protein.

The chlorella virus PBCV-1 encodes a 94-amino acid protein named Kcv that produces a K+-selective and slightly voltage-sensitive conductance when expressed in heterologous systems. As reported herein, (i) Northern analysis of kcv expression in PBCV-1-infected cells revealed a complicated pattern suggesting that the gene might be transcribed as a di- or tri-cistronic mRNA both at early and late times after virus infection. (ii) The protein kinase inhibitors H-89, A3, and staurosporine inhibited PBCV-1 Kcv activity in Xenopus oocytes, suggesting that Kcv activity might be controlled by phosphorylation or dephosphorylation. (iii) The PBCV-1 genomic sequence revealed a gene encoding a putative protein kinase (pkx) adjacent to kcv. These findings prompted us to examine the kcv flanking regions in 16 additional chlorella viruses and transcription in two of these viruses, as well as the effect of the three protein kinase inhibitors on two Kcv homologs in Xenopus oocytes. The results indicate (i) pkx is always located 5' to kcv, but the spacing between the two genes varies from 31 to 1588 nucleotides. More variation occurs in the kcv 3' flanking region of the 16 viruses. (ii) The kcv gene is expressed as a late mono-cistronic mRNA. (iii) Unlike the affect on PBCV-1 Kcv, the three protein kinase inhibitors have little or no effect on the activity of the two Kcv homologs in oocytes. (iv) A comparison of the kcv 5' upstream sequences from the 16 viruses identified a highly conserved 10-nucleotide sequence that is present in the promoter region of all of the viruses.

3' Flanking Region↗
PubMed2005

Genetic rearrangements on the Chlorovirus genome that switch between hyaluronan synthesis and chitin synthesis.

Chlorella viruses or chloroviruses form polysaccharide fibers on the cell wall of host Chlorella cells after infection. Such polysaccharides are either hyaluronan synthesized by virus-encoded hyaluronan synthase (HAS) or chitin synthesized by viral chitin synthase (CHS). Some chloroviruses synthesize both hyaluronan (HA) and chitin simultaneously. To understand the relationship between "HA-synthesizing" and "chitin-synthesizing" viruses, we characterized the CVK2 genomic regions, one flanking chs and the other corresponding to PBCV-1 has and found that on CVK2 DNA, a single ORF (PBCV-1 A330R) was replaced with a 5 kbp region containing chs, ugdh2 (the second gene for UDP-glucose dehydrogenase) and two other ORFs, and that has was replaced with another chs gene. In some chloroviruses, ugdh was lost. These results suggest that chlorovirus types changed from "has viruses" to "chs viruses" or from "chs viruses" to "has viruses" by exchanging the genes.

Base Sequence↗
PubMed2006

C-terminal repetitive motifs in Vp130 present at the unique vertex of the Chlorovirus capsid are essential for binding to the host Chlorella cell wall.

Previously, Vp130, a chloroviral structural protein, was found to have host-cell-wall-binding activity for NC64A-viruses (PBCV-1 and CVK2). In this study, we have isolated and characterized the corresponding protein from chlorovirus CVGW1, one of Pbi-viruses that have a different host range. In NC64A-viruses, Vp130 consists of a highly conserved N-terminal domain, internal repeats of 70-73 aa motifs and a C-terminal domain occupied by 23-26 tandem repeats of a PAPK motif. However, CVGW1 was found to have a slightly different Vp130 construction where the PAPK repeats were not in the C-terminus but internal. Immunofluorescence microscopy with a specific antibody revealed that the C-terminal region containing the Vp130 repetitive motifs from PBCV-1 and CVK2 was responsible for binding to Chlorella cell walls. Furthermore, by immunoelectron microscopy and immunofluorescence microscopy, Vp130 was localized at a unique vertex of the chlorovirus particle and was found to be masked through binding to the host cells. These results suggested that Vp130 is localized at a unique vertex on the virion, with the C-terminal repetitive units outside for cell wall binding.

Amino Acid Sequence↗
PubMed2006

Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway.

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+-binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses.

Amidohydrolases↗
PubMed2002

The short N-terminus is required for functional expression of the virus-encoded miniature K(+) channel Kcv.

