SARS-CoV-2 is a coronavirus that leads to the respiratory disease COVID-19 and is the cause of a global pandemic currently gripping the world. SARS-CoV-2, like all viruses, relies on many components of the infected cell to drive viral replication. One component of all eukaryotic cells that coronaviruses rely on for their replication are lipid membranes that separate from different regions of a cell. Coronaviruses hijack host cell proteins for viral replication, as well as some of the host cell membranes, restructuring them to generate a unique membrane architecture that supports the production of viral RNA. However, it is currently unclear how coronaviruses accomplish this restructuring of host the cell membrane. This gap in knowledge has limited the understanding of how SARS-CoV-2 replicates in host cells and thus limited the development of therapeutics that might be useful to slow its replication. Therefore, to gain insight into the mechanisms by which coronaviruses restructure host cell membranes, this Project will investigate three coronavirus proteins (nsp3, nsp4 and nsp6) that are required for this restructuring event. As a Broader Impact, Project staff will engage with local middle and high school students to discuss the structures of different of SARS-CoV-2 components and how the structures of these viral proteins inform scientists’ understanding of viral mechanisms.
All positive sense RNA viruses restructure host cell membranes to support the assembly of the replication and transcription complexes required for viral RNA replication. Coronaviruses, including SARS and MERS, generate a series of interconnected double membrane vesicles that appear to be generated from endoplasmic reticulum membranes. The formation of these vesicles require three integral membrane nonstructural proteins, nsp3, nsp4 and nsp6, that are encoded by the virus. However, the molecular mechanisms by which these three nsp proteins work together to generate double membrane vesicles is unknown. To gain insight into these mechanisms, this Project will study the structure and function of nsp3, nsp4 and nsp6 from SARS-CoV-2. Specifically, this work will 1 ) identify the host cell proteins that interact with nsp3, nsp4 and nsp6; and 2) determine the atomic level structures of these three viral proteins. To accomplish this, these proteins will be expressed in human lung cell lines and screened for binding partners by immunoprecipitation and mass spectrometry. The structure of these proteins will be investigated using a combination of X-ray crystallography and electron microscopy. Knowledge of the structure of the nsp proteins and their interactions will provide important insight into how this virus takes advantage of its host cell, resulting in a devastating pandemic.
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.
