CHARACTERIZATION OF LARGE ALGAL VIRUSES AND THEIR GENE PRODUCTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0223502
• Project Start Date: Sep 1, 2010
• Project End Date: Aug 31, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
Plant Pathology

Non Technical Summary
There is an increasing interest in using algae as an efficient renewable source of high quality oils for biofuels. However, it is obvious that if algae are grown in large-scale raceways that pathogens, including viruses, will become a major issue. Our laboratory has as much experience as anyone in the world on algal viruses and we plan to continue studying these fascinating viruses. About 30 years ago we discovered and began to characterize the first member of what is now a rapidly increasing family of viruses (Phycodnaviridae) that infect eukaryotic algae. Phycodnaviruses and putative phycodnaviruses are huge (150 to 220 nm in diameter) icosahedral, dsDNA-containing (genomes up to 560 kb) viruses that are ubiquitous in aqueous environments throughout the world. The development of environmentally sustainable, economically viable sources of renewable biofuels is a major goal for the United States and the world. Exacerbation of global climate change associated with the use of fossil fuels is a global challenge requiring new sources of cleaner carbon-neutral fuels. Lipid-rich algae, which require CO2 for growth, are potentially an efficient renewable source of high quality oils that can serve as fuel feedstocks. However, if algae are grown on a large scale then pathogens, including viruses, will become a major issue. For the most part, the threat of viruses has been ignored by people promoting algae. Algal viruses are potentially a greater problem for algae than higher plant viruses are to higher plants because higher plant viruses are usually vectored by insects. Thus, one needs two components for a virus disease outbreak on higher plants, while algal viruses only require one component because they are not vectored. The phycodnaviruses have many additional properties that justify their continued study including: i) Some phycodnaviruses are predicted to have more than 600 protein encoding genes, which are more genes than some bacteria. ii) The viruses are sources of new and unexpected genes. Some of the chlorella virus genes encode commercially important enzymes such as DNA restriction endonucleases, whereas others encode enzymes that are the smallest in their class and may represent the minimal catalytic unit. Consequently, these small proteins often serve as models for mechanistic and structural studies. iii) The viruses are an important source of genetic elements, e.g., promoters and enhancers, as well as enzymes, for genetically engineering plants and algae. iv) The phycodnaviruses probably have a common ancestor with the poxviruses (e.g. smallpox virus) and African swine fever virus (lethal to swine and is quarantined in the USA). Thus chlorella virus properties may be relevant to these other important viruses. v) Phycodnaviruses play a dynamic, albeit largely unknown, role in regulating phytoplankton communities in aqueous environments, such as termination of massive algal blooms commonly referred to as red tides and brown tides. We are well prepared to conduct the proposed research because of our 30 years of work with the chlorella viruses, which were discovered at the University of Nebraska-Lincoln (UNL).

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	1899	1101	100%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1899 - Oilseed and oil crops, general/other;

Field Of Science
1101 - Virology;

Keywords

algae

chlorella

algal-viruses

virus structure

metacaspase

protein glycosylation

sumoylation

algal virus resistance

Goals / Objectives
1. Continue studies on virus PBCV-1 structure and the infection process using 5-fold symmetry averaging reconstruction and tomography procedures. 2. Continue to investigate glycosylation of the chlorella virus major capsid proteins. 3. Evaluate the role of SUMOylation in PBCV-1 replication, specifically cytoplasmic/nuclear transport. 4. Evaluate the role of a chlorella host metacaspase in virus PBCV-1 replication, specifically DNA packaging. 5. Explore chlorella resistance to virus infection. 6. Continue to characterize interesting PBCV-1 gene products.

Project Methods
1. The virus PBCV-1 structure experiments will be done in collaboration with the Rossmann's group at Purdue University. This is one of the best virus structure groups in the world and they have the appropriate equipment for the studies. We provide the highly purified virus. 2. Some of the virus structure work will be conducted in collaboration with the mass spectroscopy center at the University of Nebraska. A new faculty member is arriving in Sept, 2010 who has extensive expertise in analyzing post translationally modified proteins. We will provide him with the appropriate material for analysis. We will be identifying and cloning the virus-encoded genes involved in the process. 3. Objectives 3, 4, and 6 will require the growing of the virus and standard cloning and biochemical assays. 4. Objective 5 will dependent our extensive experience in growing algae and purifying the viruses that infect them.

