Determining how the evolution of the coronavirus macrodomain contributes to its biochemical and virological functions

This project explores how macrodomain proteins have evolved to balance their different multiple biochemical activities. Macrodomains are ancient enzymes that bind to and remove ADP-ribose, an important post-translation modification that is critical in several cellular stress responses, including DNA damage, ER stress, and virus infection. These enzymes are conserved through all domains of life, including archaea, bacteria, eukaryotes, and viruses, indicating that they are critical for multiple cellular processes. However, the function of macrodomain enzymes in cell biology and microbiology are just now being uncovered. Furthermore, recent research indicates that macrodomains have evolved to properly balance their biochemical functions depending on if they are expressed from a virus, a bacteria, or from a eukaryotic cell. This project will evaluate how viral macrodomains have evolved to develop the ideal biochemical properties that allow them function in the context of a virus infection. This project also has strong educational and community outreach components. Most notably, students at all training levels will participate in this project and will learn how to evaluate the evolution of proteins through workshops in phylogenetics. This project will provide new insights into the fundamental biology of macrodomain enzymes and could lead to new insights into antiviral drug-development. This project aims to define how the coronavirus macrodomain has evolved to best function in the context of a virus infection using the mouse coronavirus, murine hepatitis virus (MHV), as a model. The use of MHV, which is unable to infect humans, for creating macrodomain mutations eliminates the potential for gain-of-function research. Research over the last decade has demonstrated that the coronavirus macrodomain blocks innate immune responses, is critical for viral pathogenesis, and is a potential drug target. Furthermore, the macrodomain uses both its ADP-ribose binding and hydrolysis activities to promote virus replication, and they must be balanced for optimal replication. The PI will use recombinant proteins and a panel of recombinant viruses, developed using a bacterial artificial chromosome based reverse genetic system, to better understand how highly conserved amino acids and selective pressure induced mutations impact Mac1 biochemical activities, virus replication, and pathogenesis using well-established assays and model systems. Additionally, macrodomains from across the evolutionary spectrum will be expressed in the context of MHV infection to define the evolutionary limits of macrodomain divergence that is tolerated in MHV. This work will provide a deeper understanding of how macrodomains have evolved to counter various ADP-ribose dependent antiviral responses. This project is jointly funded by the Genetic Mechanisms Program and the Division of Molecular and Cellular Biosciences. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.