Elucidating the role of the SARS-CoV-2 ORF9b protein in virus replication

PROJECT SUMMARY Highly pathogenic human coronaviruses encode an array of accessory proteins that are crucial for their virulence, enabling them to effectively manipulate cellular machinery and subvert host defenses. Our research proposal is centered on the SARS-CoV-2 accessory protein, ORF9b, which is known to amplify viral virulence in vivo, yet its specific roles during viral infection remain to be elucidated. Transfection studies suggest that ORF9b prevents innate immune induction by hindering the assembly of the MAVS signalosome, a finding that awaits confirmation in the context of actual SARS-CoV-2 infection. The investigation of ORF9b in virus-infected cells has traditionally been hampered by its integrated localization within the nucleocapsid gene, complicating efforts to manipulate ORF9b without unintentionally interfering in the nucleocapsid functionality. We have successfully overcome this challenge by strategically relocating the ORF9b sequence away from the nucleocapsid gene, creating a SARS-CoV-2 variant that produces native ORF9b levels and replicates as efficiently as the wild-type virus. This engineered virus provides a strong platform for exploring ORF9b functions during infection without unintended impact on the nucleocapsid gene. Further advancing our methodology, we tagged ORF9b in its new locale with an HA tag and TurboID, setting the stage for pioneering investigations of ORF9b networks in SARS-CoV-2-infected cells. Our extensive validation of these viral variants confirmed their ability to replicate comparably to the unmodified virus while preserving the native characteristics of ORF9b. We will employ these advanced tools to rigorously examine the impact of ORF9b phosphorylation on SARS- CoV-2 replication and the protein's specific functions (Aim 1). Additionally, we will generate a detailed, time- resolved interaction map of ORF9b, uncovering its evolving interactions during infection (Aim 2). Achieving these goals will provide significant insights into the biological mechanisms of ORF9b and enhance our understanding of SARS-CoV-2 pathogenesis. This foundational work will also set the stage for extending similar methodologies to other viral proteins, vastly improving our knowledge of the coronaviral biology and aiding in the identification of new prophylactic and therapeutic avenues.