Research Projects Overview

Molecular Virology of Plant RNA Viruses   

Virus replication

We use Tombusviruses, small model RNA viruses of plants, to identify the viral and host players in replication and to unravel the mechanism of virus replication. RNA replication (multiplication) is the central process in virus infections, which in case of Tombusviruses is a robust process, and it leads to the production of millions of progeny viruses in a day per infected cells. Better understanding of virus replication is expected to lead to improved antiviral strategies and enhanced resistance against virus diseases in plants. The basic discoveries made with Tombusviruses are expected to influence studies on replication of important human and animal pathogens.

Virus recombination

The second area of major emphasis in my research program is to understand the mechanism of virus evolution. This area is also important since viruses can change dramatically via recombination, which, in turn, can lead to emergence of new viruses and strains. The new recombinant viruses may elude host defenses initially due to their unique features. RNA recombination in viruses can also hinder the use of viruses as gene delivery systems or gene expression vectors. We are working on the dissection of the mechanism of RNA recombination, which may lead to development of recombination predicting software, safer virus vectors and lead to improved vaccination programs.

Current Projects

Development of the TBSV-yeast system to study virus replication and virus-host interactions. 
We have developed yeast because of the advantages of using yeast as a model host. Since viruses are intracellular parasites that use the resources of eukaryotic cells, it is feasible to study virus replication in yeast (S. cerevisiae) cells. This makes the awesome power of yeast genetics, biochemistry and cell biology available for virus research. Another advantage is the knowledge on host proteins is the most comprehensive in yeast. In addition, the findings from yeast model host can then be validated in the native host as we have already shown for 20 host genes for TBSV. Altogether, the available unique combination of tools for tombusviruses, including development of the powerful yeast replication system, and a novel cell-free authentic tombusvirus replication assay, makes yeast a truly outstanding model to provide accelerated progress and to facilitate exploratory research on virus-host interactions, which will be applicable to RNA viruses of plants and animals.

• Nagy, P. D., J. Pogany, and J. Y. Lin. 2014. How yeast can be used as a genetic platform to explore virus-host interactions: from 'omics' to functional studies. Trends Microbiol 22:309-316.
• Nagy, P. D., and J. Pogany. 2006. Yeast as a model host to dissect functions of viral and host factors in tombusvirus replication. Virology 344:211-20.
• Pogany, J., M. R. Fabian, K. A. White, and P. D. Nagy. 2003. A replication silencer element in a plus-strand RNA virus. EMBO J 22:5602-11.
• Panavas, T., and P. D. Nagy. 2003. Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus. Virology 314:315-25. 

Genome-wide screens and global proteomic approaches to identify host factors affecting TBSV replication.
We have performed a dozen complementary genome-wide and global proteomics approaches that have led to the identification of ~500 host genes affecting TBSV replication in yeast. These screens are based on a yeast single-gene deletion library, over-expression library, temperature-sensitive (ts) yeast mutant library, yeast two-hybrid screen, and yeast protein arrays, making it the most complete screens among any virus-host systems. Altogether, this research led to the identification of ~500 host proteins, and many genes have been identified in multiple screens, indicating that these host genes are important factors during TBSV replication. The systems biology approach makes TBSV-yeast system as one of the best characterized for virus-host interactions among any pathogens.

• Nagy, P. D., and J. Pogany. 2012. The dependence of viral RNA replication on co-opted host factors. Nature Reviews Microbiology 10:137-149.
• Pogany, J., J. Stork, Z. Li, and P. D. Nagy. 2008. In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70. Proc Natl Acad Sci U S A 105:19956-61.
• Serviene, E., N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy. 2005. Genome-wide screen identifies host genes affecting viral RNA recombination. Proc Natl Acad Sci U S A102:10545-50.
• Panavas, T., E. Serviene, J. Brasher, and P. D. Nagy. 2005. Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses. Proc Natl Acad Sci U S A 102:7326-31.

Characterization of co-opted host factors critical for TBSV replication. 
We have characterized the detailed functions of more than 20 co-opted host proteins, which are involved in various viral processes, such as: Hsp70, elongation factor 1A and the ESCRT Vps4 AAA+ ATPase in the assembly of the viral replicase complex; eEF1A and eEF1Bgamma, DEAD-box RNA helicases and GAPDH in viral RNA synthesis. We have characterized Lipid transfer ORP proteins and VAP proteins in membrane contact site formation that is required for TBSV replication. These works revealed the complex and amazingly sophisticated interaction between TBSV and the host cells.

