Source: MONTANA STATE UNIVERSITY submitted to NRP
VIRUS DETECTION AND ANTI-VIRAL DEFENSE SYSTEMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0171019
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Plant Sciences and Plant Pathology
Non Technical Summary
The objective of this research is to expand our knowledge and understanding of the role of viruses in the ecology and evolution of life. To date, almost all known viruses infect either bacteria or eukaryotes (plants, animals, fungi, etc.). Few viruses are currently known that infect the third domain of life, Archaea. This research overcomes this scientific deficiency by directly searching for and characterizing viruses that infect Archaea. In addition, this project is focused on the molecular understanding of a newly discovered cellular anti-viral defense system (called the CRISPR/Cas defense system). The expected outcomes and impacts of this resaerch are an understanding of the CRISPR/Cas system that will lead to new methods and tools to discover and detect new viruses in any environment with direct applications in the detection of new viruses affecting plant and animal health.
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
2120199110150%
2010210110150%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0210 - Water resources; 0199 - Soil and land, general;

Field Of Science
1101 - Virology;
Goals / Objectives
The long term goal of this research is to significantly contribute to our understanding of virus diversity, ecology, and evolution. We have only a rudimentary understanding of viral diversity and their role in the environment. This is true of viruses that affect all life forms, including plants and the microbes that influence plant growth. The Specific Objectives of this project are to: 1)Dramatically expand our knowledge of archaeal virus diversity by deep sequencing of virus genomes directly from archaeal dominated environmental samples. 2)The development of spacer sequences present within host cell CRISPR/Cas loci as tools to discover previously unknown viruses. 3)Determine the role of the CRISPR/Cas anti-viral defense system in shaping both the cellular and viral population structure. The expected outcomes of this research will contribute both to advancing a fundamental understanding of host-viral interactions and viral diversity and development of new tools for virus discovers. These new tools will include metagenomic tools to isolate new viral genomes, and chip-based platforms for virus discovery. These tools should be applicable across a broad range of environments, including agricultural settings.
Project Methods
Three basic methods will be undertaken to accomplish the specific objectives of the proposed research. These include the following. To significantly expand our knowledge of virus diversity, the deep DNA sequencing of viral enriched samples across a broad spectrum of environments will be performed. The development of spacer sequences present within host cell CRISPR/Cas loci as tools to discover previously unknown viruses CRISPR spacer sequences will be used as probes for detection of new viruses. To determine the role of the CRISPR/Cas anti-viral defense system in shaping both the cellular and viral population structure a monitoring of CRISPR/cas loci dynamics will be used to identify new viruses entering an environment and as a proxy to monitor viral diversity and dynamics.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:The general target audience for this project is the scientific and higher education student community. The specific scientific audience is research virologists and microbiologists in the US and worldwide. The specific higher education student community is undergraduate students, graduate students, and postdoctoral fellows in STEM fields. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided the following opportunities for training and professional development; (i) The PhD training of 3graduate students in virology, (ii) the training of 1 postdoctoral fellows in the molecular aspects of virology, and (iii) the training of technicians on the identification of viruses. How have the results been disseminated to communities of interest?Results have been communicated to parties of interest by publications in the scientific literature, interest articles in the press,invited seminars at universities, and by speeches at national international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The activity of this program included conducting and analyzing experiments research is to contribute to our understanding of virus diversity, ecology, and evolution. In specific, the research activities for this project included 1) developing a new model for microbial host-virus interactions which took into acount the new host CRISPR anti-viral defense system, (ii) solving the high resolution structure of an archaeal virus ATPase packaging motor, (iii) and the discovery of five new viruses to science.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Munson-McGee, J., Field, E.,Bateson, M., Rooney, C., Stepanaukas, R., Young, M. Nanoarchaeota, their Sulfolobales host, and Nanoarchaeota virus distribution across Yellowstone National Park hot springs. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 81:7860-7868, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Bolduc, B., Wirth, J., Mazurie, A, Young, M. Viral assemblage composition in Yellowstone acidic hot springs assessed by network analysis. ISME J. 9:2162-2177, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hochstein, R., Bollschweiler, D., Engelhardt, H., Lawrence, M.C.., Young, M. Large Tailed Spindle Viruses of Archaea: a New Way of Doing Viral Business. J. Virol. 89:9146-9149, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Menzel, P., Gudbergsdottir, S., Ruth R., Anne G., Lin, L., Zhang, Q., Contursi, P., M., Marco K., Jakob K., Bolduc, B., Gavrilov, S., Ravin, N., Mardanov, A., Bonch-Osmolovskaya, E., Young, M., Krogh, A., Peng, X. Comparative Metagenomics of Eight Geographically Remote Terrestrial Hot Springs. Microb. Ecol. 70:411-424, 2015.


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

Outputs
Target Audience: The general target audience for this project is the scientific and higher education student community. The specific scientific audience is research virologists and microbiologists in the US and worldwide. The specific higher education student community is undergraduate students, graduate students, and postdoctoral fellows in STEM fields. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided the following opportunities for training and professional development; (i) The PhD training of 3 graduate students in virology, (ii) the training of 1 postdoctoral fellows in the molecular aspects of virology, and (iii) the training of technicians on the identification of viruses. How have the results been disseminated to communities of interest? Results have been communicated to parties of interest by publications in the scientific literature, interest articles in the press, invited seminars at universities, and by speeches at national international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Continue our research efforts according to our project plans.

Impacts
What was accomplished under these goals? The activity of this program included conducting and analyzing experiments research is to contribute to our understanding of virus diversity, ecology, and evolution. In specific, the research activities for this year included 1) developing a new model for microbial host-virus interactions which took into acount the new host CRISPR anti-viral defense system, (ii) solving the high resolution structure of an archaeal virus ATPasepackaging motor, (iii) and the discovery of four new viruses to science.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Childs, L., England, W., Young, M., Whitaker, R., Weitz, J. CRISPR-induced distributed immunity in microbial populations PLoS One 2014.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Dellas, N., Snyder, J., Bolduc, B., Young, M. Archaeal Viruses: Diversity, replication and structure. Ann. Rev Virol. 2014.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The general target audience for this project is the scientific and higher education student community. The specific scientific audience is research virologists and microbiologist in the US and worldwide. The specific higher education student community is undergraduate students, graduate students, and postdoctoral fellow in STEM fields. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided the following opportunities for training and professional develop; (i) The PhD training of 2 graduate students in virology, (ii) the training of 2 postdoctoral fellows in molecular aspects of virology, and (iii) the training technicians on the identification f viruses. How have the results been disseminated to communities of interest? Results have been communicated to parties of interest by publications in the scientific literature, interest articles in the press, invited seminars at universities, and by speeches at national international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Continue our research efforts according to our project plans.

Impacts
What was accomplished under these goals? The activity of this program included include conducting and analyzing experiments research is to contribute to our understanding of virus diversity, ecology, and evolution. In specific, the research activities for this year included 1) expanding our knowledge of archaeal virus diversity by deep sequencing of DNA and RNA virus genomes directly from archaeal dominated environmental samples, 2) the development of a microarray based detection method for viruses using spacer sequences present within host cell CRISPR/Cas loci and 3) examining the role of the CRISPR/Cas anti-viral defense system in shaping both the cellular and viral population structure.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Snyder, J., Samson, R., Brumfield, S., Bell, S., Young, M. Functional interplay between a virus and the ESCRT machinery in Archaea. Proc. Natl. Acad. Sci. USA 110:10783-10787, 2013.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: 2. Veesler, D., Ng, T-S., Sendamarai, A., Eilers, B., Lawrence, C. M., Lok, S-M., Young, M., Johnson, J. E., Fu, C.. Atomic structure of the 75 MDa extremophile Sulfolobus turreted icosahedral virus determined by CryoEM and X-ray crystallography. Proc. Natl. Acad. Sci. USA 110:5504-5509, 2013.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Manrique, P., Freire, M., Chen, C., Zadeh, H., Young, M., Suci, P. Perturbartion of the indigenous rat oral microbiome by ciprofloxacin. Mol. Oral MicroBio. 28:404-414, 2013.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Reeves, B., Young, M., Grieco, P., Suci, P. Aggregatibacter actinomycetemcomitans biofilm killing by targeted ciprofloxacin prodrug. Biofouling 29:1005-1014, 2013.
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Dellas, N., Lawrence, C.M., Young, M. Review: A Survey of Protein Structures from Archaeal Viruses. Extremophiles and Extreme Environments, 2013.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Snyder, J., Brumfield, S., Kerchner, K.,, Quax, T., Prangishvili, D., Young, M. Insights into a viral lytic pathway from an archaeal virus-host system. J. Virology. 87:2186-2192, 2013
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Snyder, J., Young, M. Lytic viruses infecting organisms from the three domains of life. Trans. BioChem. 41:309-313, 2013


