Mechanisms of Mpro autocleavage

Project Summary SARS-CoV-2, the virus that causes COVID-19, relies on a critical enzyme called Mpro (main protease) to reproduce inside infected cells. This enzyme must first "cut itself free" from a larger protein before it can function properly. This process is called autocleavage. However, we don't fully understand how this self- activation is controlled, which limits our ability to develop new antiviral treatments. Our research will investigate two key questions about Mpro activation. First, we will determine how small changes in the protein sequences surrounding Mpro affect how efficiently it cuts itself free. We suspect that certain sequence changes could make this process work better, suggesting that the virus hasn't yet evolved to be perfectly efficient. If we can identify these "better" sequences, we might discover new ways to disrupt the virus. Second, we will explore how the virus's association with cell membranes influences Mpro activation. The proteins that flank Mpro attach to cellular membranes, and we believe this placement helps coordinate the timing of enzyme activation. Understanding this spatial control could reveal new targets for antiviral drugs. We will use baker's yeast as our experimental system because it allows us to study these processes in a controlled, safe environment without working directly with the live virus. This approach lets us systematically test different protein variants and membrane interactions that would be difficult to study in other systems. We will then validate our most promising yeast findings using safe SARS-CoV-2 replicon systems that mimic viral replication without producing infectious particles. This two-step approach ensures our mechanistic results in yeast are relevant in the viral system. Our research has a strong educational component that will train the next generation of scientists. We will involve undergraduate students, graduate students, and high school students in hands-on research experiences. This work could lead to new therapeutic strategies by identifying previously unknown vulnerabilities in the virus's replication process. The principles we discover may also apply to other similar viruses, potentially informing future pandemic preparedness efforts. The findings will advance our fundamental understanding of how viruses control these essential enzymes while providing authentic research experiences that prepare students for careers in science and medicine.