Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to NRP
GENOMIC REPLICATION AND VIRUS-HOST INTERACTIONS OF POSITIVE-STRAND RNA VIRUSES: UNDERSTANDING AND CONTROL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1001997
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jan 10, 2014
Project End Date
Nov 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
School of Plant and Environmental Sciences
Non Technical Summary
The great majority of plant viruses are positive-strand RNA viruses [(+)RNA viruses], which cause tremendous agricultural losses each year. Development of crops with broad-spectrum and stable viral resistance is crucial for fighting mixed infections, which are common in the fields. For example, synergistic infection of soybean by Soybean mosaic virus and Bean pod mottle virus causes much severe infections than that of each virus. A universal feature of (+)RNA virus replication is that they assemble their replication complexes in association with host intracellular membranes, which are usually remodeled by viral proteins during infections. However, the mechanisms of virus-induced membrane rearrangements are not well-understood and the lipid microenvironment of viral replication complexes is not well-defined. A better understanding of such processes will identify novel and promising targets for designing broad-spectrum antiviral strategies. Our project targets the above knowledge gap by using Brome mosaic virus (BMV), which invaginates endoplasmic reticulum membranes to form spherular structures in barley, Nicotiana benthamiana and the baker's yeast. The primary objectives of this proposal are to determine the lipidomic alterations induced by BMV infection in various hosts, dissect the mechanisms by which BMV modulates lipid synthesis, and develop a pipeline to generate crop plants that are resistant to multiple viruses, intially by manipulating host lipid synthesis. Specifically, by using lipidomic and electron microscopic analyses, we will test the hypothesis that BMV recruits host lipid synthesis enzymes to promote phospholipid synthesis dedicated to viral replication. Building on previously work and our preliminary data that unsaturated fatty acids (UFAs) are required for replication of a number of plant viruses, we will generate crop plants that express artificial microRNAs to knock down the expression of UFA synthesis genes to achieve resistance to multiple viruses. By completion of the project, we expect to provide new knowledge on virus-host interactions and new ways to control viral infection.
Animal Health Component
15%
Research Effort Categories
Basic
75%
Applied
15%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21215501101100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1550 - Barley;

Field Of Science
1101 - Virology;
Goals / Objectives
The long-term objectives of this project are to understand virus-host interactions and viral replication mechanisms of (+)RNA viruses as well as to develop mechanism-based, broad-range antiviral strategies. The objectives for the next five years are to: 1. Examine the membrane rearrangements induced during BMV infection in plants. 2. Elucidate the interactions between cellular lipid synthesis and BMV replication. 3. Manipulate host lipid synthesis to control replication of BMV and other plant viruses.
Project Methods
To identify and characterize BMV-induced membrane rearrangements in plants, electron microscopy (EM) analysis will be employed. In addition, anti-BMV replication protein antiserum will be used to determine whether BMV replication proteins are localized with these rearranged membranes by immunogold labeling. To systemically analyze lipid compositional alterations upon BMV infection, we will use a lipidomic approach, which measure the amount of about 200 different lipids. The significance of the lipid changes will be statistically analyzed and these that are significantly altered will be further addressed. These data will enable us to identify potential key lipids that are required for BMV replication. To knock down the expression of host genes that are required for BMV and other viruses by expressing aritificial microRNAs thus to block viral infection in plants.

Progress 01/10/14 to 11/30/18

Outputs
Target Audience:Fellow scientists, Faculty, Academic researchers in the fields of plant virology, pathlogy, and cell biology; Educators (extension agents, k-12); Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have four undergraduate students (two sophomores and two seniors) working in the lab on independent projects under direct supervision of senior graduate students. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. We have cloned several groups of membrane-shaping proteins and tested the effects of knockdown or overexpression of such genes on BMV infection. These genes include Reticulons (RTNs) and Lunapark (LNP). Overexpression of one of cloned RTNs (8 total) inhibited BMV replication; Knocking down gene expression of individual or a combination of LNPs, however, didn't affect BMV replication. We will continue to work on overexpression of membrane-shaping genes and their relationships with viral replication. 2. We have cloned multiple plant genes involved in phospholipid synthesis. To identify those that are involved in BMV replication. We expressed each of them with a mCherry fluorescence protein in yeast and tested their distribution with the absence or presence of BMV 1a. We have identified 3 genes, CEK1 (choline-ethanolamine kinase 1), CEK3, and PLMT (phospholipid methyltransferase), which were redistributed by and co-localized with BMV 1a at the nuclear membrane. We will test the effect of knockdown or overexpression of these genes on BMV replication. 3. We previous found that host CHO2, whose product is involved in phosphatidylcholine synthesis, is required for BMV replication. Deleting CHO2 inhibited BMV replication but affected yeast cell growth. We have generated a Cho2p mutant, Cho2p-aia, supports normal cell growth and PC synthesis but failed to support BMV replication. We have found that Cho2paia mutant prevented BMV replication protein 1a from localizing to its normal replication sites and thus, blocked the formation of viral replication complexes.