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The outputs for this project include the activities of conducting and analyzing experiments on new methods for the detection, isolation and characterization of unknown viruses from diverse environmental samples. This included deep DNA sequencing of cellular components of environmental samples and bioinformatics extraction of CRISPR spacer sequences from the cellular DNA sequence. Oligonucleotide probes corresponding to the extracted CRISPR spacer sequences were then synthesized and then used to create to create a microarray chip. This microarray chip is currently being tested for its ability to rapidly detect previously unknown viruses in diverse environmental samples. In complementary experiments, size fraction and density gradient ultra-centrifugation was used to separate cellular and viral fractions from environmental samples. The purified viral fraction was then subjected to deep DNA sequencing. The resulting DNA sequences were then used to assemble near complete viral genomes of previously unknown viruses. The high-resolution structures of viral gene products as well as protein components of the CRISPR anti-viral defense system were examined by X ray crystallography and electron microscopy. These activities were integrated with the teaching and mentoring of undergraduate and graduate students in virus research methods and analysis. Output products included publication of the findings of this work in peer reviewed scientific journals, the popular press and at invited scientific meetings worldwide. PARTICIPANTS: Dr. Mark Young (PI) Dr. Jamie Snyder (Postdoc) Dr. Nikki Dellas (Postdoc) Ben Bolduc (Grad Student) Becky Hochstein (Grad Student) Pilar Manrique (Grad Student) TARGET AUDIENCES: The target audience for this research is the scientific community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The major outcomes during 2012 fell into three categories: detection of new viruses in the environment, the assembly of new viral genomes, and the determination of multiple high resolutions structures of viral and host anti-viral gene products. The development of the CRISPR spacer sequence based microarray platform allowed for scanning diverse environmental samples for 1000s of unknown viruses at a time. The viral metagenomic research leads to the assembly of greater than 50 new DNA and RNA viral genomes. By using the tools of X ray crystallography and cryo electron microscopy the near atomic resolution structures of 5 viral and 3 anti-viral host defense proteins were determined. The overall outcomes of the research were an expanded knowledge of viruses in our environment and a deeper understanding of the cellular antiviral defense systems used to combat virus infections. In addition, 4 graduate students and 4 undergraduate students received training in virus research.

Publications

  • Eilers, B.; Young, M.; Lawrence, C. M.. The Structure of an Archaeal Viral Integrase Reveals an Evolutionarily Conserved Catalytic Core yet Supports a Mechanism of DNA Cleavage in trans. J. Virology. 86:8309-8313, 2012.
  • Schlenker, C., Goel, A., Tripet, B., Menon, S., Willi, T., Dlakic, M., Young, M., Lawrence, C.M., Copie, V. Structural Studies of E73 from a Hyperthermophilic Archaeal Virus Identify the "RH3" Domain, an Elaborated Ribbon-Helix-Helix Motif Involved in DNA Recognition. Biochemistry. 51:2899-2910, 2012.
  • Childs, L, Held, NL, Young, MI, Whitaker, RJ and Weitz, JS Multi-scale model of CRISPR-induced co-evolutionary dynamics: diversification at the interface of Lamarck and Darwin. Evolution. 66:20152012, 2012.
  • Goel, A., Tripet, B., Menon, S., Willi, T., Dlakic, M., Young, M., Lawrence, C M., Copie, V. Structural Studies of E73 from a Hyperthermophilic Archaeal Virus Identify the "RH3" Domain, an Elaborated Ribbon-Helix-Helix Motif Involved in DNA Recognition. Biochemistry. 51: 2899-28910. 2012.
  • Gauss, George H.; Reott, Michael A.; Rocha, Edson R. Young, M., Douglas, T., Smith, C., Lawrence, C.M. Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily. J. Bacteriology. 194:15-27, 2012.
  • Bolduc, B., Shaughnessy, D., Wolf, Y.I., Koonin, E.V., Roberto, F. F., Young, M. . Identification of Novel Positive-Strand RNA Viruses by Metagenomic Analysis of Archaea-Dominated Yellowstone Hot Springs. J. Virol. 86:5562-5573 2012.
  • Maaty, W., Selvig, K., Ryder, S., Tarlykov, P., Hilmer, J., Heinemann, J., Steffens, J., Snyder, J., Ortmann, A., Movahed, N., Spicka, K., Chetia, L., Grieco, P., Dratz, E., Douglas, T., Young, M. Bothner, B. Proteomic Analysis of Sulfolobus solfataricus during Sulfolobus Turreted Icosahedral Virus Infection. J. Proteomic Res. 11:1420-1432. 2012.
  • Maaty, W., Steffens, J., Heinemann, J., Ortmann, A., Reeves, B., Biswas, S., Dratz, E., Grieco, P., Young, M., Bothner, B. Global analysis of viral infection in an archaeal model system. Frontiers in Microbiology. 3:411-419, 2012.
  • Rynda-Apple, A., Dobrinen, E., McAlpine, M., Read, A., Harmsen, A., Richert, L., Calverley, M., Pallister, K., Voyich, J., Wiley, J., Johnson, B., Young, M., Douglas, T., Harmsen, A. Virus-Like Particle-Induced Protection Against MRSA Pneumonia Is Dependent on IL-13 and Enhancement of Phagocyte Function. AMERICAN JOURNAL OF PATHOLOGY. 181:196-210, 2012.
  • Usselman, R., Russek, S., Klem, M., Allen, M., Douglas, T., Young, M., Idzerda, Y. Singel, D. Temperature dependence of electron magnetic resonance spectra of iron oxide nanoparticles mineralized in Listeria innocua protein cages. J APPL PHYS, 112, 2012.
  • Reott, M., Rocha, E., Young, M., Douglas, T., Smith, C. J., Lawrence, C. M. Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily. J. Bact. 194:15-27. 2012.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The activity of this program included include conducting and analyzing experiments research is to contribute to our understanding of virus diversity, ecology, and evolution. In specific, the research activities for this year included 1) expanding our knowledge of archaeal virus diversity by deep sequencing of virus genomes directly from archaeal dominated environmental samples, 2) the development of spacer sequences present within host cell CRISPR/Cas loci as tools to discover previously unknown viruses and 3) examining the role of the CRISPR/Cas anti-viral defense system in shaping both the cellular and viral population structure. PARTICIPANTS: Dr. Mark Young, Principle Investigator; Dr. Jamie Snyder, Postdoctoral Scholar; Dr. Nikki Dellas, Postdoctoral Scholar; Mary Bateson, Senior Research Technician; Sue Brumfield, Senior Research Technician; Ben Bolduc, Graduate Student; Becky Hochstein, Graduate Student; All individuals are located at Montana State University, Bozeman MT. TARGET AUDIENCES: The general target audience for this project is the scientific and higher education student community. The specific scientific audience is research virologists and microbiologist in the US and worldwide. The specific higher education student community is undergraduate students, graduate students, and postdoctoral fellow in STEM fields. PROJECT MODIFICATIONS: No major modifications are requested or anticipated.

Impacts
The outcomes /impacts of the past year have led to the discovery of new viruses and new techniques to discover and monitor previously unknown viruses to science. Deep DNA and RNA sequencing of environmental samples has led to discovery of >10 new viruses. The development of a CRISPR spacer sequence based microarray has led to the detection of >100 new viruses.

