VCOM Louisiana Research Day Program Book 2024

Biomedical Research: Section 1

Delaney Yu, OMS-III; Juan Guerra, OMS-III; Ariana Faraji, OMS-IV, Kasia Michalak MS, Lin Kang, PhD; Pawel Michalak, PhD; Stephen Diguiseppe, PhD VCOM Biomedical Science Laboratory 14 EVOLUTION IN SARS-CoV-2 SPIKE PROTEIN FACILITATE ADAPTATION TO HUMANS

Background: 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. Objective: 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. Methods: 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. Results: Predicted adaptive mutations have the potential to affect virus infectivity in human cells. To comprehend these adaptive mutations, we reverted amino acid sites back to ancestral amino acids to test functionality. The T372A and N519H shows significantly decreased infectivity compared to wild type spike protein. As described in previous literature, D614G shows high infectivity in cells expressing human ACE2. Conclusions: We identified adaptive mutations via selective sweep analysis and found that pseudovirus harboring ancestral mutations in SARS-CoV-2 spike protein show decreased infectivity in cells expressing human ACE2. Previously reported data suggests this is important for adaptive mutations and spillover into humans

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