Progress 09/01/10 to 08/31/15

Outputs
Target Audience:Our research is important to the following groups of people: 1. Scientists that study viruses. 2. Scientists interested in algal biofuels. 3. Scientists interested in mental health issues. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several graduate students, undergraduate students, postdocs, visiting scientists and a faculty member on a sabbatical leave have worked on various aspects of the chloroviruses at the University of Nebraska during the past 5 years. In addition, we have worked with scientists (faculty, graduate students, and postdocs) at many other laboratories both within the United States and in Europe. Scientists at the following institutions have worked with us on some aspects of the viruses: Darmstadt Technical University, Germany; University of Milano, Italy; Purdue University; University of Massachusetts - Lowell; US Navel Research Center; University of Genova, Italy; Marseille University, France; University of British Columbia; Mount Sinai Medical Center; University of California - Irvine; Nebraska Wesleyan University; University of Napoli Federico II, Italy; Johns Hopkins Medical School; Joint Genome Institute; Weizmann Institute, Israel. How have the results been disseminated to communities of interest?Primarily through publications. However, we have also given many invited seminars at various universities both in the United States, Canada, and Europe. In addition, we were invited to give keynote addresses at several scientific congresses during this time. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Slightly revised goals: 1. Continue studies on virus PBCV-1 structure and the infection process using 5-fold symmetry averaging reconstruction and tomography procedures. 2. Continue to investigate glycosylation of the chlorella virus major capsid proteins. 3. Examine PBCV-1 and host gene expression during virus infection. 4. Sequence and annotate the genomes of more chloroviruses, represent 3 different groups. 5. Explore chlorella resistance to virus infection. 6. Continue to characterize interesting PBCV-1 gene products. 7. Continue to explore the possibility that the chloroviruses possibly contribute to cognitive behavioral changes in mice and humans. Goal 1. In collaboration with Michael Rossmann's laboratory at Purdue University we produced an atomic structure of the prototype chlorovirus PBCV-1 to 8.5 Å resolution. These studies led to the finding that the icosahedral particles have one unique vertex, which contains a spike structure that makes the first contact with the host algal cell during infection. PBCV-1 attachment to its host and the initial infection process differs from all other viruses that infect eukaryotic organisms. Studies with Avi Minsky's laboratory at the Weizmann Institute of Science in Israel discovered that the origin of the internal membrane present in PBCV-1 is from the endoplasmic reticulum near the nucleus outer membrane. Work being written up for publication describes a re-examination of the crystal structure of the PBCV-1 major capsid protein. This re-evaluation corrects some errors in the original predicted structure and also includes the physical structure of four glycans that are attached to the protein. Another important and unexpected finding is that PBCV-1 attachment to its host's cell wall was reversible. Finally, a proteome analysis conducted on highly purified PBCV-1 virions revealed that the particle contained at least 148 viral-encoded proteins and 1 host-encoded protein. A separate study revealed the structure of the viral packaged DNA. These studies resulted in 7 publications in the past 5 years. Goal 2. Unlike all other glycoprotein-containing viruses, the chloroviruses, encode most, if not all, of the machinery used to glycosylate their major capsid protein. Furthermore, the glycosylation process occurs in the cytoplasm rather than in the endoplasmic reticulum - Golgi pathway used by other viruses that infect eukaryotes. In collaboration with a research group at the University of Napoli Federico II (Cristina De Castro), the 4 glycan structures attached to the PBCV-1 major capsid protein as well as the glycan structures attached to 5 more chloroviruses were determined. They all have a basic core structure that is modified among the various viruses. The glycan structures are very unusual and nothing like them has been reported previously. The crystal structure of one of the virus-encoded glycosyltransferases was also crystallized. We are still trying to determine which virus genes are involved in the glycosylation process. These studies resulted in 4 publications in the past 5 years. Goal 3. Both microarray analyses and RNA-SEQ sequencing were used to explore virus and host transcription during virus infection. The microarray analysis reveled that virus transcription can be divided into early genes and late genes, which are separated as to when virus DNA replication begins. However, the RNA-SEQ experiments, which are much more sensitive that microarray analyses indicated that all of the ~400 putative PBCV-1 genes were expressed by one hour after infection - although the initiation of gene expression was staggered, which represented early and late genes. These studies resulted in 3 publications in the last 5 years. Goal 4. Forty-one chloroviruses, representing 3 groups of chloroviruses, were sequenced and annotated. This effort produced several significant findings: i) The viruses encoded ~325 to ~415 proteins. ii) The function of ~45% of the proteins could be predicted. iii) Collectively the 41 viruses encoded members in 632 different protein families. Thus, because no one virus has more than ~400 proteins, the genetic diversity among the chloroviruses is enormous. iv) 155 protein families are present in all the viruses and they are considered to be the core genes. v) Only 7 of the 632 viral genes had their closest similarity with genes from one of the host Chlorella spp. This discovery, along with phylogenetic analyses, implies that the chloroviruses must have had at least one other host in their evolutionary history. vi) The evolutionary origin of ~50% of the chlorovirus genes have no close homologs in the databases. Therefore, the donor organism(s) for these viruse genes is completely unknown. These studies resulted in 4 publications in the last 5 years. Goal 5. We discovered a new virus/chlorella system in nature and the system is being characterized. This system is interesting because some of the viruses that infect this new host can also infect one of our previous hosts; however, they are unable to complete virus replication in the previous host. Therefore, resistance to the virus is occurring inside