Publications

  • Lintner, N., Kerou, M., Brumfield, S., Graham, S., Liu, H., Naismith, J., Sdano, M., Peng, N., She, Q., Copie, V., Young, M., White, M., Lawrence, C. M. Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE). J. Biol. Chem. 286:21643-21656. 2011.
  • Usselman, R., Walter, E., Willits, D., Douglas, T., Young, M., Singel, D. Monitoring Structural Transitions in Icosahedral Virus Protein Cages by Site-Directed Spin Labeling. JACS 133:4156-4159. 2011.
  • Wirth, J., Snyder, J., Hochstein, R., Ortmann, A., Willits, D., Douglas, T., Young, M. Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). Virology 415:6-11. 2011.
  • Snyder, J. and M. Young. Potential role of cellular ESCRT proteins in the STIV life cycle. Bichem. Soc. Trans. 39:107-110. 2011.
  • Snyder, J., Brumfield, S., Peng, N., She, Q., Young, M. Sulfolobus Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures. J. Virology 85:6287-6292. 2011.
  • Snyder, J., and M. Young. Advances in understanding archaea-virus interactions in controlled and natural environments. Curr. Opin. Microbio. 14:497-503. 2011.
  • Lintner, N., Frankel, K., Tsutakawa, S., Alsbury, D., Copie, V., Young, M., Tainer, J., Lawrence, C. M. The Structure of the CRISPR-Associated Protein Csa3 Provides Insight into the Regulation of the CRISPR/Cas System. J. Mol. Biol. 405:939-955. 2011.
  • Heinemann, J., Maaty, W.S., Gauss, G., Akkaladeyi, N., Brumfield, S., Rayaprolu, V., Young, M. Lawrence, C. M., Bothner, B. Fossil record of an archaeal HK97-like provirus. Virology 417:362-368. 2011.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Approximately 2100 viruses are known to date, with 25% of these viruses infecting plants. Most known plant viruses can cause disease on important agronomic crops. However, we still only have only a rudimentary understanding of total virus diversity associated with plants and with cellular life in general. It is estimated that we have isolated only 0.1-.01% of the viruses on earth. Considering the central role that viruses play in causing disease, controlling microbial community composition and structure, and driving evolution it seems appropriate to develop new research tools and approaches to examine the total viral diversity and the cellular defense responses to viral infection. The outcomes of this project are directed at contributing to accomplish this goal. The outcomes of this research are to generate a better fundamental understanding of viral diversity and how virus-host interactions influence disease expression, including viral disease of plants, through designed experiments, teaching and mentoring both undergraduate and graduate students in a research laboratory environment. The outcomes of this project have been to expand our knowledge of archaeal virus diversity by deep sequencing of viral genomes directly from archaeal dominated environmental samples (e.g. viral metagenomics). We have also developed the use of spacer sequences present within host cell CRISPR/Cas loci as tools to discover previously unknown viruses. This research effort has involved the teaching and training 4 undergraduate students, 3 PhD graduate students, and 1 post-doctoral fellow over the past year. PARTICIPANTS: Individuals: Dr. Mark Young, PI: Project management and design. Dr. Jamie Snyder, Post-doctoral Fellow: Developed CRISPR array. Ben Bolduc, PhD graduate student: Involved with viral metagenomics. Rebecca A. Hochstein, PhD graduate student: Involved with viral sequencing. Jennifer Fulton Wirth, PhD graduate student: Involved with CRISPR based virus detection. Mary Bateson, Technician: Involved with viral metagenomics. Susan Brumfield, Technician: Involved with virus TEM. Keshia Kerchner, Undergraduate: Developed CRISPR array. Lindsay Klouser, Undergraduate: Involved with viral sequencing. Danie Shaughnessy, Undergraduate: Involved with viral sequencing. Collaborators: Dr. Francisco Roberto, DOE-INL; Dr. J.E. Johnson, The Scripps Research Institute. TARGET AUDIENCES: The target audiences are virologist and other members of the basic life science community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The impacts of this research have been to advancing the fundamental understanding of host-viral interactions, viral diversity and the development of new tools for virus discovery. These new tools include metagenomic tools to isolate new viral genomes, and chip-based platforms for virus discovery. These tools are applicable across a broad range of environments, including agricultural settings. The direct impacts of this research over the past year include the discovery of more than 100 new archaeal viruses previously unknown to science.

Publications

  • Suci, P., Kang, S., Gmuer, R., Douglas, T., and Young, M. (2010). Targeted Delivery of a Photosensitizer to Aggregatibacter actinomycetemcomitans Biofilm. Antimicrobial Agents and Chemotherapy. 54: Klem, MT., Young, M., and Douglas, T. (2010). Biomimetic synthesis of photoactive alpha-Fe2O3 templated by thehyperthermophilic ferritin from Pyrococus furiosus. J. Materl Chem 20.
  • Broomell, CC., Birkedal, H., Oliveira, CLP., Pedersen, JS., Gertenbach, JA., Young, M., and Douglas, T. (2010). Protein cage nanoparticles as secondary building units for the synthesis of 3-dimensional coordination polymers. Soft Matter 6.
  • Lucon, J., Abedin, M. J.,,Uchida, M., Liepold, L., Jolley, C., Young, M., and Douglas, T. (2010). A click chemistry based coordination polymer inside small heat shock protein. Chem Commun 46.
  • Pool, V., Klem, M., Jolley, C., Arenholz, EA, Douglas, T., Young, M., and Idzerda, YU. (2010). Site determination and magnetism of Mn doping in protein encapsulate iron oxide nanoparticles. J. Appl Physics. 107.
  • Usselman, A.U., Klem, M.T., Russek, E., Young, M., Douglas, T., and Goldfarb, RB. (2010). Two-component magnetic structure of iron oxide nanoparticles mineralized in Listeria innocua protein cages. J. Appl Physiscs. 107.
  • Inskeep, WP., Rusch, DB., Jay, ZJ., Herrgard, MJ,Kozubal, MA, Richardson, TH, Macur, RE., Hamamura, N., Jennings, RD., Fouke, BW., Reysenbach, AL., Roberto, F., Young, M., Schwartz, A., Boyd, ES., Badger, JH., Mathur, EJ., Ortmann, AC., Bateson, M., Geesey, G., and Frazier, M . (2010). Metagenomes from High-Temperature Chemotrophic Systems Reveal Geochemical Controls on Microbial Community Structure and Function. PLOS ONE 5: e7142.
  • Snyder, JC., Bateson, MM., Lavin, M., and Young, MJ. (2010). Use of Cellular CRISPR (Clusters of Regularly Interspaced Short Palindromic Repeats) Spacer-Based Microarrays for Detection of Viruses in Environmental Samples. Appl Env. Microbio. 76: 7251-7258.
  • Fu, CY., Wang, K., Gan, L., Lanman, J., Khayat, R., and Young, MJ., Jensen, GJ., Doerschuk, PC., Johnson, JE. (2010). In Vivo Assembly of an Archaeal Virus Studied with Whole-Cell Electron Cryotomography. Structure 18.
  • Menon, S., Eilers, Brian J., Young, M., and Lawrence, CM. (2010). The Crystal Structure of D212 from Sulfolobus Spindle-Shaped Virus Ragged Hills Reveals a New Member of the PD-(D/E)XK Nuclease Superfamily. J. Virology. 84: 5890-5897.
  • Khayat, R., Fu, CY., Ortmann, AC., Young, MJ., and Johnson, JE. (2010). The Architecture and Chemical Stability of the Archaeal Sulfolobus Turreted Icosahedral Virus. J. Virology. 84: 9575-9583. Klem, MT., Young, M., and Douglas, T. (2010). Biomimetic synthesis of photoactive alpha-Fe2O3 templated by thehyperthermophilic ferritin from Pyrococus furiosus. J. Materl Chem 20.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: During the reporting period there were significant outputs in terms of activities, services and products. Activities included detailed analysis of experiments in the use of plant virus nanotemplates to provide protection against lethal doses of influenza in animal trials. In addition, the evaluation of experimental data on the ability to specifically target plant virus nanopartices to cells within a periodontal biofilms were performed. Both of these activities involved the mentoring of undergraduate and graduate students as well as postdoctoral scholars. A total of 7 individuals were involved in these activities. Classes were taught the entire year in research methods. Events included presentations describing experimental results at 10 national or international meetings and presentations at 12 universities. Services included counseling with more than 13 laboratories worldwide on the use of plant viruses in nanotechnology. PARTICIPANTS: Professor Mark Young: Principle Investigator and Project director. Designed and oversaw the all project efforts. Dr. Peter Suci: Research Assistant Professor. Implemented the use of plant viral nanoparticles for targeted cell delivery in biofilms. Dr. Jamie Snyder: Postdoctoral Fellow. Implemented chemical modifications plant viral based protein cage nanoparticles. Jenifer Fulton: PhD graduate student: Implemented genetic modifications plant viral based protein cage nanoparticles. Ben Bulduc: PhD graduate student: Performed computational studies on plant viral based protein cage nanoparticles. Becky Hochstein: PhD graduate student. Performed genetic and chemical modifications of protein cage nanoparticles. Sue Brumfield: Technician. Performed TEM and bacoulovirus based expression studies of plant virus based nanoparticles. Naomi Tanaka: Undergraduate research student. Performed purification of plant virus nanoparticles. Krysta Buska: undergraduate research student. Performed phage display on plant viral nanoparticles. TARGET AUDIENCES: The immediate target audience for this project is the scientific community. This includes the scientific community represented by academy, industry and government. The long term target for this research is the general public through dissemination of scientific knowledge and the development of new therapeutic strategies for improving human and plant health. The efforts of the project deliver science-based knowledge through both formal mechanisms (including classroom teaching and lectures) and informal education mechanisms such as national televised documentaries for the Discovery Channel and National Geographic, newspaper articles, as well as public lectures for the general community. PROJECT MODIFICATIONS: No major changes to report.

