Determining the Role of PP5 in ACE2 Expression and Cell Susceptibility to SARS-CoV-2

Hannah Giannini and Dr. Richard Honkanen, Department of Biochemistry and Molecular Biology, University of South Alabama College of Medicine, 5851 USA Drive North, Mobile, AL 36688

Laboratories worldwide are experiencing difficulties culturing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19. Resultingly, discoveries concerning virus diagnosis and treatment are being hindered. This project aims to develop a cell line that is more susceptible to infection by SARS-CoV-2 when compared to common laboratory cell lines that are currently in use. To enter a cell, SARS-CoV-2 binds to angiotensin-converting enzyme-2 (ACE2) which is expressed on the surface of some cells. The expression of ACE2 has been reported to be responsive to hypoxia-inducible factors (HIF-1). Protein phosphatase 5 (PP5) expression is also responsive to HIF-1, acting to suppress some HIF-1 induced events. The role of PP5 in the feedback inhibition of HIF-1 induced ACE2 expression is not known. Our hypothesis is that the suppression of PP5 may upregulate HIF-1 induced ACE2 expression, allowing more virus to enter the cell. To test this hypothesis, we used a CRISPR-Cas9-based method to disrupt PP5 expression in two different cell lines: lung epithelial (A549) and human embryonic kidney (HEK 293 E420K Het). Additionally, a A549 PPP2R5D (another regulatory phosphatase) knockout cell line was generated. Total knockout efficiency of each cell population was determined by genomic sequence analysis after DNA isolation, PCR, and gel electrophoresis. A similar process was performed to screen single sorted cell colonies for the desired edit. Once knockout cell colonies were identified, additional screenings are performed to verify the knockout prior to SARS-CoV-2 exposure. If protein phosphatase inhibition improves infection, these findings will further mechanistic knowledge of PP5 and ACE2, and the edited cells could serve as an improved experimental cell line for COVID-19 research.

Additional Abstract Information

Presenter: Hannah Giannini

Institution: University of South Alabama

Type: Poster

Subject: Biochemistry

Status: Approved

Time and Location

Session: Poster 1
Date/Time: Mon 1:30pm-2:30pm
Session Number: 2118