2022 VCOM Research Retreat Program

Abstracts

Intracellular Osteopontin in Pulmonary Arterial Hypertension Presenter: Rebekan Morrow, PhD Co-Authors: Reggie Skains*; Adam Morrow, PhD

Identifying Site-Specific Mutations that Increase SARS-CoV-2 Virulence using Selective Sweep Analysis Presenter: Stephen DiGiuseppe, PhD Co-Authors: Juan Guerra*; Nora Rady*; Kasia Michalak, MSc; Ariana Faraji*; Lin Kang, PhD; Pawel Michalak, PhD In 2019, the world was alerted to an emerging novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which poses a significant threat to global public health. Patients infected with SARS-CoV-2 experience mild to severe flu-like symptoms in the upper respiratory tract. While most infections will be cleared by the immune system, the virus can disseminate to the lower respiratory tract where patients may develop severe pneumonia and severe acute respiratory distress syndrome (ARDS), a life-threatening condition that requires patients to be intubated in intensive care. Fortunately, several vaccine candidates were approved for emergency use. Despite global deployment of multiple vaccines, continued outbreaks across the world suggest the pandemic is far from over. The origins of SARS-CoV-2 are still unknown, several bat- and pangolin-derived viruses were found to be closely related suggesting a zoonotic origin. The exact selective mutations that allowed SARS-CoV-2 to jump to humans is not well understood, but recent innovations in assays involving SARS-CoV-2 have provided insight into these mutations. Previous work examining these mutations in the SARS-CoV-2 genome through selective sweep analysis found that a non-synonymous change within the Spike protein receptor-binding domain increased binding to human ACE2, possibly contributing to its adaptive evolution. We hypothesize that site-specific mutations with adaptive sequence signatures in SARS-CoV-2 spike contribute to its increased affinity to human ACE2 receptor resulting in increased virulence. To safely study SARS-CoV-2 infectivity, we generated pseudovirus harboring SARS-CoV-2 Spike via a recombinant lentivirus system. The pseudovirus binds and enters cells expressing the human ACE2 receptor. Using the lentivirus system, we performed site-directed mutagenesis to mutate specific residues within the Spike gene with selective sweep signatures detected from SARS-CoV-2 genomes deposited in the GISAID EpiCov database. We generated pseudovirus harboring the wild-type or mutated Spike protein. We quantified the relative abundance of wild-type and mutant pseudovirus using specific primers by PCR. Next, we infected 293T cells expressing human ACE2 with wild-type and mutant pseudovirus and measured infectivity by relative luciferase activity and live-cell fluorescence imaging. In turn, we seek to identify specific mutations of the Spike protein that increase infectivity of SARS-CoV-2 pseudovirus to better understand how the native virus binds to ACE2 cells across different species, and determine which mutations govern changes in virulence. *VCOM Student Pulmonary arterial hypertension (PAH) is a progressive disease caused by narrowing of the pulmonary arteries, at least partially due to extracellular matrix remodeling and endothelial to mesenchymal transition (EndoMT). Osteopontin (OPN) is a protein involved in many cellular processes, including adhesion, migration, and modulation of the inflammatory response. The goal of this project was to determine whether loss of intracellular osteopontin enhances endothelial dysfunction in pulmonary artery endothelial cells (PAEC) in PAH. Previous data have shown that PAEC from an animal model of PAH exhibit lower levels of intracellular OPN than healthy controls. We generated stable OPN overexpressors and transfected PAEC with siRNA to transiently knock down OPN expression. RNA sequencing was performed as a screen for possible targets of interest. Protein levels were evaluated by western blot and ELISA. Contrary to expectations, PAH cells expressed higher levels of OPN and displayed increased markers of endothelial activation, possibly contributing to the inflammatory phenotype of this disease.