the cell and we are trying to determine the nature of this resistance. All of our previous findings of algal resistance to virus infection resulted from a change in the host cell wall receptor. The first paper on this new group of viruses has been submitted for publication. Goal 6. Nine unusual recombinant proteins were produced from chlorella virus genes and characterized. These included the chlorovirus encoded potassium ion channel proteins (9 publications), a monothiol glutaredoxin (1 publication), a calcium transporting ATPase (1 publication), an L-rhamnose synthetic enzyme (1 publication), a potassium ion transporter (1 publication), a polyamine acetyltransferase (1 publication), an aquaglycerolporin (1 publication), a Cu/Zn superoxide dismutase (1 publication) and a Skp1-binding ankyrin repeat protein (1 publication). With a couple of exceptions, all of these experiments were conducted in collaborations with other laboratories that were experts on a particular protein. For example, the potassium ion channel research, as well as the research on the other channel and two transporter proteins, was done in collaboration with scientists at the University of Milan (Anna Moroni) and Darmstadt Technical University (Gerhard Thiel). Goal 7. In collaboration with scientists at Johns Hopkins Medical School (led by Bob Yolken) we continue to explore the possibility that the chloroviruses might contribute to cognitive behavioral changes in mice and humans. One highly visible research paper was published in 2014 that described a possible correlation between cognitive behavior in humans and mice and some of the chloroviruses. The news media picked it up and gave it the name 'stupidity virus', which even made the David Letterman's show. In another recently published manuscript, we demonstrated that one chlorovirus, named ATCV-1, has a dramatic effect on mice macrophages. These studies have resulted in 3 publications in the last 5 years. Several other results were obtained in the past 5 years that deserve to be mentioned. Together with the Joint Genome Institute we sequenced and annotated two algal genomes - Chlorella variabilis and Coccomyxa subellipsoidea. These were among the first algae to have their genomes sequenced. The PBCV-1 encoded water channel protein was expressed in tobacco plants and it mitigated drought stress. Finally, we wrote nine review manuscripts on the algal viruses and other giant viruses during the past 5 years.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Thiel, G., Greiner, T., Dunigan, D.D., Moroni, A., Van Etten J.L. (2015). Large dsDNA chloroviruses encode diverse membrane transport proteins. Virology 479/480, 38-45.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Yolken, R.H., Jones-Brando, L., Dunigan, D.D., Kannan, G., Dickerson, F.B., Severance, E.G., Sabunciyan, S., Talbot, C.C., Prandovszky, E., Gurnon, J.R., Agarkova, I.V., Leister, F., Gressitt, K.L., Chen, O., Deuber, B., Ma, F., Pletnikov, M.V., Van Etten, J.L. (2015). Reply to Kjartansdottier et al.: chlorovirus ATCV-1 findings not explained by contamination. Proc. Natl. Acad. Sci. USA 112, E297.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Cosentino, C., Alberio, L., Gazzarrini, S., Aquila, M., Romano, E., Cermenati, S., Zuccolini, P., Petersen, J., Beltrame, M., Van Etten, J.L., Christie, J.M., Thiel, G., Moroni, A. (2015). Engineering of a light-gated potassium channel. Science 348, 707-710.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Milrot, E., Mutsafi, Y., Fridmann-Sirkis, Y., Shimoni, E., Rechav, K., Gurnon, J.R., Van Etten, J.L., Minsky, A. (2015). Virus-host interactions: Insights from the replication cycle of the large Paramecium bursaria chlorella virus. Cellular Microbiol. (in press) (Editors Choice).
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Petro, T.M., Agarkova, I.V., Zhou, Y., Yolken, R.H., Van Etten, J.L., Dunigan, D.D. (2015). Response of mammalian macrophages to challenge with the chlorovirus ATCV-1. J. Virol. 89, 12096-12107.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: De Castro, C., Speciale, I., Duncan, G., Dunigan, D.D., Agarkova, I., Lanzetta, R., Sturiale, L., Palmigiano, A., Garozzo, D., Molinaro, A., Tonetti, M., Van Etten, J.L. (201x). Chloroviruses N-linked glycans share a new type of conserved core architecture unprecedented in any form of life. Angewandte Chemie (in press).
• Type: Book Chapters Status: Published Year Published: 2010 Citation: Thiel, G., A. Moroni, D. Dunigan, and J.L. Van Etten. (2010). Initial events associated with virus PBCV-1 infection of Chlorella NC64A. Progress in Botany (U. L�ttge, W. Beyschlag, B. B�del, eds.) vol. 71. Springer -Verlag, Berlin. pp. 169-183.
• Type: Book Chapters Status: Published Year Published: 2010 Citation: Wilson, W.H., J.L. Van Etten, and M.J. Allen. (2010). The Phycodnaviridae: the story of how tiny giants rule the world. In "Lesser Known Large dsDNA Viruses". (J. Van Etten, ed). Springer, Berlin. p. 1-42.
• Type: Journal Articles Status: Published Year Published: 2011 Citation: Thiel, G., D. Baumeister, I. Schroeder, S.M. Kast, J.L. Van Etten, and A. Moroni. (2011). Minimal art: or why small viral K+ channels are good tools for understanding basic structure and function relations. Biochem. Biophys. Acta 1808, 580-588. [special issue on biomembranes]
• Type: Book Chapters Status: Published Year Published: 2015 Citation: Van Etten, J.L., Dunigan, D.D. (2015). Voyages with chloroviruses. In: The Phage World. Rohwer, F. (eds). p. 326-337.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: An international group of scientists who study the dynamic role of viruses in regulating phytoplankton communities in aqueous environments such as the termination of massive algal blooms commonly referred to as red tides and brown tides. People involved in the emerging algal biofuels industry also benefit from our work. Finally, assuming the reported association between some of the chlorella viruses and cognitive behavior in mammals is verified, a whole new audience will be interested in these viruses. Changes/Problems: No significant project modification to report during this reporting period. What opportunities for training and professional development has the project provided? See accomplishments. How have the results been disseminated to communities of interest? Published in professional journals, invited talks and participation at national meetings, and giving invited seminars. What do you plan to do during the next reporting period to accomplish the goals? Continue the research together with our collaborators as we have done for the last 35 years. We will also continue efforts to obtain external funding to support our research efforts.

