Structure of SARS-CoV-2 Mpro reveals mechanism of protein cleavage

Researchers at Penn State University have made significant progress in understanding the cleavage process of the SARS-CoV-2 virus. The virus, along with other RNA viruses, translates its RNA genome into a polyprotein, which is then cleaved into individual proteins by a protease enzyme. This cleavage process is crucial for virus replication and is a target for antiviral drugs like Paxlovid.

In a study published in the Journal of Biological Chemistry, the researchers used cryo-electron microscopy to visualize the protease, called Mpro, in complex with the polyprotein. They aimed to determine the order in which the proteins are cleaved, as this order remained unclear.

Previous studies had used X-ray crystallography to investigate Mpro, but the researchers at Penn State wanted a more representative form of the polyprotein and higher resolution. They found that previous studies had used only small peptides to mimic the polyprotein, limiting the understanding of how Mpro interacts with different recognition sites on the polyprotein.

By using cryo-electron microscopy, the researchers obtained detailed 3D visualizations of Mpro and the polyprotein, shedding light on the cleavage process. This new information could aid in the development of more effective drugs for COVID-19 treatment by optimizing the binding of antiviral drugs or identifying new ways to inhibit the cleavage process.

The researchers discovered that the protease Mpro primarily associates with the recognition site at the cleavage location of the polyprotein, making minimal contact with the rest of the polyprotein. This finding suggests that the cleavage order may be dictated by the structure of the polyprotein itself.

According to Narwal, if Mpro was solely responsible for selecting the preferred recognition site, it would be expected to make additional contacts with the polyprotein for stability and energetic reasons. However, the lack of these additional contacts led the researchers to suspect that the polyprotein’s structure influences the cleavage order. Certain recognition sites within the polyprotein may be more accessible to Mpro, and as each protein is cleaved and separates from the polyprotein, the next preferred site becomes exposed, leading to a consistent cleavage order.

The use of cryo-electron microscopy (cryo-EM), an imaging technique providing atomic resolution structures, allowed the researchers to obtain these new insights. While X-ray crystallography is commonly used for smaller targets like Mpro, cryo-EM was employed in this study due to its ability to investigate larger molecules. Despite the challenges of obtaining high-resolution structures of Mpro, the researchers successfully focused on Mpro using cryo-EM, thanks to meticulous sample preparation and advanced data-processing techniques.

The initial experiments were conducted at the Penn State Huck Institutes of the Life Sciences’ Cryo-EM Facility, and further imaging was carried out at the National Cancer Institute’s National Cryo-EM Facility in Frederick, Maryland.

While the researchers achieved a clear image of Mpro when bound to the polyprotein, the image of the polyprotein itself was less distinct. Ongoing efforts are focused on visualizing the entire complex and exploring other regions of the polyprotein to gain a comprehensive understanding of the cleavage process. These insights are expected to facilitate future research on viral replication and contribute to the development of efficient antiviral drugs.

Source: Pennsylvania State University

Leave a Comment