Plant Pathology Commons^™

Expression Of Chlorovirus Mt325 Aquaglyceroporin (Aqpv1) In Tobacco And Its Role In Mitigating Drought Stress, Saadia Bihmidine, Mingxia Cao, Ming Kang, Tala Awada, James L. Van Etten, David Dunigan, Thomas E. Clemente

James Van Etten Publications

Main conclusions A Chlorovirus aquaglyceroporin 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 …

Viruses As Nanoparticles: Structure Versus Collective Dynamics, S. Sirotkin, A. Mermet, M. Bergoin, V. Ward, James L. Van Etten

James Van Etten Publications

In order to test the application of the “nanoparticle” concept to viruses in terms of low-frequency dynamics, large viruses (140–190 nm) were compared to similar-sized polymer colloids using ultra-small-angle x-ray scattering and very-low-frequency Raman or Brillouin scattering. While both viruses and polymer colloids show comparable highly defined morphologies, with comparable abilities of forming self-assembled structures, their respective abilities to confine detectable acoustic vibrations, as expected for such monodisperse systems, differed. Possible reasons for these different behaviors are discussed.

Viruses Infecting Marine Picoplancton Encode Functional Potassium Ion Channels, Fenja Siotto, Corinna Martin, Oliver Rauh, James L. Van Etten, Indra Schroeder, Anna Moroni, Gerhard Thiel

James Van Etten Publications

Phycodnaviruses are dsDNA viruses, which infect algae. Their large genomes encode many gene products, like small K+ channels, with homologs in prokaryotes and eukaryotes. Screening for K+ channels revealed their abundance in viruses from fresh-water habitats. Recent sequencing of viruses from marine algae or from salt water in Antarctica revealed sequences with the predicted characteristics of K+ channels but with some unexpected features. Two genes encode either 78 or 79 amino acid proteins, which are the smallest known K+ channels. Also of interest is an unusual sequence in the canonical α-helixes in K+ channels. Structural prediction algorithms indicate that the …

The Voltage-Sensing Domain Of A Phosphatase Gates The Pore Of A Potassium Channel, Cristina Arrigoni, Indra Schroeder, Giulia Romani, James L. Van Etten, Gerhard Thiel, Anna Moroni

James Van Etten Publications

The modular architecture of voltage-gated K⁺ (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv_Synth1, a functional voltage-gated, outwardly rectifying K⁺ channel. Kv_Synth1 displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V1/2 = +56 mV; z of approx. 1), including the depolarizationinduced mode shift. The degree of outward rectification of the channel is critically …

A Virus-Encoded Potassium Ion Channel Is A Structural Protein In The Chlorovirus Paramecium Bursaria Chlorella Virus 1 Virion, Giulia Romani, Adrianna Piotrowski, Stefan Hillmer, James Gurnon, James L. Van Etten, Anna Morani, Gerhard Thiel, Brigitte Hertel

James Van Etten Publications

Most chloroviruses encode small K+ channels, which are functional in electrophysiological assays. The experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral K+ channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. This hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1). Here, we used a mAb raised against the functional K+ channel from chlorovirus MA-1D to search for the viral K+ channel …

Potassium Ion Channels: Could They Have Evolved From Viruses?1[W], Gerhard Thiel, Anna Moroni, Guillaume Blanc, James L. Van Etten

James Van Etten Publications

Cells communicate among themselves by electrical activity. Sophisticated membrane-embedded proteins, called ion channels, catalyze rapid, selective, and regulated ion fluxes across membranes (Hille, 2001). The resulting membrane currents are responsible for neuronal activity and the systemic propagation of electrical signals in animals. The activity of some channels is important for muscle movement in animals or growth in plants; other channels sense the concentration of physiological signals and modulate key processes in all kinds of eukaryotic cells. Among the many diverse ion channels in higher organisms, K+ channels are among the most important. One feature of K+ channels is that they …

A Virus-Encoded Potassium Ion Channel Is A Structural Protein In The Chlorovirus Paramecium Bursaria Chlorella Virus 1 Virion, Giulia Romani, Adrianna Piotrowski, Stefan Hillmer, James Gurnon, James L. Van Etten, Anna Moroni, Gerhard Thiel, Brigitte Hertel

James Van Etten Publications

Most chloroviruses encode small K+ channels, which are functional in electrophysiological assays. The experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral K+ channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. This hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1). Here, we used a mAb raised against the functional K+ channel from chlorovirus MA-1D to search for the viral K+ channel …

Giant Viruses, James L. Van Etten

James Van Etten Publications

The common view of viruses, mostly true, is of tiny burglars that sneak into cells, grab the biosynthetic controls and compel the cell to make huge numbers of progeny that break out of the cell and keep the replication cycle going. Viruses are supposed to be diminutive even compared to cells that are just a micrometer (1,000 nanometers) in diameter. They are supposed to travel light, making do with just a few well-adapted genes.

