University of Kentucky

UK College of Agriculture

UK Plant Pathology

Peter Nagy's Laboratory

          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

1. 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 as 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.