Impacts
What was accomplished under these goals? This project is to characterize large dsDNA viruses, commonly referred to as chlorella viruses that infect freshwater algae. These viruses and their evolutionary relatives contain more protein encoding genes (i.e., 350 to 2500) than any other known viruses. The chorella viruses are ubiquitous in freshwater from around the world and they can reach titers of thousands of infectious particles per ml of indigenous water. We study many aspects of the chlorella viruses and their gene products, often with collaborators from other institutions. Some of our current projects include: i) In collaboration with Michael Rossmann's laboratory at Purdue University we continue to attempt to produce an atomic structure of the prototype chlorella virus PBCV-1. PBCV-1 attachment to its host and the initial infection process differs from all other viruses that infect eukaryotic organisms. We published one paper on this subject this year indicating that PBCV-1 attachment to its host's cell wall is reversible. ii) Unlike all other glycoprotein-containing viruses, the chlorella viruses, encode most, if not all, of the machinery used to glycosylate their major capsid protein. Furthermore, the glycosylation process occurs in the cytoplasm rather than in the endoplasmic reticulum - Golgi pathway used by eukaryotes. In collaboration with a research group at the University of Naples, the glycan structures from several more chlorella viruses have been determined. They all have a basic core structure that is modified in the various viruses. The glycan structures are very unusual and nothing like them has been reported previously. iii) We have discovered a new virus/chlorella system in nature and the system is being characterized. This system is interesting because some of the viruses that infect this new host can also infect one of our previous hosts; however, they are unable to complete virus replication in the old host. iv) Three unusual recombinant proteins were produced from chlorella virus genes and characterized; manuscripts describing these properties were published this year. These included a Cu/Zn superoxide dismutase, a Skp1-binding ankyrin-repeat protein, and the smallest known potassium ion channel. The potassium ion channel research was done in collaboration with scientists at the University of Milan and Darmstadt University. v) Together with a research group at the University of Marseille, we completed a transcriptome analysis during the first hr of PBCV-1 infection, which resulted in two publications. vi) We published a highly visible research paper this year in collaboration with scientists at Johns Hopkins Medical School, The paper described a possible correlation between cognitive behavior in humans and mice and some of the chlorella viruses. The news media picked it up and gave it the name stupidity virus, which even made the David Letterman's show.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Rowe, J.M., Jeanniard, A., Gurnon, J.R., Xia, Y., Dunigan, D.D., Van Etten, J.L., Blanc, G. (2014). Global analysis of Chlorella variabilis NC64A mRNA profiles during the early phase of Paramecium bursaria chlorella virus-1 infection. PLoS ONE 9, e90988.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Blanc, G., Mozar, M., Agarkova, I.V., Gurnon, J.R., Yanai-Balser, G., Rowe, J.M., Xia, Y., Riethoven, J.J., Dunigan, D.D., Van Etten, J.L. (2014). Deep RNA sequencing reveals hidden features and dynamics of early gene transcription in Paramecium bursaria chlorella virus 1. PLoS ONE 9, e90989.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Bihmidine, S., Cao, M., Kang, M., Awada, T., Van Etten, J.L., Dunigan, D.D., Clemente, T.E. (2014). Expression of chlorovirus MT325 aquaglyceroporin (aqpv1) in tobacco and its role in mitigating drought stress. Planta 240, 209-221.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Siotto, F., Martin, C., Rauh, O., Van Etten, J.L., Schroeder, I., Moroni, A., Thiel, G. (2014). Viruses infecting marine picoplancton encode functional potassium ion channels. Virology 466/467, 103-111.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Agarkova, I., Hertel, B., Zhang, X., Lane, L., Tchourbanov, A., Dunigan, D.D., Thiel, G., Rossmann, M.G., Van Etten, J.L. (2014). Dynamic attachment of chlorovirus PBCV-1 to Chlorella variabilis. Virology 466/467, 95-102.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Kang, M., Duncan, G., Kuszynski, C., Oyler, G., Zheng, J., Becker, D.F., Van Etten, J.L. (2014). Chlorovirus PBCV-1 encodes an active copper-zinc superoxide dismutase. J. Virol. 88, 12541-12550.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Noel, E.A., Kang, M., Adamec, J., Van Etten, J.L., Oyler, G.A. (2014). Chlorovirus Skp1-binding ankyrin-repeat protein interplay and mimicry of cellular ubiquitin ligase machinery. J. Virol. 88, 13798-13810.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Sirotkin, S., Mermet, A., Bergoin, M., Ward, V., Van Etten, J.L. (2014). Viruses as nanoparticles: structure versus collective dynamics. Physical Review E 90, 022718.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Yolken, R.H., Jones-Brando, L., Dunigan, D.D., Kannan, G., Dickerson, F.B., Severance, E.G., Sabunciyan, S., Talbot, C.C., Prandovszky, E., Gurnon, J.R., Agarkova, I.V., Leister, F., Gressitt, K.L., Chen, O., Deuber, B., Ma, F., Pletnikov, M.V., Van Etten, J.L. (2014). Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice. Proc. Natl. Acad. Sci. USA 111, 16106-16111.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: An international group of scientists who study the dynamic role of viruses in regulating phytoplankton communities in aqueous environments such as the termination of massive algal blooms commonly referred to as red tides and brown tides. People involved in the emerging algal biofuels industry also benefit from our work. Changes/Problems: No significant project modifications to report during this reporting period. What opportunities for training and professional development has the project provided? See accomplishments. How have the results been disseminated to communities of interest? See accomplishments. What do you plan to do during the next reporting period to accomplish the goals? See accomplishments.