Publications


    Progress 10/01/17 to 09/30/18

    Outputs
    Target Audience:Researchers, fellow scientists, and growers in the fields of plant virology, pathlogy, and cell biology. Changes/Problems:No changes. What opportunities for training and professional development has the project provided?We have four undergraduate students (two sophomores and two seniors) working in the lab on independent projects under direct supervision of senior graduate students. One undergraduate student attended Annual Meeting of American Society for Virology. How have the results been disseminated to communities of interest?Three papers have been published in PLoS Pathogens, Plant Direct, and PLoS One. Students also attended Annual Meeting of ASV with posters. What do you plan to do during the next reporting period to accomplish the goals? Continue to test possible roles of RTNs and LNPs in BMV replication. We plan to clone another group of ER membrane-shaping proteins, RHDs (root hair defective), and test their involvement in BMV replication. Continue to identify plant host phospholipid synthesis genes that are involved in BMV replication and test the potential to control viral replication by manipulating critical lipid synthesis genes. To take advantage of model plants for examining virus-host interactions, we will start working on Brochypodium distachon, which is a model for cereal plants, and spring beauty latent virus (SBLV), which infects Arabidopsis.

    Impacts
    What was accomplished under these goals? We have cloned several groups of membrane-shaping proteins and tested the effects of knockdown or overexpression of such genes on BMV infection. These genes include Reticulons (RTNs) and Lunapark (LNP). Overexpression of one of cloned RTNs (8 total) inhibited BMV replication; Knocking down gene expression of individual or a combination of LNPs, however, didn't affect BMV replication. We will continue to work on overexpression of membrane-shaping genes and their relationships with viral replication. We have cloned multiple plant genes involved in phospholipid synthesis. To identify those that are involved in BMV replication, we expressed each of them with a mCherry fluorescence protein in yeast and tested their distribution with the absence or presence of BMV 1a. We have identified 3 genes, CEK1 (choline-ethanolamine kinase 1), CEK3, and PLMT (phospholipid methyltransferase), which were redistributed by and co-localized with BMV 1a at the nuclear membrane. We will test the effect of knockdown or overexpression of these genes on BMV replication. We previous found that host CHO2, whose product is involved in phosphatidylcholine synthesis, is required for BMV replication. Deleting CHO2 inhibited BMV replication but affected yeast cell growth. We have generated a Cho2p mutant, Cho2p-aia, that supports normal cell growth and PC synthesis but failed to support BMV replication. We have found that Cho2p-aia mutant prevented BMV replication protein 1a from localizing to its normal replication sites and thus, blocked the formation of viral replication complexes.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhang Z., He G., Han G.S., Zhang J., Catanzaro N., Diza A., Wu Z., Carman G.M., Xie L. and Wang X. (2018) Host Pah1p Phosphatidate Phosphatase Limits Viral Replication by Regulating Phospholipid Synthesis. PLoS Pathogens 14: e1006988. doi: 10.1371/journal.ppat.1006988
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Liu H.*, Soyars C.L.*, Li J.*, Fei Q., He G., Peterson, B.A., Meyers B.C, Nimchuk Z. L., and Wang X. (2018) CRISPR-Cas9-mediated resistance to cauliflower mosaic virus. Plant Direct, 2: 1-9. doi: 10.1002/pld3.47 *: Equal contribution.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Sibert B.S., Navine A.K., Pennington J., Wang X., and Ahlquist P. (2018) Cowpea chlorotic mottle bromovirus replication proteins support template-selective RNA replication in S. cerevisiae PLoS One


    Progress 10/01/16 to 09/30/17

    Outputs
    Target Audience:Researchers in the fields of plant virology, pathology, and cell biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?An undergradaute student, Richard Samule Herron, worked in the lab for 2 years. He went on to become a graduate student at the University of Pittsburgh. How have the results been disseminated to communities of interest?Publications and presentations at conferences. What do you plan to do during the next reporting period to accomplish the goals?1. BMV replication protein 1a is responsible for VRC formation and double-stranded RNA is replication intermediate. We will use anti-BMV 1a and anti-dsRNA anti-antibodies to further confirm membrane-bound VRCs in BMV-infected barley in a immunogold labeling approach. 2. Responses of cellular PAP to BMV replication: accumulation and distribution. 3. Continue to work on plant phospholipid synthesis enzymes that are recruited to VRCs to detemine whether they are required for BMVreplication in plants.

