Progress 04/14/10 to 04/13/15
Outputs
OUTPUTS: We have made good progress in achieving our objectives: Objective 1. BMV replication mechanism in plants: we identified an new interaction between BMV and host lipid synthesis that may contribute to BMV-promoted lipid synthesis. We have made more mutants in 1a's amphipathic alpha-helix to strengthen our understanding on how the helix is involved in host membrane rearrangemnts. Objective 2: Functional roles of host acyl-CoA binding protein (ACBP) in BMV replication. We have found that supplementing unsaturated fatty acids can largerly complement BMV replication. We have purified ACBP protein and tested its ability to bind phospholipids in vitro. The above have resulted in a peer-reviewed paper and several presentations. PARTICIPANTS: 1 post-doctoral associate, 1 undergraduate student, and 2 high school students have been trained in the project with significant contributions from all participants. TARGET AUDIENCES: Target audience was research peers and the plant virology research community PROJECT MODIFICATIONS: This research project is being terminated due to the investigator's move to Virginia Tech University.
Impacts
Using the BMV as a model system, we are understanding the replication of RNA viruses in the host cell in order to define control mechanisms and strategies.
Publications
- Zhang J., Diaz A., Mao L., Alquist P., and Wang X. (2012). Host Acyl Coenzyme A binding protein regulates replication complex assembly and activity of a positive-strand RNA virus. Journal of Virology 86: 5110-5121.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities: Conducted and continued the experiments according to our research objectives and last year's findings. mentored graduate student, post-doctoral associate, and high school students. Events: Attended 30th annual meeting of American Society of Virology, 20th Noble Foundation Virology Retreat, 1st Huazhong Agricultural University and TAMU bilateral symposium on plant pathology and biology. Services: Ad hoc reviewer for NSF, Journal of Visualized Experiments PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have made good progress in achieving our objectives: Objective 1. BMV replication mechanism in plants: we identified an new interaction between BMV and host lipid synthesis that may contribute to BMV-promoted lipid synthesis. We have made more mutants in 1a's amphipathic alpha-helix to strengthen our understanding on how the helix is involved in host membrane rearrangemnts. Objective 2: Functional roles of host acyl-CoA binding protein (ACBP) in BMV replication. We have found that supplementing unsaturated fatty acids can largerly complement BMV replication. We have purified ACBP protein and tested its ability to bind phospholipids in vitro. Objective 3: Controlling BMV replication by manipulating lipid synthesis. We have generated Nicotianan benthamiana transgenic plants expressing artificial microRNAs targeting the expressing of plant Stearoyl-ACP desaturase. We are in the process testing virus infection and fatty acid compositions. The above have resulted in a peer-reviewed paper and several presentations. 1 post-doctoral associate, 1 graduate student, and 2 high school students have been trained in the project with significant contributions from all participants.
Publications
- Publication: Wang X., Diaz A., Hao L., Gancarz B., den Boon J.A., and and Ahlquist P. 2011. Intersection of the multivesicular body pathway and lipid homeostasis in RNA replication by a positive-strand RNA virus. Journal of Virology 85:5494-5503, Co-corresponding authors: Wang X. and Ahlquist P., (Featured in SPOTLIGHT.
- Presentations: Wang X., Diaz A., Mao L., Zhang J. and Ahlquist P. 2011 Host acyl-CoA binding protein is required for genomic replication of a positive-strand RNA virus. 30th ASV annual meeting, Minneapolis, Minnesota, July 16-20.
- Diaz A., Wang X., and and Ahlquist P. 2011 Host ESCRT components are required for bromovirus RNA replication compartment assembly. 30th ASV annual meeting, Minneapolis, Minnesota, July 16-20.
- Hao L., Wang X., and Ahlquist P. 2011 Altering host metabolic fluxes changes fate of virus infections. 30th ASV annual meeting, Minneapolis, Minnesota, July 16-20.
- Wang X., Diaz A., Mao L., Zhang J. and Ahlquist P. 2011 Host acyl-CoA binding protein is required for genomic replication of a positive-strand RNA virus. 20th Virology Retreat, Noble Foundation, OK, Apr. 29-May 1.