Impacts
What was accomplished under these goals? This project is to characterize large dsDNA viruses, commonly referred to as chlorella viruses that infect freshwater algae. These viruses and their evolutionary relatives, which include the poxviruses (e.g. smallpox and the gigantic amoeba viruses called mimiviruses and pandoraviruses), contain more protein encoding genes (i.e. 350 to 2500) than any other known viruses. The chlorella viruses are ubiquitous in freshwater from around the world and they can reach titers of 100,000 infectious particles per ml of indigenous water. We study many aspects of the chlorella viruses and their gene products, often with collaborators from other institutions. Some of our current projects include: i) In collaboration with Michael Rossmann’s laboratory at Purdue University we continue to attempt to produce an atomic structure of the prototype chlorella virus PBCV-1. Currently, we are at about 8 Å resolution. PBCV-1 attachment to its host and the initial infection process differs from all other viruses that infect eukaryotic organisms. ii) Unlike all other glycoprotein-containing viruses, the chlorella viruses encode most, if not all, of the machinery used to glycosylate their major capsid protein. Furthermore, the glycosylation process occurs in the cytoplasm rather than in the traditional endoplasmic reticulum - Golgi pathway used by eukaryotes. During the past year we have identified several virus mutants that contribute to the glycosylation process and several of them have been sequenced and are awaiting annotation. iii) In collaboration with a research group at the University of Naples (Italy), the structures of two of the glycans attached to the major capsid protein of virus PBCV-1 have been solved. As predicted, the structures are very unusual and nothing like them have been seen previously. A manuscript describing these exciting results was recently published in the Proc. Natl. Acad. Sci. iv) We have conducted an intensive transcriptome analysis during the first hour of PBCV-1 infection. This project in collaboration with scientists at the University of Marseille has led to one published manuscripts and two more manuscripts have been submitted for publication. v) Forty-one chlorella viruses were sequenced in the past couple of years. Bioinformatic analyses of these sequences has provided many unexpected results and a manuscript describing these results was recently published in BMC Genomics. One of the most surprising findings is that these viruses have tremendous genetic diversity – probably more than any other group of viruses. vi) We have recently discovered another virus/chlorella system in nature and we are just beginning to characterize this system.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Colson, P., De Lamballerie, X., Yutin, N., Asgari, S., Bigot, Y., Bideshi, D.K., Chang, X,W., Federici, B.A., Van Etten, J.L., Koonin, E.V., La Scola, B., Raoult, D. (2013). Megavirales, a proposed new order for eukaryotic nucleocytoplasmic large DNA viruses. Arch. Virol. (in press).
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Thiel, G., Moroni, A., Blanc, G., Van Etten, J.L. (2013). Potassium ion channels: could they have evolved from viruses. Plant Physiol. 162, 1215-1224.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Romani, G., Piotrowski, A., Hillmer, S., Gazzarrini, S., Gurnon, J.R., Van Etten, J.L., Moroni, A., Thiel, Hertel, B. (2013). Viral encode potassium ion channel is a structural protein in the chlorovirus PBCV-1 virion. J. Gen. Virol. 94, 2549-2556.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: De Castro, C., Molinaro, A., Piacente, F., Gurnon, J.R., Sturiale, L., Palmigiano, A., Lanzetta, R., Parrilli, M., Garosso, D., Tonetti, M., Van Etten, J.L. (2013). Structure of the N-linked oligosaccharides attached to virus PBCV-1 major capsid protein: an unusual class of complex N-glycans. Proc. Natl. Acad. Sci. USA 110, 13956-13960.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Arrigoni, C., Schroeder, I., Romani, G., Van Etten, J.L., Thiel, G., and Moroni, A. (2013). The voltage-sensing domain of a phosphatase gates the pore of a potassium channel. J. Gen Physiol. 141, 389-395. [Highlighted article]
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Jeanniard, A., Dunigan, D.D., Gurnon, J.R., Agarkova, I.V., Kang, M., Vitek, J., Duncan, G., McClung, O.W., Larsen, M., Claverie, J.M., Van Etten, J.L., Blanc, G. (2013). Towards defining the chloroviruses: a genomic journey through a genus of large DNA viruses. BMC Genomics 14, 158.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Rowe, J.M., Dunigan, D.D., Blanc, G., Gurnon, J.R., Xia, Y., Van Etten, J.L. (2013). Evaluation of higher plant virus resistance genes in the green alga, Chlorella variabilis NC64A, during the early phase of infection with Paramecium bursaria chlorella virus-1. Virology 442, 101-113. [Highlighted article]