Kcv (K(+) Chlorella virus) is a miniature virus-encoded K(+) channel. Its predicted membrane-pore-membrane structure lacks a cytoplasmic C-terminus and it has a short 12 amino acid (aa) cytoplasmic N-terminus. Kcv forms a functional channel when expressed in human HEK 293 cells. Deletion of the 14 N-terminal aa results in no apparent differences in the subcellular location and expression level of the Kcv protein. However, the truncated protein does not induce a measurable current in transfected HEK 293 cells or Xenopus oocytes. We conclude that the N-terminus controls functional properties of the Kcv channel, but does not influence protein expression.

Base Sequence↗
PubMed2003

Are chlorella viruses a rich source of ion channel genes?

Plaque-forming dsDNA (>330 kb) viruses that infect certain unicellular, eukaryotic chlorella-like green algae contain approximately 375 protein-encoding genes. These proteins include a 94 amino acid K+ channel protein, called Kcv, as well as two putative ligand-gated ion channels. The viruses also encode other proteins that could be involved in the assembly and/or function of ion channels, including protein kinases and a phosphatase, polyamine biosynthetic enzymes and histamine decarboxylase.

Amino Acid Sequence↗
PubMed2003

Possible function for virus encoded K+ channel Kcv in the replication of chlorella virus PBCV-1.

The K+ channel Kcv is encoded by the chlorella virus PBCV-1. There is evidence that this channel plays an essential role in the replication of the virus, because both PBCV-1 plaque formation and Kcv channel activity in Xenopus oocytes have similar sensitivities to inhibitors. Here we report circumstantial evidence that the Kcv channel is important during virus infection. Recordings of membrane voltage in the host cells Chlorella NC64A reveal a membrane depolarization within the first few minutes of infection. This depolarization displays the same sensitivity to cations as Kcv conductance; depolarization also requires the intact membrane of the virion. Together these data are consistent with the idea that the virus carries functional K+ channels in the virion and inserts them into the host cell plasma membrane during infection.

Animals↗
PubMed2003

The viral potassium channel Kcv: structural and functional features.

The chlorella virus PBCV-1 was the first virus found to encode a functional potassium channel protein (Kcv). Kcv is small (94 aa) and basically consists of the M1-P-M2 (membrane-pore-membrane) module typical of the pore regions of all known potassium channels. Kcv forms functional channels in three heterologous systems. This brief review discusses the gating, permeability and modulation properties of Kcv and compares them to the properties of bacterial and mammalian K+ channels.

Amino Acid Sequence↗
PubMed1995

Analysis of 45 kb of DNA located at the left end of the chlorella virus PBCV-1 genome.

Forty-five kilobases of DNA, including the previously sequenced 2.2-kb inverted repeat region, located at the left termini of the 330-kb Chlorella virus PBCV-1 genome were sequenced and analyzed. Eighty-five complete open reading frames (ORFs) larger than 195 nucleotides were identified. Thirty-seven of the 85 ORFs, which are densely packed on both strands of the DNA, were considered major ORFs. Fifteen of the major ORFs have similarity to genes in the databases, including bacterial glycerophosphoryl diester phosphodiesterase, bacteriophage T4 endonuclease V, D-isomer specific 2-hydroxyacid dehydrogenases, and beta-alanine synthetase and bacterial nitrilases. Two major ORFs resemble the virus major capsid protein. Three major ORFs contain three or more ankyrin-like repeat elements and four ORFs encode proline-rich proteins.

Amino Acid Sequence↗
PubMed1997

Chlorella virus SC-1A encodes at least five functional and one nonfunctional DNA methyltransferases.

Chlorella virus SC-1A encodes at least six DNA methyltransferases (MTases): four N6-methyldeoxyadenine (m6A) MTases, M x CviSI (TGCmA), M x CviSII (CmATG), M x CviSIII (TCGmA) and M x CviSIV (GmATC), one 5-methyldeoxycytosine (m5C) MTase, M x CviSV (approximately RCmCG), and one nonfunctional m5C MTase, M x CviSVI, which is homologous to the MTase M x CviJI [RGmC(T/C/G)] produced by another chlorella virus IL-3A. Genes encoding three of the SC-1A m6A MTases (M x CviSI, M x CviSII, and M x CviSIII) and the nonfunctional m5C MTase were cloned and sequenced. Neither M x CviSI nor M x CviSIII genes hybridized to genes for their respective isomethylomers, M x CviRI and M x CviBIII, from other chlorella viruses. However, the M x CviSII gene hybridized strongly to its M x CviAII isomethylomer gene from virus PBCV-1. Like the prototype chlorella virus PBCV-1, the SC-1A genome contains inverted terminal repeats, one of which is adjacent to the nonfunctional m5C MTase. The three cloned m6A MTase genes are distributed throughout the approx. 345 kb SC-1A genome.