Impacts
Both changes in knowledge and changes in actions have occurred over the reporting period. The changes in knowledge includes new fundamental knowledge on the use of plant virus nanoparticles as bio templates for applications in human health. This new knowledge includes the use of plant virus nanoparticles to provide protection against lethal doses of a wide variety of respiratory viruses. It also includes new knowledge on how to combine genetic and chemical modifications of plant virus nanoparticles to target specific cell types within a complex consortium of cells to deliver antimicrobial or non-invasive imaging capacity. New knowledge was also created on how to build 3D branched polymers within a plant virus nanoparticle for imparting chemical stability and functionality, by design, to the nanoparticle. As a result of this new knowledge there was a change in action to expand animal trials in the use of plant virus nanoparticles with the long term goal of producing new therapeutic treatments using plant virus nanoparticles with direct benefits for human and plant health.

Publications

  • Wiley JA, Richert LE, Swain SD, Harmsen A, Barnard DL, Randall TD, Jutila M, Douglas T, Broomell C, Young M, Harmsen A. (2009). Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses. PLoS One 4(9):e7142.
  • Szymczyna, B., Taurog, R., Young, M., Snyder, J., Johnson, J., Williamson, J. (2009). Synergy of NMR, computation, and X-ray crystallography for structural biology. Structure 17: 499-507.
  • Varpness Z, Suci PA, Ensign D, Young MJ, Douglas T. (2009). Photosensitizer efficiency in gentically modified protein cage architectures. Chem Commun 25:3726-8.
  • Li, HY, Klem, MT., Sebby, KB., Singel, DJ., Young, M., Douglas, T., Idzerda, YU. (2009). Determination of anisotropy constants of protein encapsulated iron oxide nanoparticles by electron magnetic resonance.J. Mag. Magn Mater. 321: 175.
  • Suci PA, Kang S, Young M, Douglas T. (2009). A streptavidin-protein cage Janus particle for polarized targeting and modular functionalization. J Am Chem Soc. 131(26):9164-5.
  • Kang S, Jolley CC, Liepold LO, Young M, Douglas T. (2009). From metal binding to nanoparticle formation: Monitoring biomimetic iron oxide synthesis within protein cages using mass spectrometry. Angew Chem Int Ed.48(26):4772-6.
  • Kang S, Suci PA, Broomell CC, Iwahori K, Kobayashi M, Yamashita I, Young M, Douglas T. (2009). Janus-like protein cages: Spatially controlled dual-functioanl surface modifications of protein cages. Nano Lett. (6):2360-6.
  • Liepold, L., Oltrogge, L., Suci, P., Douglas, T., and M. Young. (2009). Accurate mass assignment of noncovalent complexes by electrospray mass spectrometry. J. Amer. Society for Mass Spec. 20 435-442.
  • Uchida, M., D. A. Willits, K. Muller, A. F. Willis, L. Jackiw, M. Jutila, M. J. Young, A. E. Porter, and T. Douglas. (2009). Intracellular Distribution of macrophage targeting ferritin-iron oxide nanocomposite. Advanced Materials 21, 458-468.
  • Aniagyei, SE., Kennedy, CJ., Stein, B., Willits, DA., Douglas, T., Young, MJ., De, M., Rotello, VM., Srisathiyanarayanan, D., Kao, CC., Dragnea, B. (2009). Synergistic effects of mutations and nanoparticle templating in the self-assembly of Cowpea chlorotic mottle virus capsids. Nano Letters 9: 393-398.
  • Klem, M., Suci, P., Britt, D., Young, M., and T. Douglas (2009). In-plane ordering of a genetically engineered viral protein cage. J. Adhesion 85:69-77.
  • Liepold, L., Abedin, M., Buckhouse, E., Frank, J., M.. Young and Douglas, T. (2009). Supramolecular protein cage composite MR contrast agents with extremely efficient relaxivity properties. Nano Lett. 9: 4520-4526.
  • Abedin MJ, Liepold L, Suci P, Young M, Douglas T. (2009). Synthesis of a cross-linked branched polymer network in the interior of a protein cage. J Am Chem Soc. 131(12):4346-54. Lawrence, C. M., S. Menon, B. J. Eilers, B. Bothner, R. Khayat, T. Douglas, and M. J. Young. (2009). Structural and functional studies of archaeal viruses. J Biol Chem 284: 12599-12603.


Progress 01/01/07 to 12/31/07

Outputs
The outputs of this project over this reporting period have been disseminated by five independent mechanisms to the scientific community and the general public. Theses output mechanisms to the scientific community include invited presentations/talks at national and international scientific meetings (13 presentations), invited presentations at other universities or research institutions (6 presentations), and publications in peer-reviews scientific journals (see below). Dissemination to the general public has been though public lectures to community groups (2) informal teaching in K-12 environments (9 presentations), and radio, newspaper, and television interviews and articles (4 total).

Impacts
The long-term outcome of this project is the develop the use of plant viruses as biotemplates for nanomaterials fabrication with beneficial applications in agriculture, medicine, and materials sciences. The objective of this research is to use plant viruses as protein cage architectures for nanomaterials synthesis. The goal is to move towards direct applications in medicine, catalysis and electronics. In the past year we have made significant new progress in producing a new animal cell/tissue targeted high performance drug delivery and imaging technology based our plant virus protein cage biotemplate. We have demonstrated the ability to specifically target, image and deliver therapeutic activity to selected pathogenic bacteria and animal cell and tissue types. In addition, we have made a new type of nanomaterials synthesized within a plant virus capsid that acts as a catalyst for the production of hydrogen from protons (water). The impacts of the research accomplished in the past year are significant. The advances in the past year demonstrated that we can modify plant viruses and other protein cage architectures to impart novel function by design to the assembled cages-like architectures which greatly expands the utility of cage-like architectures as nano templates for nano materials fabrication with applications in nanotechnology, including biomedicine, catalysis and electronics. We have made significant advancements in demonstrating the practical applications of this science in therapeutic delivery and MR imaging as well as a new method for hydrogen production These discoveries significantly contribute the Montana's and the nation's effort in nanotechnology.

Publications

  • M. T. Klem, D. A. Resnick, K. Gilmore, M. Young, Y. U. Idzerda and T. Douglas Synthetic control over magnetic moment and exchange bias in all-oxide materials encapsulated within a spherical protein cage. J Am Chem Soc (2007) 129: 197-201.
  • B.Wiedenheft, M. Flenniken, M. A. Allen, M. Young and T. Douglas Bioprospecting in high temperature environments; application of thermostable protein cages. Soft Matter (2007) 3(9):1091-1098.
  • P. A. Suci, D. L. Berglund, L. Liepold, S. Brumfield, B. Pitts, W. Davison, L. Oltrogge, K. O. Hoyt, S. Codd, P. S. Stewart, M. Young and T. Douglas High-density targeting of a viral multifunctional nanoplatform to a pathogenic, biofilm-forming bacterium. Chemistry & Biology (2007) 14(4): 387-398.
  • M. Uchida, M. T. Klem, M. Allen, P. Suci, M. Flenniken, E. Gillitzer, Z. Varpness, L. O. Liepold, M. Young and T. Douglas Biological containers: Protein cages as multifunctional nanoplatforms. Advanced Materials (2007) 19(8): 1025-1042.
  • L.O. Liepold, S. Anderson, D. Willits, L. Oltrogge, J.A. Frank, T. Douglas, and M. Young Viral Capsids as MRI contrast agents Magnetic Resonance in Medicine (2007) 58(5):871-880.
  • Klem, M. T., D. A. Resnick, K. Gilmore, M. Young, Y. U. Idzerda, and T. Douglas Synthetic control over magnetic moment and exchange bias in all-oxide materials encapsulated within a spherical protein cage. Journal of the American Chemical Society (2007) 129:197-201.
  • C. R. Kaiser, M. Flenniken, E. Gillitzer, A. G.Harmsen, A. L.Harmsen, M. Jutila, T.Douglas, and M. J. Young Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo. Int J Nanomedicine (2007) 2(4):1-18.
  • Larson, E.T., B.J. Eilers, D. Reiter, A.C. Ortmann, M.J. Young, and C.M. Lawrence A new DNA binding protein highly conserved in diverse crenarchaeal viruses. Virology (2007) 363:387-396.
  • Roberto, F., Watkins, M., Grogan, D., and Young, M. Initial characterization of a 32-kb plasmid from a Yellowstone strain of Sulfolobus islandicus. Plasmid 57 (2) 226-227, 2007.
  • Larson, E.T., B. Eilers, S. Menon, D. Reiter, A.C. Ortmann, M.J. Young, and C.M. Lawrence A winged-helix protein from Sulfolobus turreted icosahedral virus points toward stabilizing disulfide bonds in the intracellular proteins of a hyperthermophilic virus. Virology (2007) 368:249-261. P.Suci, Z. Varpness, E. Gillitzer, T. Douglas, and M. Young Photodynamic killing of a microbial pathogen using protein cage architectures functionalized with a photosenistizer. Langmuir (2007) 23: 12280-12286.
  • Snyder JC, Wiedenheft B, Lavin M, Roberto FF, Spuhler J, Ortmann AC, Douglas T, Young M Virus movement maintains local virus population diversity. Proc Natl Acad Sci U S A. 2007 27;104(48):19102-7.