    Impacts
    What was accomplished under these goals? 1. We have shown that deleting a host gene encoding physphatidate phosphotase (PAP) greatly promoted membrane-bound viral replication complexes (VRCs) due to the enhanced synthesis of phospholipids. 2. Host PAP directs lipid synthesis to storage lipids, away from the synthesis of phospholipids, which are used for active cell growth and membrane synthesis. We demonstrated that PAP limits BMV replication because it restricts phospholipid synthesis. Because both virus and host cell competes for limited phosphlipids and thus, PAP restricts both virus replication and cell growth. 3. We previously showed that BMV recruits host phosphatidylcholine (PC) synthesis enzyme Cho2p to VRCs to produce VRC-localized PC for BMV replication. We have identified a Cho2p mutant that inhibited BMV replication but support PC synthesis in general. This provides proof of concept that modulating host lipid synthesis controls viral replication.

    Publications


      Progress 10/01/15 to 09/30/16

      Outputs
      Target Audience:Researchers in the fields of plant virology, pathology, and cell biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student attended and presentated at two conferences. Undergraduate students presented posters on campus. How have the results been disseminated to communities of interest?Our results have been disseminated via publication, seminars, and conferences. What do you plan to do during the next reporting period to accomplish the goals?1. To elucidate the mechanisms by which Erv14 and Cornichon are involved in targeting BMV replication protein 1a to the nuclear membrane to initiate membrane remodeling. 2. To dissect the mechanism by which phosphatidate (PA) regulates phospholipid synthesis for supporting viral replication. 3. To confirm weather PLM is required for BMV replication by knocking down PLM expression in Nicotiana benthamiana and test the effect on BMV replication.

      Impacts
      What was accomplished under these goals? 1. New host mutants have been identified that support more viral replication complexes and viral replication. New host proteins in the coat protein complex II (COPII) vesicles have been confirmed that are required for targeting BMV replication protein 1a to the nuclear membrane; These include Erv14 in yeast and Cornichon in plants. 2. We have shown in yeast and plant that BMV promotes cellular phosphatidylcholine (PC) synthesis at viral replication complexes. In collaborating with Dr. George Belov at University of Maryland, and Dr. Glenn Randall at University of Chicago, we have shown that poliovirus and Hepatitis C virus also induced phosphatidylcholine synthesis at viral replication in the infected human cells. 3. We have identified plant lipid synthesis enzymes that interact with and recruited by BMV replication protein 1a. These include phospholipid methyltransferase (PLM) and choline/ethanolamine kinase.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang J., Zhang Z., Chukkapalli V., Nchoutmboube J., Li j., Randall G., Belov G., and Wang X. * (2016) Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites. Proc. Natl. Acad. Sci. USA. 113: E1064-1073. doi:10.1073/pnas.1519730113. *: corresponding author
      • Type: Journal Articles Status: Published Year Published: 2016 Citation: Li J., Fuchs S., Zhang J., Wellford S., Schuldiner M., and Wang X. * (2016) An unrecognized function for COPII components in recruiting a viral replication protein to the perinuclear endoplasmic reticulum. J. Cell Science. ePub 08/18/2016 doi:�10.1242/jcs.190082 * corresponding author.


      Progress 10/01/14 to 09/30/15

      Outputs
      Target Audience:Researchers in the fields of plant virology and pathology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post-doctoral associate and graduate students presented their results at several conferences. How have the results been disseminated to communities of interest?Our results have been disseminated via publication, seminars, and conferences. What do you plan to do during the next reporting period to accomplish the goals?1. For objective 1, we are in the process of developing an immunogold electron microscopic assay to detect the localization of key viral and host proteins in relation to viral replication complexes. 2. We are testing more host lipid metabolism proteins in BMV replication. 3. We are screening Cho2p mutants that disrupt the 1a-Cho2p interaction and use the Cho2p mutant to block BMV replication, providing proof-of-concept evidence.