- Zhang J. Garcia-Raiz H., and Wang X. 2011 Lipid composition of cell membranes is critical for Brome mosaic virus replication in plants. 20th Virology Retreat, Noble Foundation, OK, Apr. 29-May 1.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Activities: All positive-strand RNA viruses replicate on intracellular membranes in association with vesicular spherules or other membrane rearrangements. BMV replication induces formation of viral replication complexes termed viral spherules in perinuclear ER membranes. BMV 1a is necessary and sufficient to induce formation of the viral spherules. We proposed to study how BMV replication protein 1a induces viral spherule formation in plants (Objective 1), to study the roles of acyl-CoA binding protein in BMV replication (Objective 2), and control replication of BMV and other positive-strand RNA viruses by manipulating host lipid synthesis (Objective 3). We have made good progress in the first and outstanding progress in the last 2 objectives. Objective 1: BMV replication mechanism in plant. We proposed to identify regions and amino acids in BMV 1a that are critical for spherule induction. We have made all the constructs expressing BMV 1a fragments, mutants with mutations at NTPase/helicase domain and the amphipathic alpha-helix domain. BMV replication was inhibited in all these mutants. We are in the process to test spherule formation. Objective 2: Functional roles of host acyl-CoA binding protein (ACBP) in BMV replication. Deleting ACBP inhibited BMV replication up to 30-fold and resulted in formation of the aberrant viral spherules that are smaller but 5-fold more than those in the wild type (wt) cells. We found that BMV 1a specifically interacts with ACBP in the yeast split-ubiquitin system (SUS), which is designed to study protein-protein interactions in the native environment. We have hypothesized that 1a may recruit ACBP to BMV replication by the 1a-ACBP interaction. We analyzed and compared lipid compositions between wt and cells lacking ACBP. Our data showed that BMV induced a ~28% increase in phophatidylcholine (PC) content. Increased PC synthesis has been found to be associated with flock house virus replication, indicating increased PC synthesis is a common feature of (+)RNA virus replication. The BMV-stimulated PC synthesis did not occur in cells with ACBP deleted, suggesting ACBP is required for the process. Objective 3: Controlling BMV replication by manipulating lipid synthesis. We proposed to knock down expression of genes involved in unsaturated fatty acid (UFA) synthesis using artificial miRNA approach. Collaborating with Dr. Hernan Garcia-Reiz at Oregon State University, we designed amiRNAs targeting each of 2 members of Stearoyl-ACP desaturase (SACPD). Dr. Jiantao Zhang, a post-doctoral researcher in the lab, transiently expressed these amiRNA in Nicotiana benthamian plants using agroinfitration. Based on the amount of amiRNA produced, and accumulation of transcript of target genes, he has chosen the best amiRNA from 3 candidates. Dr. Zhang then made transgenic N. benthamiana plants expressing each of the efficient amiRNAs. He is in the process characterizing the transgenic plants in terms of lipid composition and resistance to BMV infection. PARTICIPANTS: 1. Dr. Lan Mao duration: Feb - Sep, 2010 2. Dr. Jiantao Zhang duraiton: March 2010-present 3. Ms Alejandra Gutierrez duration: Jan 2010-present 4. Andrew Hernandes Jan 2010-May 2010, Sep 2010-present 5. Gabriela Ontiveros duration: April 2010-present TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: No major modifications
Impacts
Mentored 2 post-doctoral researchers and 2 high school students. Events: Attended 3 conferences, Noble Foundation Virology Retreat; LIPID MAPS Annual meeting; Annual conference of American Society for Virology. Services: Invited reviewer for NSF grant proposal; invited speaker at the Department of Plant Pathology and Microbiology, TAMU.
Publications
- Wang X., Diaz A., Mao L., and Ahlquist P. (2010). Deletion of Host Acyl-coA Binding Protein Alters Membrane Curvature and Inhibits Genomic Replication of a Positive-Strand RNA Virus. LIPIDS MAPS Annual meeting 2010, San Diego, California. 05/04-08/2010, poster
- Wang X, Diaz A, Bates P., Hao L., Browse J., and Ahlquist P. (2010). Lipid composition of cell membranes is critical for positive-strand RNA virus genome replication in plants. 29th Annual meeting American Society for Virology, 07/17-21/2010. Bozeman, Montana. Presentation
- Wang X. (2010). Functional roles of host acyl-CoA binding protein in brome mosaic virus replication. Invited seminar. Department of Plant Pathology and Microbiology, TAMU. 11/04/2010
- Peer reviewed journal: Diaz A. Wang X. and Ahlquist P. (2010). Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function. Proc. Natl. Acad. Sci. USA, 107: 16291-16296
- Presentations and Posters: Wang. X. (2010). Cellular lipid synthesis/composition and replication of positive-strand RNA viruses: understanding and virus control, Noble Foundation 19th Annual Virology Retreat, 04/30-05/03/2010, presentation.
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