In 1992, a new microorganism was isolated from a power-plant cooling tower in Bradford, England, where Timothy Robotham, a microbiologist at Leeds Public Health Laboratory, was …

Minimal Art: Or Why Small Viral K+ Channels Are Good Tools For Understanding Basic Structure And Function Relations, Gerhard Thiel, Dirk Baumeister, Indra Schroeder, Stefan M. Kast, James L. Van Etten, Anna Moroni

James Van Etten Publications

Some algal viruses contain genes that encode proteins with the hallmarks of K+ channels. One feature of these proteins is that they are less than 100 amino acids in size, which make them truly minimal for a K+ channel protein. That is, they consist of only the pore module present in more complex K+ channels. The combination of miniature size and the functional robustness of the viral K+ channels make them ideal model systems for studying how K+ channels work. Here we summarize recent structure/function correlates from these channels, which provide insight into functional properties such as gating, pharmacology and …

Functional Hak/Kup/Kt-Like Potassium Transporter Encoded By Chlorella Viruses, Timo Greiner, José Ramos, Maria C. Alvarez, James Gurnon, Ming Kang, James L. Van Etten, Anna Moroni, Gerhard Thiel

James Van Etten Publications

Chlorella viruses are a source of interesting membrane transport proteins. Here we examine a putative K+ transporter encoded by virus FR483 and related chlorella viruses. The protein shares sequence and structural features with HAK/KUP/KT-like K+ transporters from plants, bacteria and fungi. Yeast complementation assays and Rb+ uptake experiments show that the viral protein, termed HAKCV (high-affinity K+ transporter of chlorella virus), is functional, with transport characteristics that are similar to those of known K+ transporters. Expression studies revealed that the protein is expressed as an early gene during viral replication, and proteomics data indicate that it is not packaged in …

A Functional Calcium-Transporting Atpase Encoded By Chlorella Viruses, Maria Cristina Bonza, Holger Martin, Ming Kang, Gentry L. Lewis, Timo Greiner, Sonia Giacometti, James L. Van Etten, Maria Ida De Michelis, Gerhard Thiel, Anna Moroni

James Van Etten Publications

Calcium-transporting ATPases (Ca²⁺ pumps) are major players in maintaining calcium homeostasis in the cell and have been detected in all cellular organisms. Here, we report the identification of two putative Ca²⁺ pumps, M535L and C785L, encoded by chlorella viruses MT325 and AR158, respectively, and the functional characterization of M535L. Phylogenetic and sequence analyses place the viral proteins in group IIB of P-type ATPases even though they lack a typical feature of this class, a calmodulin-binding domain. A Ca²⁺ pump gene is present in 45 of 47 viruses tested and is transcribed during virus infection. Complementation analysis of …

Identification Of An L-Rhamnose Synthetic Pathway In Two Nucleocytoplasmic Large Dna Viruses, Madhu Parakkottil Chothi, Garry A. Duncan, Andrea Armirotti, Chantal Abergel, James R. Gurnon, James L. Van Etten, Cinzia Bernardi, Gianluca Damonte, Michela Tonetti

James Van Etten Publications

Nucleocytoplasmic large DNA viruses (NCLDVs) are characterized by large genomes that often encode proteins not commonly found in viruses. Two species in this group are Acanthocystis turfacea chlorella virus 1 (ATCV-1) (family Phycodnaviridae, genus Chlorovirus) and Acanthamoeba polyphaga mimivirus (family Mimiviridae), commonly known as mimivirus. ATCV-1 and other chlorovirus members encode enzymes involved in the synthesis and glycosylation of their structural proteins. In this study, we identified and characterized three enzymes responsible for the synthesis of the sugar L-rhamnose: two UDP-D-glucose 4,6-dehydratases (UGDs) encoded by ATCV-1 and mimivirus and a bifunctional UDP-4-keto-6-deoxy-D-glucose epimerase/reductase (UGER) from mimivirus. Phylogenetic …

Crystal Structure Of A Virus-Encoded Putative Glycosyltransferase, Yi Xiang, Ulrich Baxa, Ying Zhang, Alasdair C. Steven, Gentry L. Lewis, James L. Van Etten, Michael G. Rossmann

James Van Etten Publications

The chloroviruses (family Phycodnaviridae), unlike most viruses, encode some, if not most, of the enzymes involved in the glycosylation of their structural proteins. Annotation of the gene product B736L from chlorovirus NY-2A suggests that it is a glycosyltransferase. The structure of the recombinantly expressed B736L protein was determined by X-ray crystallography to 2.3-Å resolution, and the protein was shown to have two nucleotide-binding folds like other glycosyltransferase type B enzymes. This is the second structure of a chlorovirus-encoded glycosyltransferase and the first structure of a chlorovirus type B enzyme to be determined. B736L is a retaining enzyme and belongs …