Progress 01/01/06 to 12/31/06

Outputs
The aim of this project is to develop the use of plant viruses as biotemplates for nanomaterials fabrication with beneficial applications in agriculture, medicine, and materials sciences. The purpose of this research is to use plant viruses as protein cage architectures for nanomaterials synthesis. The goal is to move towards direct applications in medicine, catalysis and electronics. In the past year we have made significant progress in producing a new high performance drug delivery and imaging technology based our plant virus protein cage biotemplate. We have move forward in testing these materails in in vivo model sytems. In addition, we have made significant progress in making high performance nanomaterial catalysts for hydrogen synthesis.

Impacts
The impacts of the research accomplished in the past year are likely to be significant. The advances in the past year demonstrated that we can modify plant viruses and other protein cage architectures to impart novel function by design to the assembled cages-like architectures which greatly expands the utility of cage-like architectures as nano templates for nano materials fabrication with applications in nanotechnology, including biomedicine, catalysis and electronics. We have made significant advancements in demonstrating the practical applications of this science in drug delivery and MR imaging as well as a new method for hydrogen production These discoveries significantly contribute the Montana's and the nation's effort in nanotechnology.

Publications

  • Uchida M, Flenniken ML , Allen M, Willits DA, Crowley BE, Brumfield S, Willis AF, Jackiw L, Jutila M, Young MJ, Douglas T. Targeting of cancer cells with ferrimagnetic ferritin cage nanoparticles. JACS 128 (51): 16626-16633, 2006.
  • Douglas T, Young M. Viruses: Making friends with old foes Science 312 (5775): 873-875, 2006.
  • Suci, PA, Klem, MT, Arce, FT, Douglas, T and, Young, M. Assembly of multilayer films incorporating a viral protein cage architecture. Langmuir 22 (21): 8891-8896, 2006.
  • Ortmann, A., Wiedenheft, B., Douglas, T. and M. Young. Hot Archaeal Viruses Reveal Deep Evolutionary Connections. Nature Reviews Microbiology 4 (7): 520-528, 2006.
  • Flenniken, M.L., Willits, D.A., Harmsen, A.L., Liepold, L.O., Harmsen, A.G., Young, M. J., Douglas., T. Melanoma and lymphocyte cell-specific targeting incorporated into a heat shock protein cage architecture. Chemistry and Biology. 13(2):161-70, 2006.
  • Tang J, Johnson JM, Dryden KA, Young MJ, Zlotnick A, Johnson JE. The role of subunit hinges and molecular "switches" in the control of viral capsid polymorphism.J Struct Biol. 154(1):59-67, 2006.
  • Ramsay B, Wiedenheft B, Allen M, Gauss GH, Martin Lawrence C, Young M, Douglas T. Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus. J Inorg Biochem.. 100 (5-6): 1061-1068, 2006.
  • Resnick DA, Gilmore K, Idzerda YU, Klem MT, Allen M, Douglas T, Arenholz E, Young M. Magnetic properties of Co3O4 nanoparticles mineralized in Listeria innocua Dps. J. Appl. Phys. 99 (8):. No. 08Q501 2006.
  • Speir, J., Bothner, B., Qu, C., Willits, D., Young, M., and John E. Johnson. Enhanced local symmetry interactions globally stabilize a mutant virus capsid that maintains infectivity and capsid dynamics. J. Virol. 80:3582-3591, 2006.
  • Maaty, W., Ortmann, A., Dlakic, M., Schulstad, K., Hilmer, J., Liepold, L., Weidenheft, B., Douglas, T., Young, M., and B. Bothner. Characterization of the archaeal thermophile Sulfolobus turreted icosahedral virus validates an evolutionary link among double-stranded DNA viruses from all domains of life J. Virol. 80:7625-7635. 2006.
  • Gauss, G., Bena, P., Wiedenheft, B., Young, M., Douglas, T., and C. M. Lawrence. Structure of the DPS-like protein from Sulfolobus solfataricus reveals a bacterioferritin-like dimetal binding site within a DPS-like dodecameric assembly. Biochemistry 45 (36): 10815-10827, 2006.
  • Larson, E., Reiter, D., Young, M., and C. M. Lawrence. The structure of A197 from Sulfolobus 10. Turreted Icosahedral Virus: a crenarchaeal viral glycosyltransferase exhibiting the GT-A fold. J. Virol. 80 (15): 7636-7644, 2006.
  • Gillitzer, E., Suci, P., Douglas T., and M. Young. Controlled ligand display on a symmetrical protein cage architecture through mixed assembly. Small 2 (8-9): 962-966, 2006.
  • Barry, R., Young, M., Stedman, K., E. Dratz. Proteomic mapping of the hyperthermophilic and acidophilic archaeon Sulfolobus solfataricus P2. Electrophoresis 27 (14): 2970-2983, 2006.


Progress 01/01/05 to 12/31/05

Outputs
The aim of this project is to develop the use of plant viruses as biotemplates for nanomaterials fabrication with beneficial applications in agriculture, medicine, and materials sciences. The purpose of this research is to use plant viruses as protein cage architectures for nanomaterials synthesis. The goal is to move towards direct applications in medicine, catalysis and electronics. In the past year we have made significant progress in producing a new high performance drug delivery and imaging technology based our plant virus protein cage biotemplate.

Impacts
The impacts of the research accomplished in the past year are likely to be significant. The advances in the past year demonstrated that we can modify plant viruses and other protein cage architectures to impart novel function by design to the assembled cages-like architectures which greatly expands the utility of cage-like architectures as nano templates for nano materials fabrication with applications in nanotechnology, including biomedicine, catalysis and electronics. We have made significant advancements in demonstrating the practical applications of this science in drug delivery and MR imaging as well as a new method for hydrogen production These discoveries significantly contribute the Montana's and the nation's effort in nanotechnology.

Publications

  • Khayat R, Tang L, Larson ET, Lawrence CM, Young M, Johnson JE.. Structure of an archaeal virus capsid protein reveals a common ancestry to eukaryotic and bacterial viruses. P NATL ACAD SCI USA 102 (52): 18944-18949, 2005.
  • Wiedenheft B, Mosolf J, Willits D, Yeager M, Dryden KA, Young M, Douglas T.An archaeal antioxidant: Characterization of a Dps-like protein from Sulfolobus solfataricus. P NATL ACAD SCI USA 102 (30): 10551-10556, 2005.
  • Botero LM, D'Imperio S, Burr M, McDermott TR, Young M, Hassett DJ. Poly(A) polymerase modification and reverse transcriptase PCR amplification of environmental RNA. APPL ENVIRON MICROBIO 71 (3): 1267-1275, 2005.
  • Johnson JM, Tang J, Nyame Y, Willits D, Young MJ, Zlotnick A. Regulating self-assembly of spherical oligomers.Nano Lett. 5(4):765-70, 2005.
  • Varpness Z, Peters JW, Young M, Douglas T. Biomimetic synthesis of a H-2 catalyst using a protein cage architecture NANO LETTERS 5 (11): 2306-2309, 2005.
  • Klem MT, Willits D, Solis DJ, Belcher AM, Young M, Douglas T. Bio-inspired synthesis of protein-encapsulated CoPt nanoparticles ADV FUNCT MATER. 15 (9): 1489-1494, 2005.
  • Flenniken ML, Liepold LO, Crowley BE, Willits DA, Young MJ, Douglas T. Selective attachment and release of a chemotherapeutic agent from the interior of a protein cage architecture Chem Commun (4): 447-449, 2005.