      Impacts
      What was accomplished under these goals? 1. We have developed a protocol to detect viral and host proteins in cell wall-free protoplasts prepared from virus-infected leaf tissues by immunofluorescence confocal microscopy. The advantage of using protoplasts over plant tissues is the fast infiltration of fixatives and much cleaner data. We are working on immunogold electron microscopic analysis using protoplasts. 2. We further confirmed that host PC synthesis enzyme, Cho2p, is recruited by BMV replication protein 1a to the viral replication site, where PC accumulation increases. Deleting the CHO2 gene blocked BMV replication more than 30-fold. In collaboration with Drs. Glenn Randal and George Belov, we showed that PC accumulation enhancesthe viral replication sites of hepatitis virus C and poliovirus. 3. We will disrupt the 1a-Cho2p interaction to block the viral replication site-localized PC synthesis. This approach only blocks viral replication site-localized PC synthesis but not the PC synthesis in general so host growth should not be affected.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2015 Citation: Diaz A., Zhang J., Ollwerther A., Wang X*., and Ahlquist P*. (2015) Host ESCRT proteins are required for Bromovirus RNA replication compartment assembly and function. PLoS Pathogens 11: e1004742. doi:10.1371/journal.ppat.1004742 *: Co-corresponding authors
      • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cao X., Jin X., Zhang X., Li Y, Wang C., Wang X., Hong J., Wang X., Li D., and Zhang Y. (2015) Morphogenesis of the ER membrane-invaginated vesicles during Beet black scorch virus infection: The role of auxiliary replication protein and new implications of three-dimensional architecture. Journal of Virology, 89: 6184-6195. doi:10.1128/JVI.00401-15 (Featured in SPOTLIGHT).
      • Type: Journal Articles Status: Published Year Published: 2015 Citation: Liu H., Tolin S., Bush E., Creswell T., Hansen M.A., and Wang X. 2015 First report of Tomato spotted wilt virus on Pittosporum tobira in the United States. Plant Disease. ePub 09/01/2015.
      • Type: Journal Articles Status: Published Year Published: 2015 Citation: Odokonyero D., Mendoza M.R., Alvarado V.Y., Zhang J., Wang X., and Scholthof H.B. 2015 Transgenic down-regulation of ARGONAUTE2 expression in Nicotiana benthamiana interferes with several layers of antiviral defenses. Virology 486: 209-218.


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

      Outputs
      Target Audience: Researchers in the fields of plant virology and pathology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The post-doc researcher and graduate student have participated and presented posters in 4 conferences. How have the results been disseminated to communities of interest? Our results have been published in journals, presented in conferences and invited seminars. What do you plan to do during the next reporting period to accomplish the goals? In Objective 1, we have started to process BMV-infected barley and N. benthamiana as well as SBLV-infected various plants to examine the remodeled membrane structures. In objective 2, we will test whether viral replication site-associated and BMV-promoted PC accumulation is also associated with viral replication sites in plants. This can be done by make protoplasts from healthy and BMV- or SBLV-infected leaf samples. Protoplasts will then be processed for immunofluorescence microscopic analysis by using anti-1a or anti-PC antibodies. Plant enzymes in the PC synthesis will be tested for their possible interactions with BMV 1a. In objective 3, we are in the processing generating more transgenic N. benthamiana plants and knocking down one or few host genes to test the effectiveness of virus resistance.

      Impacts
      What was accomplished under these goals? We have made strides in the Objective 2. It has been previously found that several viruses enhance host phosphatidylcholine (PC), however, it is not known whether the enhanced PC is associated with viral replication complexes. Using a monoclonal antibody specifically recognizing PC, we showed that BMV promotes the synthesis and accumulation of PC at sites colocalizing with viral replication sites in yeast. BMV does so by interacting and recruiting a PC synthesis enzyme, Cho2p, to the viral replication sites. For the Objective 1, we have developed a yeast system for another plant (+)RNA virus, Spring beauty latent virus (SBLV). SBLV infects Arabidopsis so we could compare host membrane rearrangement in yeast, Arabidopsis, and N. benthamiana. Many available Arabidopsis mutants defective in lipid synthesis can be quickly screened. In the Objective 3, we unexpectedly found that knocking down a specific member of stearoyl-ACP desaturase (SACPD) family, which converts saturated fatty acids to unsaturated fatty acids, affected ovule development, leading to sterile phenotype. This is the first report that SACPD is required for ovule development. On the other hand, there is no other growth phenotypes at the vegetative stage.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2014 Citation: Diaz A., and Wang X. 2014 Bromovirus-induced remodeling of host membranes during viral RNA replication. Current Opinion in Virology. 9:104-110.
      • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang J. , Li J., Garcia-Ruiz H., Bates P., Mirkov T., and Wang X. 2014 A stearoyl-acyl carrier protein desaturase, NbSACPD-C, is critical for ovule development in Nicotiana benthamiana. The Plant Journal. 80: 489-502.
      • Type: Journal Articles Status: Published Year Published: 2014 Citation: Hao L., Lindenbach B., Wang X., Dye B., Kushner D., He Q., Newton M., and Ahlquist P. 2014 Genome-wide analysis of host factors in Nodavirus RNA replication. PLoS One 9: e95799.