DEVELOPMENT OF CELLULAR PLATFORMS FOR THE FUNCTIONAL AND PHYSIOPATHOLOGICAL STUDY OF SARS-COV-2 NON-STRUCTURAL PROTEINS AND SCREENING OF ANTIVIRAL COMPOUNDS

COVID-19, caused by the SARS-CoV-2 virus, presents ongoing challenges due to emerging variants and the lack of targeted treatments. Understanding the virus's mechanisms and developing antiviral therapies are crucial for combating this disease. SARS-CoV-2 is a positive-sense RNA virus with a 30 kb genome encoding non-structural proteins such as 3CL^pro and PL^pro proteases, which are essential for viral replication. These proteases also contribute to immunoevasion; 3CL^pro inhibits IFN signaling, and PL^pro modulates cellular metabolism and interferon-stimulated gene (ISG) expression. Further research is needed to explore how these proteins influence immune pathways, both transcriptionally and functionally. We aim to develop cellular models expressing SARS-CoV-2 non-structural proteins to study viral-host interactions and the resulting metabolic and transcriptional changes. We aim to establish stable cell lines expressing these viral proteins to investigate their roles in viral pathophysiology. This will help elucidate the pathways and interactions that enable SARS-CoV-2 to infect and cause severe disease in host cells. Our previous LC-MS analysis in Calu-3 and Vero E6 cells indicates that 3CL^pro interacts with host proteins like HMGB1, whose elevated levels correlate with COVID-19 severity. Additionally, 3CLpro associates with enzymes such as pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). To further explore these interactions, techniques like co-immunoprecipitation, immunofluorescence microscopy, flow cytometry, qRT-PCR, and enzyme-linked immunosorbent assays (ELISA) will be employed for a comprehensive analysis of the cellular and molecular mechanisms underlying SARS-CoV-2 pathogenesis. Identifying these critical molecular interactions and mechanisms will help uncover potential therapeutic targets for more effective COVID-19 treatments. Additionally, the developed cellular platforms, based on A549 cells transfected with 3CL^pro and PL^pro genes, will be used to screen antiviral compounds. This builds on our group's prior in vitro tests with recombinant 3CL^pro and PL^pro proteins. By elucidating the interactions between viral proteins and host cellular processes, we aim to contribute to novel therapeutic strategies and a deeper understanding of SARS-CoV-2 virus-host interactions and preparing for future viral outbreaks.