Progress 01/01/04 to 12/31/04

Outputs
The central research focus of this project continues to be the molecular and chemical basis of plant virus assembly and disassembly. This year we examined the role of the viral nucleic acid in controlling the viral assembly and disassembly process. Results from this work have provided new scientific insights that have that led to the conceptual design of new plant virus control strategies based on interrupting the viral assembly pathway. We have extended these studies beyond plant viruses to include other protein cage architectures found in biology. Like plant viruses, these other protein cage architectures are assembled from subunits that form precisely defined cage-like architectures. In the past year we have demonstrated that we can both genetically and chemically modified plant viruses and other protein cage architectures to impart novel function by design to the assembled cages-like architectures. We have shown that theses modified cage-like architectures can be used as nano templates for nano materials fabrication useful for a broad rage of applications in nanotechnology, including biomedicine, catalysis and electronics.

Impacts
The impacts of the research accomplished in the past year are likely to be significant. The discovery of the role that the viral nucleic acid in affecting plant virus assembly and disassembly is a significant step in our goal of developing new control measures caused by plant viruses and our efforts to design small molecules that specifically block the virus assembly and disassembly process. The advances in the past year demonstrated that we can modify plant viruses and other protein cage architectures to impart novel function by design to the assembled cages-like architectures which greatly expands the utility of cage-like architectures as nano templates for nano materials fabrication with applications in nanotechnology, including biomedicine, catalysis and electronics. These discoveries significantly contribute the Montana's and the nation's effort in nanotechnology.

Publications

  • Wiedenheft, B., Stedman , K., Roberto, F., Willits, D., Gleske, A., Zoeller, L., Snyder, 13. J., Douglas, T., and M. Young. Comparative genomic analysis of hyperthermophilic archaeal Fuselloviridae viruses. J. Virology 78 1954-1961. 2004.
  • Johnson, J., Willits, D., Young, M., and A. Zlotnick. Interactions with capsid protein alters RNA structure and the pathway for in vitro assembly of Cowpea chlorotic mottle virus. J. Mol. Bio 335:455-464. 2004.
  • Brumfield, S., Willits, D., Tang, L., Johnson, J.E., Douglas, T., and M Young. Heterologous expression of modified CCMV coat protein results in the assembly of protein cages with altered architectures and function. J. Gen Virol. 85:1048-1053. 2004.
  • Ensign, D., Young, M., and T. Douglas. Photocatalytic synthesis of copper colloids from Cu (II) by the ferrihydrite core of ferritin. Inorg. Chem. 43:3441-3446. 2004.
  • Botero, L., Brown, K., Brumefield, S., Burr, M., Castenholz, R., Young, M., and T. McDermott. Thermobaculum terrenum gen. nov., sp. Nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia. Arch Microbiol 181:269-277. 2004.
  • Rice, G., Tang, L, Stedman, K., Roberto, F., Sphuler, J., Johnson, J.E., Douglas, T., and M. Young. The structure of a thermophilic archaeal virus shows that a dsDNA viral capsid type spans all three domains of life. Proc. Natl. Acad. Sci. USA 101:7716-7720. 2004.
  • Kraft, P., Oeckinghaus, A., Kummel, D., Gauss, G., Gilmore, J., Wiedenheft, B., Young, M., and M. Lawrence. Crystal structure of F-93 from Sulfolobus spindle-shaped Virus 1, a winged-helix DNA binding Protein. J. Virol. 78:11544-11550. 2004.
  • Kraft, P., A., Kummel, D., Oeckinghaus, A., Gauss, G., Wiedenheft, B., Young, M., and M. Lawrence. Structure of D-63 from Sulfolobus spindle-shaped virus 1: Surface properties of the dimeric four-helix bundle suggest an adaptor protein function J. Virol. 78:7438-7442. 2004.
  • Flenniken, M., Allen, M., Young, M., and T. Douglas. Viruses as host assemblies. Encyclopedia of Supramolecular Chemistry. 2004.


Progress 01/01/03 to 12/31/03

Outputs
The overall goal of this project is to elucidate the fundamental biological and chemical process dictating plant virus assembly and disassembly. These studies combine the tools of molecular biology, biochemistry and structural biology to accomplish this goal. This past year our efforts have focused on understanding the non-covalent interactions that are used between the protein subunits that control the self-assembly of the Cowpea chlorotic mottle virus (CCMV). One hundred and eighty coat protein subunits self assembly around the viral nucleic acid to from the virus particle. In the past year, we have also focused our attention on the mechanism by which the viral RNA is released from the CCMV particle once it reaches a susceptible plant. Significant advances have been made on both fronts. Our understanding of plant virus assembly has progressed significantly in the past year. Two major discoveries were made. The first describes in chemical detail the pathway for CCMV assembly. We discovered that virus assembly is initiated from a non-covalent dimer of the coat protein. This dimer then assembles into a pentamer of dimers and addition of more coat protein dimers to this nucleating center results in the assembled virus particle with 180 subunits. We have learned how to manipulate the interaction of the subunits such that we can dictate the type of morphology the final assembly product. This has had significant impact on our use of these assembled viral cages as constrained reaction vessels for nanomaterials synthesis with significant applications in medicine, catalysis and electronics. The second major are of advancement has been to elucidate the structure of the viral RNA-coat protein interaction that nucleates the CCMV assembly process. We have discovered two high affinity binding sites on the viral RNA that initiate the assembly process. The discovery of these regions is a significant step in our goal developing new control measures caused by plant viruses by designing small molecules that specifically block the virus assembly process. Significant progress was also accomplished in understanding the disassembly of CCMV upon reaching a host plant cell. The disassembly mechanism is not simply the reverse of the assembly mechanism discussed above. In fact, a completely new disassembly mechanism has been discovered. This disassembly mechanism is based on structural transitions at the five-fold axis of the virus particle that forms a channel for the release of the viral genome into the cells. The discovery of this new mechanism is a significant step in our goal developing new control measures caused by plant viruses by designing small molecules that specifically block the virus disassembly process.

Impacts
The impacts of discoveries made in the past year are likely to be significant. The discovery of new virus assembly and disassembly mechanisms is a significant step in our goal developing new control measures caused by plant viruses by designing small molecules that specifically block the virus assembly and disassembly process. These discoveries provide new targets for the development of effective control strategies for agriculturally important plant viruses in Montana and nationally. In addition, the progress made this year contributes significantly to our development of assembled viral cages as constrained reaction vessels for nanomaterials synthesis with significant applications in medicine, catalysis and electronics.

Publications

  • Willits, D., Zhao,X., Olson, N., Baker, T.S., Zlotnick, A., Johnson, J.E., Douglas, T., and M. J. Young. Effects of the Cowpea chlorotic mottle bromovirus beta-hexamer structure on virion assembly. Virology 306:280-288, 2003.
  • Snyder, J., Stedman, K., Rice, G., Wiedenheft, B., Sphuler, J., and M. J. Young. Viruses of hyperthermophilic Archaea. Research in Microbiology 154:446-473, 2003.
  • T. Douglas, M. Allen, M. Young Self-assembling protein cage systems and applications in nanotechnology, Biopolymers 8:405-426, 2003.
  • Klem, M., Willits, D., Young, M., and T. Douglas. 2-D Array formation of genetically engineered viral cages on Au surfaces and imaging by atomic force microscopy. JACS 125:1056-1057, 2003.
  • Flenniken, M., Willits, D., Brumfield, S., Young, M. and T. Douglas. The small heat shock protein from Methanococcus jannaschii is a versatile nano-scale platform for genetic and chemical modifications. Nano Letters 3:1573-1556, 2003.
  • Allen, M., Willits, D., Young, M., and T. Douglas. Constrained synthesis of colbalt oxide nano materials in the 12-subunit protein cage from Listeria innocua. Organic Chemistry 42:6300-6305, 2003.
  • Basu, G., Allen, M., Willits, D., Young, M., and T. Douglas. Metal binding to cowpea chlortic mottle virus using terbium (III) fluorescence. J. Biol Inorg Chem 8:721-725, 2003.
  • Schneemann, A., and M. Young. 2003 Viral assembly using heterologous expression systems and cell extracts. Adv. Protein Chem. W. Chui and J. Johnson eds., Elsevier Press. New York.
  • Johnson, J., Willits, D., Young, M., and A. Zlotnick. Interactions with capsid protein alters RNA structure and the pathway for in vitro assembly of Cowpea chlorotic mottle virus. J. Mol. Bio 2003.


