Fehr Research Project Summary

The highly-conserved coronavirus (CoV) macrodomain is essential for CoV pathogenesis in multiple animal models of infection, and thus it is a potential drug target for emerging CoVs that cause severe disease, such as Middle-East Respiratory Syndrome (MERS)-CoV or Porcine Epidemic Diarrhea Virus (PEDV). The macrodomain binds and hydrolyzes ADP-ribose from proteins, however, the contribution of specific residues in the ADP-ribose binding pocket to these activities are unknown. This gap in knowledge is a significant hurdle for identifying compounds that inhibit its activity. The long-term goal is to discern the mechanism by which the CoV macrodomain binds and cleaves ADP-ribose from proteins, determine its functions during infection, and identify compounds that inhibit its activity. The overall objective in this proposal is to determine the relative contribution of specific residues to ADP-ribose binding and hydrolysis, and correlate these results to their impact on virus replication. The central hypothesis is that specific residues in the macrodomain separately impact ADP-ribose binding vs catalysis, and also have distinct roles in virus replication. The rationale for this project is that a better understanding of the mechanism by which the CoV macrodomain functions both in vitro and in cell culture will provide a framework for the development of novel anti-viral therapeutics for highly pathogenic or emerging CoVs. The central hypothesis will be tested by pursuing the following two specific aims: 1) Determine the contribution of highly conserved macrodomain residues in ADP-ribose binding and hydrolysis; and 2) Correlate the level of macrodomain ADP-ribose binding and hydrolysis in vitro to virus replication in cell culture. Under the first aim, several recombinant macrodomain proteins with mutations in conserved residues will be tested in ADP-ribose binding and hydrolysis assays. These assays will determine the relative contribution of these residues to either binding or hydrolysis. Additionally, x-ray crystallography will be applied to select mutant proteins to determine how the mutation altered the structure of the protein to impact its activity. For the second aim, the same mutations made in aim 1 will be incorporated into recombinant MHV and MERS-CoV and test their roles in virus replication and ultimately correlate the biochemical activity of each residue to its effect on virus replication. The research proposed in this proposal is innovative, in the applicant’s opinion, because it studies the impact of specific macrodomain residues on macrodomain biochemistry and their role during infection in a cohesive manner, which has not previously been attempted. The proposed research is significant because they will provide unique insight into both the biochemistry and the function of the CoV macrodomain. These results will have a positive impact in the design and development of compounds that can inhibit this protein and potentially be developed into novel therapeutics to prevent CoV-induced disease.