Microarray Analysis Of Paramecium Bursaria Chlorella Virus 1 Transcription, Giane M. Yanai-Balser, Garry A. Duncan, James D. Eudy, Dong Wang, Xiao Li, Irina V. Agarkova, David D. Dunigan, James L. Van Etten

James Van Etten Publications

Paramecium bursaria chlorella virus 1 (PBCV-1), a member of the family Phycodnaviridae, is a large doublestranded DNA, plaque-forming virus that infects the unicellular green alga Chlorella sp. strain NC64A. The 330-kb PBCV-1 genome is predicted to encode 365 proteins and 11 tRNAs. To monitor global transcription during PBCV-1 replication, a microarray containing 50-mer probes to the PBCV-1 365 protein-encoding genes (CDSs) was constructed. Competitive hybridization experiments were conducted by using cDNAs from poly(A)- containing RNAs obtained from cells at seven time points after virus infection. The results led to the following conclusions: (i) the PBCV-1 replication cycle is temporally …

Transmembrane Domain Length Of Viral K+ Channels Is A Signal For Mitochondria Targeting, Jorg Balss, Panagiotis Papatheodorou, Mario Mehmel, Dirk Baumeister, Brigitte Hertel, Nicolas Delaroque, Frank C. Chatelain, Daniel L. Minor Jr., James L. Van Etten, Joachim Rassow, Anna Moroni, Gerhard Thiel

James Van Etten Publications

K⁺ channels operate in the plasma membrane and in membranes of organelles including mitochondria. The mechanisms and topogenic information for their differential synthesis and targeting is unknown. This article describes 2 similar viral K⁺ channels that are differentially sorted; one protein (Kesv) is imported by the Tom complex into the mitochondria, the other (Kcv) to the plasma membrane. By creating chimeras we discovered that mitochondrial sorting of Kesv depends on a hierarchical combination of N- and C-terminal signals. Crucial is the length of the second transmembrane domain; extending its C terminus by >2 hydrophobic amino acids redirects Kesv …

Differential Role Of Nadp+ And Nadph In The Activity And Structure Of Gdp-D-Mannose 4,6-Dehydratase From Two Chlorella Viruses, Floriana Fruscione, Laura Sturla, Garry Duncan, James L. Van Etten, Paola Valbuzzi, Antonio De Flora, Eleonopra Di Zanni, Michela Tonetti

James Van Etten Publications

GDP-D-mannose 4,6-dehydratase (GMD) is a key enzyme involved in the synthesis of 6-deoxyhexoses in prokaryotes and eukaryotes. Paramecium bursaria chlorella virus-1 (PBCV-1) encodes a functional GMD, which is unique among characterized GMDs because it also has a strong stereospecific NADPH-dependent reductase activity leading to GDP-D-rhamnose formation (Tonetti, M., Zanardi, D., Gurnon, J., Fruscione, F., Armirotti, A., Damonte, G., Sturla, L., De Flora, A., and Van Etten, J.L. (2003) J. Biol. Chem. 278, 21559–21565). In the present study we characterized a recombinant GMD encoded by another chlorella virus, Acanthocystis turfacea chlorella virus 1 (ATCV-1), demonstrating that it has the expected dehydratase …

Structure And Function Of A Chlorella Virus Encoded Glycosyltransferase, Ying Zhang, Ye Xiang, James L. Van Etten, Michael G. Rossmann

James Van Etten Publications

Paramecium bursaria chlorella virus-1 encodes at least 5 putative glycosyltransferases that are probably involved in the synthesis of the glycan components of the viral major capsid protein. The 1.6 Å crystal structure of one of these glycosyltransferases (A64R) has a mixed α/β fold containing a central, six-stranded β-sheet flanked by α-helices. Crystal structures of A64R, complexed with UDP, CMP, or GDP, established that only UDP bound to A64R in the presence of Mn2+, consistent with its high structural similarity to glycosyltransferases which utilize UDP as the sugar carrier. The structure of the complex of A64R, UDP-glucose, and Mn2+ showed that …

Sequence And Annotation Of The 288-Kb Atcv-1 Virus That Infects An Endosymbiotic Chlorella Strain Of The Heliozoon Acanthocystis Turfacea, Lisa A. Fitzgerald, Michael V. Graves, Xiao Li, James Hartigan, Artur J.P. Pfitzner, Ella Hoffart, James L. Van Etten

