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New Strategies to Induce Resistance against Tombusviruses Based on Host Factors
Department of Plant Pathology
Viruses cause major losses in agriculture. Most plant viruses contain RNA genomes that must be replicated inside host cells. Therefore, replication of RNA viruses in infected hosts is the central part of viral pathogenesis. Pioneering work led by the PI will contribute key knowledge to understanding how host factors participate in viral pathogenesis. The work will unravel the functions of several key host proteins, which have been implicated in replication of a large number of viruses.
The proposed innovative approaches including yeast and plant assays developed for tombusviruses as well as the powerful cell-free TBSV replication assay in yeast extracts will likely lead to major new insights into virus replication and viral pathogenesis and will have a considerable impact on our understanding of the mechanism that transforms the host cells into "viral factories".
The innovative research on tombusvirus replication could immensely help other scientists working with less tractable, but devastating viral pathogens for which similar studies are currently not yet feasible. Dissection of the role of the selected host proteins in viral pathogenesis and disease development will facilitate future antiviral approaches and treatments to reduce the effect of viral diseases. The research holds promise of benefiting society and agriculture by leading to groundbreaking results in the area of virus replication and pathogenesis.
Due to the central role of the viral replicase in virus replication, studies on protein factors present in the tombusvirus replicase complex promise not only to help elucidate the viral replication process in tombus- and related viruses, but also to lead to novel strategies to interfere with virus replication/infection. The presence of conserved host proteins in the viral replicase makes this goal attractive, possibly providing means to induce resistance against many plant viruses, and, likely helping animal virus research.
2011 Project Description
Events: The PI was: a plenary speaker at the ASM Virus replication meeting in Banff, Canada, 2011; State-of-the-art workshop speaker at the International Congress of Virology, Sapporro, Japan, 2011.
Products: 1. Two Ph. D. students obtained their degrees in Plant Pathology (Dr. Sharma and Pathak) from the PI's lab in 2011.
Dissemination: The PI gave a class and seminar to undergraduate students in genetics, Quernavaca, Mexico, 2011.
We made major progress in understanding the role of the host in virus-plant interactions. Briefly, replication of Tomato bushy stunt virus (TBSV) depends on cellular factors, such as Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an abundant metabolic enzyme. We found that GAPDH is recruited to the viral replicase complex.
By using an in vitro TBSV replication assay based on recombinant p33 and p92pol viral replication proteins and cell-free yeast extract, We find that addition of purified recombinant GAPDH to the cell-free extract prepared from GAPDH-depleted yeast results in increased plus-strand RNA synthesis and asymmetric production of viral RNAs. Our data also demonstrate that GAPDH interacts with p92pol viral replication protein, which may facilitate the recruitment of GAPDH into the viral replicase complex in the yeast model host.
In addition, we have identified a dominant-negative mutant of GAPDH, which inhibits RNA synthesis and RNA recruitment in vitro. Moreover, this mutant also exhibits strong suppression of tombusvirus accumulation in yeast and in virus-infected Nicotiana benthamiana. Overall, the obtained data support the model that the co-opted GAPDH plays a direct role in TBSV replication by stimulating (+)-strand synthesis by the viral replicase.
Pathak, K. B., J. Pogany, K. Xu, K. A. White, and P. D. Nagy. 2012. Defining the Roles of cis-Acting RNA Elements in Tombusvirus Replicase Assembly In Vitro. J Virol 86:156-71.
Stork, J., N. Kovalev, Z. Sasvari, and P. D. Nagy. 2011. RNA chaperone activity of the tombusviral p33 replication protein facilitates initiation of RNA synthesis by the viral RdRp in vitro. Virology 409:338-47.
Sharma, M., Z. Sasvari, and P. D. Nagy. 2011. Inhibition of phospholipid biosynthesis decreases the activity of the tombusvirus replicase and alters the subcellular localization of replication proteins. Virology 415:141-52.
Sasvari, Z., L. Izotova, T. G. Kinzy, and P. D. Nagy. 2011. Synergistic Roles of Eukaryotic Translation Elongation Factors 1Bgamma and 1A in Stimulation of Tombusvirus Minus-Strand Synthesis. PLoS Pathog 7:e1002438.
Nagy, P. D., R. Y. Wang, J. Pogany, A. Hafren, and K. Makinen. 2011. Emerging picture of host chaperone and cyclophilin roles in RNA virus replication. Virology 411:374-82.
Nagy, P. D., and J. Pogany. 2011. The dependence of viral RNA replication on co-opted host factors. Nat Rev Microbiol.
Nagy, P. D. 2011. The roles of host factors in tombusvirus RNA recombination. Adv Virus Res 81:63-84.
Li, Z., and P. D. Nagy. 2011. Diverse roles of host RNA binding proteins in RNA virus replication. RNA Biol 8:305-15.
Jaag, H. M., Q. Lu, M. E. Schmitt, and P. D. Nagy. 2011. Role of RNase MRP in viral RNA degradation and RNA recombination. J Virol 85:243-53.
Huang, T. S., and P. D. Nagy. 2011. Direct inhibition of tombusvirus plus-strand RNA synthesis by a dominant negative mutant of a host metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, in yeast and plants. J Virol 85:9090-102.