Progress 01/01/02 to 12/31/02

Outputs
The overall objective of this project is to investigate the chemical and molecular basis of spherical virus assembly and disassembly. Through these studies we have developed the novel use of viral protein cages (devoid of their viral genomes) as constrained reaction vessels. This development has had a significant impact on the emerging field of nanotechnology by providing new materials for application in biomedical and material sciences. Our analysis of virus assembly and disassembly in the past year has led to the important discoveries on the nature of the intermediate building blocks essential for virus particle assembly. Previous to our studies, it was not known that CCMV virus assembly occurs through dimmer formation of the coat protein subunit and that these dimmers assemble into a pentamer of dimmers. Genetic analysis reveals that blocking either dimmer formation or the formation of pentamers blocks infectious virus particle assembly. These studies have allowed us to develop strategies for the genetic and chemical modifications of the coat protein building blocks without affecting their ability to assemble into cage-like structures. This has allowed us to develop methods to target these protein cages to filled with therapeutic drugs and/or imaging agents to cells of medical interest (e.g. metastasizing breast cancer cells). In the past year we have also investigated the role of metals in the assembly process. Previous studies had suggested that Ca binding was essential for virion assembly and infectivity. Contrary to these predictions, we have shown that Ca binding is not essential for virus particle formation. Genetic analysis of the predicted CCMV metal binding sites demonstrates that virus assembly does occur in the absence of Ca binding. However, metal binding is essential for controlling a structural transition in the virus particle. We have taken advantage of our knowledge of metal binding to the viral protein cage to alter metal binding specificity. For example, we have altered the Ca metal binding site to bind Gd with high affinity. By doing so we have created a MRI bioimaging agent which is currently being evaluated by NIH for imaging in humans.

Impacts
As a result of this research, we have pioneered the use of empty viral protein cages (devoid of their nucleic acid) as constrained reaction vessels for nano materials synthesis and/or entrapment. This work has significant implications for both biomedical and material sciences.

Publications

  • Douglas, T., Strable, E., Willits, D., Aitouchen, A., Libera, M., and M. Young. Protein engineering of a viral cage for constrained nano-materials synthesis. Adv. Mater 14:123-133. 2002.
  • Gillitzer, E., Willits, D., Young, M., and T Douglas. Chemical modification of a viral cage for multivalent presentation. ChemComm. 2390-2391. 2002.
  • Willits, D., Zhao, X., Olson, N., Baker, T.S., Zlotnick, A., Johnson, J.E., Douglas, T., and M. J. Young. Effects of the Cowpea chlorotic mottle bromovirus B-hexamer structure on virion assembly. Virology 2002.
  • Snyder, J., Stedman, K., Rice, G., Wiedenheft, B., Spuhler, J., and M. J. Young. Viruses of hypertehrmophilic Archaea. Extremophiles 2002.
  • Douglas, T., Allen, M., and M. Young. Self-assembling protein cage systems and applications in nanotechnology. Biopolymer 2002.
  • Allen, M., Willits, D., Mosolf, J., Douglas, T., and M. J. Young. Protein cage constrained synthesis of ferromagnetic iron oxide nanoparticles. Adv Mater. 14: 1562-1565. 2002.


Progress 01/01/01 to 12/31/01

Outputs
The overall objective of this project is to investigate the chemical and molecular basis of spherical virus assembly and disassembly. It is reasoned that a fundamental understanding of the molecular process involved in virus assembly and disassembly will provide new basic knowledge that will be useful in devising new anti-viral strategies for protecting agriculturally important crops. Our analysis of virus assembly and disassembly in the past year has led to the important discoveries on the nature of the intermediate building blocks essential for virus particle assembly. Previous to our studies, it was not known that CCMV virus assembly occurs through dimmer formation of the coat protein subunit and that these dimmers assemble into a pentamer of dimmers. Genetic analysis reveals that blocking either dimmer formation or the formation of pentamers blocks infectious virus particle assembly. This information provides essential insight into virion assembly and the design of anti-viral strategies based on blockage of assembly intermediates. In the past year we have also investigated the role of metals in the assembly process. Previous studies had suggested that Ca binding was essential for virion assembly and infectivity. Contrary to these predictions, we have shown that Ca binding is not essential for virus particle formation. Genetic analysis of the predicted CCMV metal binding sites demonstrates that virus assembly does occur in the absence of Ca binding. However, metal binding is essential for controlling a structural transition in the virus particle. This structural transition causes swelling of the virus particle. The swelling process is essential for the infection process because swelling allows for the release of the viral nucleic acid during disassembly of the virus. These results provide additional targets for new classes of anti-viral agents based on blockage of virion swelling. Our studies of virus particle assembly and disassembly have led to a completely novel use of viruses. We have pioneered the use of empty viral protein cages (devoid of their nucleic acid) as constrained reaction vessels for nano materials synthesis and/or entrapment. This work has significant implications for both biomedical and material sciences. For example, we have demonstrated that the CCMV protein cage can be used to bind Gd, a metal used as an MRI biocontrast agent. Preliminary analysis of the CCMV protein cage with bound Gd has been performed by the NIH Bioimaging laboratory. They report that the virus protein cage with bound Gd is the best experimental MRI bioimaging agent they have tested to date. In a second example, we have demonstrated that we can synthesize magnetic materials within the CCMV protein cage. These magnetic materials are being tested as a new class of materials for applications in magnetic storage devices.

Impacts
As a result of this research, we have discovered new targets for developing new anti-viral strategies to protect crop plants from viral infection. In addition, we have pioneered the use of empty viral protein cages (devoid of their nucleic acid) as constrained reaction vessels for nano materials synthesis and/or entrapment. This work has significant implications for both biomedical and material sciences.

Publications

  • Douglas, T., Strable, E., Willits, D., Aitouchen, A., Libera, M., and M. Young. Protein engineering of a viral cage for constrained nano-materials synthesis. Adv. Mater 14:123-133. 2001.
  • Schneemann, A., and M. Young. Viral assembly using heterologous expression systems and cell extracts. Adv. Protein Chem. 2001.
  • Elumalai, S., Brey, C., Dyer, W., Talbert, L., Young, M., Qu,R. Resistance to wheat streak mosaic virus mediated by an RNA replicase gene in transgenic wheat. Transgenic Research 2001.
  • Baley, G., Talbert, L., Martin, J., Young, M., Habernicht, D., Kushnak, G., Berg, J., Lanning, S., and P. Bruckner. Agronomic and end-use qualities of Wheat streak mosaic virus resistant spring wheat. Crop Sci. 41 2001.


Progress 01/01/00 to 12/31/00

Outputs
Over the past year we have expanded our understanding of the assembly and disassembly of spherical plant viruses. We have described the chemical and molecular events in the assembly and disassembly in the model plant virus system, Cowpea chlorotic virus (CCMV). We have discovered that the CCMV virus assembly proceeds though the sequential subunit assembly of dimers, followed by the assembly of pentamers of dimer subunits. These finding are in contrast to previously held models that viral assembly proceeds through hexamers intermediates. The assembly through pentameric intermediates allows the viral assembly a higher degree of flexibility in the size and architecture of the final assembled viral protein cage. We have also discovered that the disassembly of CCMV is not simply the reverse of the assembly pathway. Instead, virion disassembly proceeds through a novel mechanism requiring large structural changes in the viral protein cage. Disassembly is caused by a large structural transition of the protein shell of the virus that releases the viral RNA into the cell through a newly created pore in the viral protein shell. These results have allowed us to develop a completely new use of viruses as constrained reaction vessels for nano material synthesis and release. These results have greatly expanded our use of viral protein shells for applications in biotechnology and nano engineering.

Impacts
Our studies continue to proved the basic research needed to develop novel approaches for the design and testing of new anti-viral drugs/chemicals for use in both plant and animal systems. Strategies based on interference with viral assembly and disassembly are likely to be some of the most effective antiviral drugs. The use of assembled protein cages as constrained reaction vessels (i.e. nano `cooking pots') has had significant impacts. For example, we are developing this technology to create a combined non-invasive cancer MRI imaging agent and drug delivery system. In addition, we are exploring the synthesis of nano materials of great interest to the electronics industry using viral protein cages as organic templates.