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: This project is to characterize large dsDNA viruses, commonly referred to as chlorella viruses that infect freshwater algae. These viruses and their evolutionary relatives, which include the poxviruses (e.g. smallpox and the gigantic amoeba viruses called mimiviruses), contain more protein encoding genes (i.e. 350 to 1100) than any other known viruses. The chlorella viruses are ubiquitous in freshwater from around the world and they can reach titers of >100,000 infectious particles per ml of indigenous water. We study many aspects of the chlorella viruses and their gene products, often with collaborators from other institutions. Some of our current projects include: i) In collaboration with Michael Rossmann's laboratory at Purdue University we continue to attempt to produce an atomic structure of the prototype chlorella virus PBCV-1. Currently, we are at about 8 angstron resolution. PBCV-1 attachment to its host and the initial infection process differs from all other viruses that infect eukaryotic organisms. ii) Unlike all other glycoprotein-containing viruses, the chlorella viruses encode most, if not all, of the machinery used to glycosylate their major capsid protein. Futhermore, the glycosylation process occurs in the cytoplasm rather than in the traditional endoplasmic reticulum - Golgi pathway used by eukaryotes. During the past year we have identified several virus mutants that contribute to the glycosylation process and we are trying to the nature of the mutations. iii) In collaboration with a research group at the University of Naples (Italy), the structures of two of the glycans attached to the major capsid protein of virus PBCV-1 have been solved. As predicted, the structures are very unusual and nothing like them have been seen previously. iv) We have conducted an intensive transcriptome analysis during the first hour of PBCV-1 infection. The sequences are currently being analyzed by bioinformatics collaborators at the University of Marseille. A manuscript describing these results is being prepared for submission. v) Forty-one chlorella viruses were sequenced in the past couple of years. Bioinformatic analyses of these sequences are providing many unexpected results and a manuscript describing these results has been submitted for publication. One of the most surprising findings is that these viruses have tremendous genetic diversity - probably more than any other group of viruses. vi) We led the effort to have the green alga Coccomyxa subellipsoidea sequenced by the Joint Genome Institute. This was the first eukaryotic microorganism from a polar environment to be sequenced and a manuscript describing this work was published this past year. PARTICIPANTS: Mr. Jim Gurnon, technologist at UNL, Dr. Dave Dunigan, research associate professor at UNL, Dr. Irina Agarkova, research assistant professor at UNL, Cristian Quispe and Eric Noel, graduate students at UNL. As indicated above we also collaborate with several laboratories located around the world. TARGET AUDIENCES: An international group of scientists who study the dynamic role of viruses in regulating phytoplankton communities in aqueous environments such as the termination of massive algal blooms commonly referred to as red tides and brown tides. People involved in the emerging algal biofuels industry also benefit from our work. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Chlorella species are considered to be excellent candidates for large-scale algal biofuels production and we led the effort to sequence the genomes of two of these green algae. The manuscript describing the first algal sequence, which was published in 2010, generated considerable interest in the scientific community. A manuscript describing the sequence and annotation of the second green alga genome (48.8 Mb) Coccomyxa subellipsoidea was published this year. This alga was the first eukaryotic microorganism from a polar (Antarctica) environment to have its genome sequenced.

