Mechanism of Inhibition of RNA Virus Replication by Host WW-domain Proteins

Co-opted host factors are key determinants of RNA virus pathology, host - virus interactions, and evolution of viruses. Recent genome-wide screens with several viruses have identified many host proteins, which directly facilitate (+)RNA virus accumulation. Among the identified host proteins are the family of WW-domain proteins, which are critical for the replication of several important human viruses. WW-domain proteins are important in protein interactions and the WW-domain represents one of the ancient motifs. The WW-domain proteins are involved in several diseases, such as human cancers, inherited diseases and viral infections. Determining the mechanism of WW-domain-driven inhibition of viral replication could help developing novel antiviral approaches with broad antiviral effects against many related RNA viruses as demonstrated for HIV earlier. Progress in our understanding of the mechanisms of subverted host factors is slow due to functional redundancy or lack of knowledge about their functions. However, easily tractable virus - host systems, such as Tomato bushy stunt virus (TBSV) and yeast as a model host can contribute to our understanding of the functions of these host proteins. This project will likely advance our understanding of the role of WW-domain proteins in virus-host interactions. This advance could immensely help other scientists working with less tractable, but devastating viral pathogens for which similar studies are currently not yet feasible. TBSV is the system of choice for these studies, because recent genome-wide screens (including ~95% of all host genes) and several proteomics approaches by the PI to catalog host genes affecting replication have made tombusvirus-host interaction one of the most complete among RNA viruses. Accordingly, in-depth analyses with a dozen host factors have shown elegantly that the identified host factors play various roles in TBSV replication in both yeast and plant hosts. These works from the PI's lab have led to the identification of new antiviral targets and successful application of pharmacological compounds to inhibit TBSV replication in plants. The gained knowledge will not only be useful for TBSV, but to other significant human pathogens, such as Hepatitis C virus, West Nile virus, Dengue virus and other flaviviruses and pestiviruses, due to the similarity of their replicase proteins. Collectively, the major advances with TBSV will stimulate development of new approaches for studying RNA replication and host - pathogen interactions for important human pathogens. The PI will unravel the multiple roles of WW-domain proteins in TBSV replication. The combination of yeast and authentic cell-free assay developed by the PI is currently the most potent for studying the mechanism of host factors involvement in viral RNA replication and viral pathogenesis. The proposal includes 4 specific aims: Aim 1. Aim 1. Testing the inhibitory effects of WW-domain proteins on the activity of the tombusvirus replicase using a cell-free TBSV replication system. Aim 2. Effect of host WW-domain proteins on the activity of the purified tombusvirus replicase on dsRNA templates. Aim 3. The role of host WW-domain proteins in the tombusvirus replicase. Aim 4. The effect of host WW-domain proteins on tombusvirus replication in plants. The proposed work is based on unique yeast model host, the awesome power of yeast genetics in combination with biochemical assays with a novel cell-free TBSV replication system developed for tombusviruses by the PI. The proposed work will likely lead to major new insights into (+)RNA virus replication and viral pathogenesis. The research holds promise of benefiting society by leading to groundbreaking results in the area of virus replication, host-virus interactions and the adaptation of viruses to their hosts.