Publications

  • Zlotnic, A., Aldrich, R. Johnson, J., Ceres, P., and M. Young. Mechanism of capsid assembly for an icosahedral plant virus. Virology 277, 450-456 (2000).
  • Young, M. and D. Willits. Virus Structure In Encyclopedia of Plant Pathology, (O. Maloy and T. Murry Eds) John Wiley & Sons. (2000).


Progress 01/01/99 to 12/31/99

Outputs
The main focus during this reporting period has been a detailed chemical understanding of spherical virus assembly and disassembly. An understanding of virus assembly and disassembly is critcal to our virus transmission within infected plants and between host plants. Our model system for these studies has been Cowpea chlorotic mottle virus (CCMV). We have demonstrated that CCCMV viral assembly and disassembly proceed by two distinct mechanisms. Assembly proceeds through the assembly of a pentamer of dimers while disassembly proceeds via channel formation of coat protein N-termini.

Impacts
Two major impacts of this research include (1) the novel use of viral protein cages as constrained reaction vessels and (2)the use of viral protein cages as a platform for expression of heterologus proteins for applications in biomedical and material sciences.

Publications

  • Douglas, T., Young, M. J., Virus particles as templates for nanophase materials synthesis, Adv. Mater., 11: 679-681, 1999.
  • Shenton, W., Douglas, T., Young, M., Stubbs, G., Mann, S., Inorganic-organic nanotube composites from template mineralization of tobacco mosaic virus, Adv. Mater., 11: 253-256, 1999.
  • Young, M., Flichkin, S., Luteovirus interactions with aphid vector cellular components. Trends MicroBio. 7:466-348, 1999.
  • Young, M., Willits, D., Plant virus structure. Encylp. PlantPath., Academic Press, New York. 1999.
  • Young, M. J., Filichkin. Luteovirus interactions with aphid vector cellular components. Trends MicroBiol. 7:346-347. 1999.
  • Elumalai, S., Brey, C., Dyer, W., Talbert, L., Young, M., Qu,R. Resistance to wheat streak mosaic virus mediated by an RNA replicase gene in transgenic wheat. Phytopathology, 1999.


Progress 01/01/98 to 12/31/98

Outputs
Significant progress has been accomplished in the past year concerning our understanding of spherical virus assembly and disassembly. This includes the discovery of the critical role of metals in virus assembly and disassembly and how metal biding signals the movement of virus from one plant cell to an adjacent plant cell. We have also analyzed the role different regions of the viral structural protein play in determining the architecture of the virus. As a result of these studies we developed a completely new application for viral protein cages as containers for nanophase material synthesis and entrapment. This application has been patented and provides the basis for the design and delivery of materials with important application in both the biological and material sciences.

Impacts
(N/A)

Publications

  • Douglas, T., M.J. Young. Host -guest encapsulation of materials by assembled virus protein cages, Nature 393:152-155, 1998.
  • Douglas, T., M.J. Young. Virus particles as templates for nanophase materials synthesis, Adv. Mater., In press 1998.
  • Shenton, W., T. Douglas, M.J. Young, G. Stubbs, and S. Mann. Inorganic-organic nanotube composites from template mineralization of tobacco mosaic virus, Adv. Mater., In press 1998.
  • Fox, J., G. Wang, J. Speir, N. Olson, J. Johnson, T. Baker, and M.J. Young. Comparison of the native CCMV virion with in vitro assembled CCMV virions by cryo-electron microscopy and image reconstruction. Virology, 224:212-218, 1998.
  • Young, M.J. 1998. CCMV assembly:Chemical switches and material synthesis. Annual Meeting of American Society for Biochemistry and Molecular Biology. May 16-20, Washington, D.C. (Abst. 139).
  • Young, M.J. and T. Douglas. 1998. CCMV as a constrained reaction vessel for inorganic and organic material synthesis. Fifth International Symposium on Positive Strand RNA Viruses. May 23-28, St. Petersburg, Fl (Abst. S3-06).
  • Young M.J. 1998. Lessons from virus assembly and disassembly studies. 17th Annual Meeting American Society of Virology. July 11-15, Vancouver, British Columbia, Canada (Invited 'State of the Art' paper).
  • Filichkin, S., S. Brumfield and M.J. Young 1998. Cowpea chlorotic mottle virus: Evidence of covalent bond formation between N-terminal portions of two coat protein subunits. 17th Annual Meeting American Society of Virology. July 11-15, Vancouver, British Columbia, Canada 9Abst. P12-2).
  • Li, N., S. Brumfield and M.J. Young. 1998. Analysis of CCMV Co-translational disassembly. 17th Annual Meeting American Society of Virology. July 11-15, Vancouver, British Columbia, Canada (Abst W28-3).
  • Filichkin, S., S. Brumfield, T. Filichkin and M.J. Young. 1998. Identification of capsid protein domains involved in transmission specificity of barley yellow dwarf virus. 17th Annual Meeting American Society of Virology. July 11-15, Vancouver, British Columbia, Canada (Abst. P6-25).


Progress 01/01/97 to 12/31/97

Outputs
During 1997 we further established cowpea chlorotic mottle virus (CCMV) as a model system for examining spherical virus assembly and disassembly. We identified and chemically characterized multiple RNA-protein and protein-protein interactions that dictate virus particle stability, architecture and assembly. We continued to gather experimental data to support a new a model for virus disassembly that we proposed last year that has broad implications for plant, animal, and insect viruses. We also completed analysis of the virion structural domains of barley yellow dwarf virus (BYDV) that control virus transmission by its aphid vectors. Our results demonstrated that it is the N-terminal domain of the BYDV coat protein that controls aphid transmission specificity.

Impacts
(N/A)

Publications

  • FILICHKIN, S.A., S. BRUMFIELD, T.P. FILICHKIN, and M.J. YOUNG. 1997. In vitro interactions of the aphid endosymbiotic SymL chaperonin with barley yellow dwarf virus. J. Virology 71:569-577. ALBERT, F.A., J.M. FOX, and M.J. YOUNG. 1997. Virion swelling is not required for cotranslational disassembly of cowpea chlortic mottle virus in vitro. J. Virology 71:4296-4299. FOX, J.M., F.A. ALBERT, J.A. SPEIR, and M.J. YOUNG. 1997. Characterization of a disassembly deficient mutant of cowpea chlorotic mottle virus. Virology 227:229-233.
  • FILICHKIN, T., S. BRUMFIELD, S. FILICHKIN, and M.J. YOUNG, 1997. Barley yellow dwarf virus: transmission properties of the virions with a mosaic coatprotein. 16th Annual Meeting American Society for Virology, July 19-23, Bozeman MT (Abst w16-1).
  • FILICHKIN, S., S. BRUMFIELD, T. FILICHKIN, and M.J. YOUNG.1997. Protein-protein interactions of the Sym L chaperonin with barley yellow dwarf virus. 16th Annual Meeting American Society for Virology, July 19-23, Bozeman MT (Abst w16-5).
  • BRUMFIELD, S., T. CARROLL, M.J. YOUNG, and E. SMIDANSKY. 1997. A morphological examination of the brown wheat mite and its relationship to the barley yellow streak mosaic virus. 16th Annual Meeting American Society for Virology, July 19-23, Bozeman MT (Abst


Progress 01/01/96 to 12/30/96

Outputs
During 1996 we further established cowpea chlorotic mottle virus (CCMV) as a model system for examining spherical virus assembly and disassembly. We identified and chemically characterized multiple RNA-protein and protein-protein interactions that dictate virus particle stability, architecture and assembly. As a result of these studies we developed a new model for virus disassembly that has broad implications for plant, animal, and insect viruses.

Impacts
(N/A)

Publications

  • Fox, J., Albert, F., Speir, J., and M. J. Young. 1997. Characterization of a disassembly deficient mutant of cowpea chlorotic mottle virus. Virology 227:229-233.
  • Albert, F., Fox, J., and M. J. Young. 1997. Virion swelling is not required for cotranslational disassembly of cowpea chlorotic mottle virus in vitro. J. Virology 227:229-235.
  • Fox, J., Zhao, X., and M. J. Young. 1996. Analysis of a salt stable mutant of cowpea chlorotic mottle virus. Virology 222:115-122.
  • Zhao, X., Kuhn. R., and M. J. young. 1996. Mutational analysis of the putative Ca binding sites in the cowpea chlorotic mottle virus capsid. 15th Ann Mtg ASV. Univ of W Ontario, London, Ontario, Canada July 13-17, 1996 (Abst. W5-2). Young,.