• Nagy, P. D. 2015. Viral Sensing of the Subcellular Environment Regulates the Assembly of New Viral Replicase Complexes during the Course of Infection. J Virol 89:5196-5199.
• Pogany, J., and P. D. Nagy. 2015. Activation of Tomato Bushy Stunt Virus RNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by Phospholipids In Vitro. J Virol 89:5714-23.
• Kovalev, N., and P. D. Nagy. 2014. The Expanding Functions of Cellular Helicases: The Tombusvirus RNA Replication Enhancer Co-opts the Plant eIF4AIII-Like AtRH2 and the DDX5-Like AtRH5 DEAD-Box RNA Helicases to Promote Viral Asymmetric RNA Replication. PLoS Pathog 10:e1004051.
• Barajas, D., K. Xu, I. F. de Castro Martin, Z. Sasvari, F. Brandizzi, C. Risco, and P. D. Nagy. 2014. Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites. PLoS Pathog 10:e1004388.
• Barajas, D., I. F. Martin, J. Pogany, C. Risco, and P. D. Nagy. 2014. Noncanonical Role for the Host Vps4 AAA+ ATPase ESCRT Protein in the Formation of Tomato Bushy Stunt Virus Replicase. PLoS Pathog 10:e1004087.
• 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.
• Li, Z., J. Pogany, S. Tupman, A. M. Esposito, T. G. Kinzy, and P. D. Nagy. 2010. Translation elongation factor 1A facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis. PLoS Pathog 6:e1001175.

Characterization of subverted lipids and membranes required for TBSV replication. 
By using artificial vesicles (liposomes), we have shown that phosphatidylethanolamine (PE) is required for TBSV replication in vitro. In addition, the combination of PE and sterols in liposomes lead to the highest level TBSV replication in vitro. Lipidomic analysis of yeast and plant cells showed increased PE level in cells replicating TBSV. Interestingly, TBSV manipulates the cells that leads to enrichment of PE at the sites of TBSV replication. Altogether, phospholipid and sterol levels are major determinants of TBSV replication, which is supported by intensive membrane proliferation.

• Xu, K., and P. D. Nagy. 2015. RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes. Proc Natl Acad Sci USA112:E1782-E1791.  
• Chuang, C., D. Barajas, J. Qin, and P. D. Nagy. 2014. Inactivation of the Host Lipin Gene Accelerates RNA Virus Replication through Viral Exploitation of the Expanded Endoplasmic Reticulum Membrane. PLoS Pathog 10:e1003944.
• Sharma, M., Z. Sasvari, and P. D. Nagy. 2010. Inhibition of sterol biosynthesis reduces tombusvirus replication in yeast and plants. J Virol 84:2270-81. 

Discovery of cell-intrinsic restriction factors against TBSV replication. 
We have discovered 70 cell-intrinsic restriction factors (CIRFs), which inhibit TBSV replication. We have characterized cyclophilins, nucleolin, ribonucleases, WW-domain and TPR-domain containing proteins and co-chaperones as CIRFs. This should help opening up new antiviral approaches and our understanding antiviral responses of the host cell.

• Barajas, D., N. Kovalev, J. Qin, and P. D. Nagy. 2015. Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins. J Virol 89:2064-79.
• Sasvari, Z., P. Alatriste Gonzalez, and P. D. Nagy. 2014. Tombusvirus-yeast interactions identify conserved cell-intrinsic viral restriction factors. Front Plant Sci 5:383.
• Kovalev, N., and P. D. Nagy. 2013. Cyclophilin a binds to the viral RNA and replication proteins, resulting in inhibition of tombusviral replicase assembly. J Virol 87:13330-42.
• Lin, J. Y., V. Mendu, J. Pogany, J. Qin, and P. D. Nagy. 2012. The TPR Domain in the Host Cyp40-like Cyclophilin Binds to the Viral Replication Protein and Inhibits the Assembly of the Tombusviral Replicase. PLoS Pathog 8:e1002491. 

Discovery of host factors regulating viral RNA recombination. 
We have also discovered 100 host proteins that greatly affect viral RNA recombination, which is a driving force in virus evolution. The host enzymes includes cellular ion pumps, ribonucleases and DEAD-box helicases. TBSV is currently the only system, where viral RNA recombination factors are characterized at the system level and also mechanistically. This progress indicates well the elegance and power of an outstanding model system that can greatly help scientific progress.

• Chuang, C., K. R. Prasanth, and P. D. Nagy. 2015. Coordinated Function of Cellular DEAD-Box Helicases in Suppression of Viral RNA Recombination and Maintenance of Viral Genome Integrity. PLoS Pathog 11:e1004680.
• Prasanth, K. R., D. Barajas, and P. D. Nagy. 2015. The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex. J Virol 89:2750-63.
• Jaag, H. M., J. Pogany, and P. D. Nagy. 2010. A host Ca2+/Mn2+ ion pump is a factor in the emergence of viral RNA recombinants. Cell Host Microbe 7:74-81.
• Jaag, H. M., and P. D. Nagy. 2010. The combined effect of environmental and host factors on the emergence of viral RNA recombinants. PLoS Pathog 6:e1001156.