Publications

• Van Etten, J.L. and D. Dunigan(2012). Chloroviruses: not your everyday plant virus. Trends Plant Sci. 17, 1-8. (Cover photo).
• Van Etten, J.L. (2012). Les virus geants. Pour la Science no 415, 22-28. (Cover photo)
• Wulfmeyer, T., C. Polzer, G. Hiepler, K. Hamacher, R. Shoeman, D.D. Dunigan, J.L. Van Etten, M. Lolicato, A. Moroni, G. Thiel, and T. Meckel. 2012. Structural organization of DNA in chlorella viruses. PLoS One 7, e30133.
• Charlop-Powers, Z., J. Jakoncic, J. Gurnon, J.L. Van Etten, and M.M. Zhou. (2012). Paramecium bursaria chlorella virus 1 encodes a polyamine acetyltransferase. J. Biol. Chem. 287, 9547-9551.
• Greiner, T., Ramos, J., Alvarez, M., Gurnon, J.R., Kang, M. Van Etten, J.L., Moroni, A., and Thiel, G. (2011). A functional HAK/KUP/KT-like potassium transporter encoded by chlorella viruses. Plant J. 68, 977-986.
• Blanc, G., Agarkova, I., Grinwood, J., Kuo, A., Brueggeman, A., Dunigan, D.D., Gurnon, J., Ladunga, I., Lindquist, E., Lucas, S., Pangilinan, J., Proschold, T., Salamov, A., Schumtz, J., Weeks, D., Yamada, T., Claverie, J.M., Grigoriev, I.V., Van Etten, J.L. (2012). The genome of the polar eukaryotic microalga Coccomyxa subellipsoidea reveals traits of cold adaptation. Genome Biology 13, R39.
• Hamacher, K., Greiner, T., Ogata, H., Van Etten, J.L., Gebhardt, M., Villarreal, L.P., Cosentino, C., Moroni, A., and Theil, G. (2012). Phycodnavirus potassium ion channel proteins question the virus molecular piracy hypothesis. PLoS One 7, e38826.
• Dunigan, D.D., Cerny, R.L., Bauman, A.T., Roach, J.C., Lane, L.C., Agarkova, I.V., Wulser, K., Yanai-Balser, G.M., Gurnon, J.R., Vitek, J.C., Kronschnabel, B.J., Jeannard, A., Blanc, G., Upton, C., Duncan, G.A., McClung, O.W., Ma, F., Van Etten, J.L. (2012). Paramecium bursaria chlorella virus 1 proteome reveals novel architectural and regulatory features of a giant virus. J. Virol. 86, 8821-8834. [highlighted article and cover photo].
• Gebhardt, M., Henkes, L.M., Tayefeh, S., Hertel, B., Greiner, T., Van Etten, J.L., Baumeister, D., Cosentino, C., Moroni, A., Kast, S.M., and Thiel, G. (2012). Relevance of lysine snorkeling in the outer transmembrane domain of small viral potassium ion channels. Biochemistry 51, 5571-5579.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: This project is to characterize large dsDNA viruses, commonly referred to as chlorella viruses that infect freshwater algae. These viruses and their evolutionary relatives, which include the poxviruses (e.g. smallpox and the gigantic amoeba virus called megavirus), contain more protein encoding genes (i.e. 350 to 1100) than any other virus. The chlorella viruses are ubiquitous in freshwater from around the world and they can reach titers of >100,000 infectious particles per ml of native water. We study many aspects of the chlorella viruses and their gene products, often with collaborators at other institutions. Some of our current projects include: i) Continue to attempt to produce an atomic structure of the prototype chlorella virus PBCV-1. Currently, we are at about 8 angstron resolution. PBCV-1 attachment to its host and the initial infection process differs from all other viruses that infect eukaryotic organisms. Unexpectedly, we discovered that chlorella virus PBCV-1 has a unique vertex with a spike structure; this was published this year. ii) Unlike all other glycoprotein-containing viruses, the chlorella viruses encode most, if not all, of the machinery used to glycosylate their major capsid protein. Futhermore, the glycosylation process occurs in the cytoplasm rather than in the traditional endoplasmic reticulum - Golgi pathway used by eukaryotes. During the past year we have identified several virus mutants that contribute to the glycosylation process. iii) We are determining the DNA structural organization in PBCV-1 virions. The DNA is neutralized by basic proteins rather than cations and polyamines that is typical for bacterial viruses. iv) We have conducted an intensive transcriptome analysis during the first hour of PBCV-1 infection. The sequences are currently being analyzed by bioinformatics collaborators at the University of Marseille. v) A functional virus-encoded potassium transporter was expressed and biochemically characterized (manuscript in press). vi) We led the effort to have the green alga Coccomyxa subellipsoidea sequenced by JGI. This is the first eukaryotic microorganism from a polar environment to be sequenced and a manuscript describing its annotation is about ready for submission. PARTICIPANTS: Mr. Jim Gurnon, technologist at UNL, Dr. Dave Dungian, research associate professor at UNL, Dr. Irina Agarkova, research assistant professor at UNL. Dr. Janet Rowe, Postdoctoral Researcher at UNL who left in March of this year. Cristian Quispe, graduate student at UNL. As indicated above we also collaborate with several laboratories around the world. TARGET AUDIENCES: An international group of scientists who study the dynamic role of viruses in regulating phytoplankton communities in aqueous environments such as the termination of massive algal blooms commonly referred to as red tides and brown tides. People involved in the emerging algal biofuels industry will also benefit from our work. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The 46.2 Mb genome of one of the chlorella virus hosts, Chlorella variabilis, was sequenced and annotated. Chlorella species are considered to be excellent candidates for large-scale algal biofuels production and this is the first Chlorella isolate to have its genome sequenced and annotated. This paper generated quite a bit of interested in the scientific community. We are completing the sequence and annotation of another 48.8 Mb green alga genome, Coccomyxa subellipsoidea, which represents the first eukaryotic microorganism from a polar (Antarctica) environment to have its genome sequenced.