James Van Etten Publications

Acanthocystis turfacea chlorella virus (ATCV-1), a prospective member of the family Phycodnaviridae, genus Chlorovirus, infects a unicellular, eukaryotic, chlorella-like green alga, Chlorella SAG 3.83, that is a symbiont in the heliozoon A. turfacea. The 288,047-bp ATCV-1 genome is the first virus to be sequenced that infects Chlorella SAG 3.83. ATCV-1 contains 329 putative protein-encoding and 11 tRNA-encoding genes. The protein-encoding genes are almost evenly distributed on both strands and intergenic space is minimal. Thirty-four percent of the viral gene products resemble entries in the public databases, including some that are unexpected for a virus. For example, these …

Chlorella Viruses Contain Genes Encoding A Complete Polyamine Biosynthetic Pathway, Sascha Baumann, Adrienne Sander, James Gurnon, Giane M. Yanai-Balser, James L. Van Etten, Markus Piotrowski

James Van Etten Publications

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 …

Chlorella Virus Mt325 Encodes Water And Potassium Channels That Interact Synergistically, Sabrina Gazzarrini, Ming Kang, Svetlana Epimashko, James L. Van Etten, Jack Dainty, Gerhard Thiel, Anna Moroni

James Van Etten Publications

Fast and selective transport of water through cell membranes is facilitated by water channels. Water channels belonging to the major intrinsic proteins (MIPs) family have been found in all three domains of life, Archaea, Bacteria, and Eukarya. Here we show that Chlorella virus MT325 has a water channel gene, aqpv1, that forms a functional aquaglyceroporin in oocytes. aqpv1 is transcribed during infection together with MT325 kcv, a gene encoding a previously undescribed type of viral potassium channel. Coexpression of AQPV1 and MT325-Kcv in Xenopus oocytes synergistically increases water transport, suggesting a possible concerted action of the two channels in …

Chlorella Viruses, Takashi Yamada, Hideki Onimatsu, James L. Van Etten

James Van Etten Publications

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 ∼366 protein‐encoding genes and 11 tRNA genes. The predicted gene products of ∼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 …

The Structure Of A T=169d Algal Virus, Pbcv-1, At 15Å Resolution, X. Yan, V. Bowman, N. H. Olson, James Gurnon, James L. Van Etten, Michael G. Rossmann, T. S. Baker

James Van Etten Publications

Paramecium bursaria chlorella virus 1 (PBCV-1) (genus Chlorovirus, family Phycodnaviridae), infects certain unicellular, exsymbiotic, chlorella-like green algae [1]. The PBCV-1 virion (~1x109 Da) has a linear dsDNA genome (330kbp) and contains at least 100 different proteins [1, unpublished data]. A published cryo-electron microscopy and three-dimensional (3D) image reconstruction of PBCV-1 (26Å resolution) revealed that the outer icosahedral glycoprotein capsid, with a maximum diameter of 1,900Å along the five-fold axis, consists of 1680 trimeric capsomers (trimer) and 12 pentameric capsomers (pentamer) [2]. These capsomers are arranged on a T=169d (h=7, k=8) quasi-equivalent lattice. The trimers and pentamers are organized into 20 …

Functional Implication Of The Trna Genes Encoded In The Chlorella Virus Pbcv-L Genome, Da Young Lee, Michael V. Graves, James L. Van Etten, Tae-Jin Choi

James Van Etten Publications

The prototype Chlorella virus PBCV-l encodes 11 tRNA genes and over 350 protein-encoding genes in its 330 kbp genome. Initial attempts to overexpress the recombinant A189/192R protein, a putative virus attachment protein, in E. coli strain BL21(DE3) SI were unsuccessful, and multiple protein bands were detected on Western blots. However, the full-length A189/192R recombinant protein or fragments derived from it were detected when they were expressed in E. coli BL21 CodonPlus (DE3) RIL, which contains extra tRNAs. Codon usage analysis of the a1891192r gene showed highly biased usage of the AGA and AUA codons compared to genes encoded by E. …

Glycosyltransferases Encoded By Viruses, Nicolas Markine-Goriaynoff, Laurent Gillet, James L. Van Etten, Haralambos Korres, Naresh Verma, Alain Vanderplasschen

James Van Etten Publications

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 …

Can Large Dsdna-Containing Viruses Provide Information About The Minimal Genome Size Required To Support Life?, James L. Van Etten

James Van Etten Publications

The genomes of a few viruses, such as Bacillus megaterium phage G (670 kb) and the chlorella viruses (330 to 380 kb), are larger than the predicted minimal genome size required to support life (ca. 320 kb). A comparison of the 256 proteins predicted to be required for life with the putative 376 proteins encoded by chlorella virus PBCV-1, as well as those encoded by other large viruses, indicates that viruses lack many of these “essential” genes. Consequently, it is unlikely that viruses will aid in determining the minimal number and types of genes required for life. However, viruses may …