Amino Acid Sequence↗
PubMed1998

Microbial glycoconjugates.

The surfaces of all microbes are 'sugar coated' with molecules such as lipopolysaccharides in Gram-negative bacteria, capsular polysaccharides in bacteria, lipoarabinomannans in mycobacteria and lipophosphoglycan in Leishmania. The basic structures of these glycoconjugates are known and, in the case of pathogens, they can function as virulence determinants. Recent publications have refined some of these structures and have elucidated interesting genes and proteins responsible for their biosynthesis.

Animals↗
PubMed2002

Site-specific DNA cleavage by Chlorella virus topoisomerase II.

The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.

Animals↗
PubMed2005

Chlorella virus Marburg topoisomerase II: high DNA cleavage activity as a characteristic of Chlorella virus type II enzymes.

Although the formation of a covalent enzyme-cleaved DNA complex is a prerequisite for the essential functions of topoisomerase II, this reaction intermediate has the potential to destabilize the genome. Consequently, all known eukaryotic type II enzymes maintain this complex at a low steady-state level. Recently, however, a novel topoisomerase II was discovered in Paramecium bursaria chlorella virus-1 (PBCV-1) that has an exceptionally high DNA cleavage activity [Fortune et al. (2001) J. Biol. Chem. 276, 24401-24408]. If robust DNA cleavage is critical to the physiological functions of chlorella virus topoisomerase II, then this remarkable characteristic should be conserved throughout the viral family. Therefore, topoisomerase II from Chlorella virus Marburg-1 (CVM-1), a distant family member, was expressed in yeast, isolated, and characterized. CVM-1 topoisomerase II is 1058 amino acids in length, making it the smallest known type II enzyme. The viral topoisomerase II displayed a high DNA strand passage activity and a DNA cleavage activity that was approximately 50-fold greater than that of human topoisomerase IIalpha. High DNA cleavage appeared to result from a greater rate of scission rather than promiscuous DNA site utilization, inordinately tight DNA binding, or diminished religation rates. Despite the fact that CVM-1 and PBCV-1 topoisomerase II share approximately 67% amino acid sequence identity, the two enzymes displayed clear differences in their DNA cleavage specificity/site utilization. These findings suggest that robust DNA cleavage is intrinsic to the viral enzyme and imply that chlorella virus topoisomerase II plays a physiological role beyond the control of DNA topology.

Amino Acid Sequence↗
PubMed2005

DNA methylation impacts the cleavage activity of Chlorella virus topoisomerase II.

Topoisomerase II from Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) displays an extraordinarily high in vitro DNA cleavage activity that is 30-50 times higher than that of human topoisomerase IIalpha. This remarkable scission activity may reflect a unique role played by the type II enzyme during the viral life cycle that extends beyond the normal control of DNA topology. Alternatively, but not mutually exclusively, it may reflect an adaptation to some aspect of the viral environment that differs from the in vitro conditions. To this point, the genomes of many chlorella viruses contain high levels of N6-methyladenine (6mA) and 5-methylcytosine (5mC), but the DNA employed in vitro is unmodified. Therefore, to determine whether methylation impacts the ability of chlorella virus topoisomerase II to cleave DNA, the effects of 6mA and 5mC on the PBCV-1 and CVM-1 enzymes were examined. Results indicate that 6mA strongly inhibits DNA scission mediated by both enzymes, while 5mC has relatively little effect. At levels of 6mA and 5mC methylation comparable to those found in the CVM-1 genome (10% 6mA and 42% 5mC), the level of DNA cleavage decreased approximately 4-fold. As determined using a novel rapid quench pre-equilibrium DNA cleavage system in conjunction with oligonucleotide binding and ligation assays, this decrease appears to be caused primarily by a slower forward rate of DNA scission. These findings suggest that the high DNA cleavage activity of chlorella virus topoisomerase II on unmodified nucleic acid substrates may reflect, at least in part, an adaptation to act on methylated genomic DNA.

Animals↗