Publications

• Greiner, T., Ramos, J., Alvarez, M., Gurnon, J.R., Kang, M. Van Etten, J.L., Moroni, A., and Thiel, G. (2011). A functional HAK/KUP/KT-like potassium transporter encoded by chlorella viruses. Plant Journal (in press).
• Van Etten, J.L. (2011). Giant viruses. Am. Scientist. 99, 304-311.
• Van Etten, J.L. and D. Dunigan(2012). Chloroviruses: not your everyday plant virus. Trends Plant Sci. (in press) (Cover photo).
• Wulfmeyer, T., C. Polzer, G. Hiepler, K. Hamacher, R. Shoeman, D.D. Dunigan, J.L. Van Etten, M. Lolicato, A. Moroni, G. Thiel, and T. Meckel. 2012. Structural organization of DNA in chlorella viruses. PloS One (in press).
• Wilson, W. H., J.L. Van Etten, D.S. Schroeder, K. Nagasaki, C. Brussaard, G. Bratbak, and C. Suttle. (2012). Phycodnaviridae. In: Virus Taxonomy, IXth Report of the ICTV (A.M.Q. King, M.J. Adams, E.B. Carstens, E.J. Lefkowitz, eds), pp. 219-262. Elsevier/Academic Press, Amsterdam.
• Van Etten, J.L. (2012). Genus Chloroviruses (Phycodnaviridae). In: The Springer Index of Viruses, 2nd edition. C. A. Tidona and G. Darai (eds). pp. 1242-1252. Springer-Verlag, Berlin.
• Smith, D.R., F. Burki, T. Yamada, I.V. Grigoriev, J.L. Van Etten, and P.J. Keeling. (2011). The GC-rich mitochondrial and plastid genomes of the green alga Coccomyxa give insight into the evolution of organelle DNA nucleotide landscape. PloS One 6, e23624.
• Zhang, X., Y. Xiang, D.D. Dunigan, T. Klose, P.R. Chipman, J.L. Van Etten, and M.G. Rossmann. (2011). Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid. Proc. Natl. Acad. Sci. USA 108, 14837-14842.
• Van Etten, J.L. (2011). Another really, really big virus - Commentary. Viruses 3, 32-46.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: This project is to characterize large dsDNA viruses, commonly referred to as chlorella viruses that infect freshwater algae. These viruses and their evolutionary relatives, which include the poxviruses (e.g. smallpox and the gigantic amoeba virus called mimivirus), contain more protein encoding genes (i.e. 350 to 900) than any other viruses. The chlorella viruses are ubiquitous in freshwater from around the world and they can reach titers of >100,000 infectious particles per ml of native water. We study many aspects of the chlorella viruses and their gene products, often with collaborators at other institutions. Some of our current projects include: i) Attempts to produce an atomic structure of the prototype chlorella virus PBCV-1 continue. PBCV-1 attachment to its host and the initial infection process differs from all other viruses that infect eukaryotic organisms. Unexpectedly, we discovered that chlorella virus PBCV-1 has a unique vertex that contains a spike structure. These structural studies are contributing to our understanding of virus infection. ii) Unlike all other known glycoprotein-containing viruses, the chlorella viruses encode most, if not all, of the machinery used to glycosylate their major capsid protein. Futhermore, the glycosylation process occurs in the cytoplasam rather thatn the traditional endoplasmic reticulum - Golgi pathway used by eukaryotes. Thus we believe we are studying a novel and previously unknown glycosylation process. The crystal structure of a virus-encoded glycosyltransferase was solved and a virus-encoded UDP-glucose 4,6 dehydratase was expressed and biochemically characterized. iii) Virus infection immediately results in depolarization of the host membrane. This depolarization produces two effects. It alters secondary active transport of solutes and it prevents multiple virus infections. iv) Microarray analysis of PBCV-1 infection revealed that 99% of the virus genes are expressed during virus replication in the laboratory. Furthermore, the PBCV-1 replication cycle is temporarily programmed and tightly regulated. v) A functional virus-encoded calcium transporting ATPase was expressed and biochemically characterized. We also led the effort to have the genome of one of the virus hosts, Chlorella NC64A, sequenced and annotated by the Joint Genome Institute run by the United States Department of Energy. PARTICIPANTS: Jim Gurnon, technologist at the University of Nebraska-Lincoln, Dr. Dave Dungian, Research Associate Professor at the University of Nebraska, Dr. Janet Rowe, Postdoctoral Researcher at the University of Nebraska. TARGET AUDIENCES: An international group of scientists who sequence and annotate viruses as well as the Joint Genome Institute and the algal biofuels group will all benefit from this work. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Global virus transcription was monitored by microarray analysis during infection of its host by the 330 kb chlorella virus. The results led to the following conclusions: (i) the PBCV-1 replication cycle is temporally programmed and regulated; (ii) 360 (99%) of the arrayed PBCV-1 CDSs were expressed at some time in the virus life cycle in the laboratory; (iii) 227 (62%) of the CDSs were expressed before virus DNA synthesis begins; (iv) these 227 CDSs were grouped into two classes: 127 transcripts disappeared prior to initiation of virus DNA synthesis (considered early), and 100 transcripts were still detected after virus DNA synthesis begins (considered early/late); (v) 133 (36%) of the CDSs were expressed after virus DNA synthesis begins (considered late); and (vi) expression of most late CDSs is inhibited by adding the DNA replication inhibitor, aphidicolin, prior to virus infection. 2. The 46.2 Mb genome of one of the chlorella virus hosts, Chlorella NC64A, was sequenced and annotated. Chlorella species are considered to be excellent candidates for large scale algal biofuels production and this is the first Chlorella isolate to have its genome sequenced and annotated.

Publications

• Van Etten, J.L., J. Gurnon, G. Yanai-Balser, D. Dunigan, M.V. Graves. (2010). Chlorella viruses encode most, if not all, of the machninery to glycosylate their glycoproteins independent of the endoplasmic reticulum. Biochem. Biophys. Acta 1800, 152-159. [special issue on nucleo-cytoplasmic glycosylation].
• Yanai-Balser, G.M., G.A. Duncan, J.D. Eudy, D. Wang, X. Li, I.V. Agarkova, D.D. Dunigan, and J.L. Van Etten. (2010). Microarray analysis of chlorella virus PBCV-1 transcription. J. Virol. 84, 532-542. [highlighted article]
• Fitzgerald, L.A., P.K. Wu, J.R. Gurnon, J.C. Biffinger, B.R. Ringeisen, and J.L. Van Etten. (2010). Isolation of the phycodnavirus PBCV-1 by biological laser printing (BioLP). J. Viol. Meth. 167, 223-225.
• Bonza, M.C., H. Martin, M. Kang, G. Lewis, T. Greiner, S. Giacometti, J.L. Van Etten, M.I. De Michelis, G. Thiel, and A. Moroni. (2010). A functional calcium transporting ATPase encoded by chlorella viruses. J. Gen. Virol. 91, 2620-2629.
• Chothi, M.P., G.A. Duncan, A. Armirotti, C. Abergel, J.R. Gurnon, J.L. Van Etten, C. Bernardi, G. Damonti, and M. Tonetti. (2010). Identification of an L-rhamnose synthetic pathway in two nucleocytoplasmic large DNA viruses. J. Virol. 84, 8829-8838.
• Van Etten, J.L., L.C. Lane, and D.D. Dunigan. (2010). DNA viruses - the really big ones (giruses). Ann. Rev. Microbiol. 64, 83-99.
• Blanc, G., G.A. Duncan, I. Agarkova, M. Borodovsky, J. Gurnon, A. Kuo, E. Lindquist, S. Lucas, J. Pangilinan, A. Salamov, A. Terry, T. Yamada, D.D. Dunigan, I.V. Grigoriev, J.M. Claverie, and J.L. Van Etten. (2010). Chlorella sp. NC64A genome reveals adaptation to photosymbiosis, coevolution with viruses and cryptic sex. Plant Cell 22, 2943-2955.
• Xiang, Y., U. Baxa, Y. Zhang, A.C. Steven, G.L. Lewis, J.L. Van Etten, and M.G. Rossmann. (2010). Crystal structure of a virus encoded glycosyltransferase. J. Virol. 84, 12265-12273.