Juhi Jha
Research Mentor: Katherine Barnett
Mentor Department: Department of Microbiology and Immunology, Medicine
Author(s): Juhi Jha, Luiza Castro, Katherine Barnett
Session: Session 1 (9:00 AM – 9:50 AM)
Presentation Type: Poster 102
Abstract
Viral infections, such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), activate immune responses in the host cell that can include programmed cell death pathways, which have a critical effect on viral pathogenesis. Inflammasomes, protein complexes involved in the innate immune response, detect pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Recognition of these molecules triggers the formation of the inflammasome, which causes the release of signaling proteins, known as cytokines, and pyroptosis, an inflammatory form of cell death. In this project, we investigate the role of key genes related to inflammasome activation, including CASP1, GSDMD, IL1B, NLRP3, and NLRP1, during SARS-CoV-2 infection. To address this question, we generated plasmids to make CRISPR-Cas9 knockout clones in the human lung epithelial A549 cell line. Plasmids encoding the guide RNAs for these genes were linearized through restriction digest, purified via gel extraction, and transformed into STBL3 competent cells to amplify the plasmid DNA. The extracted plasmid DNA was then used to produce lentiviral vector particles in the 293T cell line, which was later harvested and used to infect A549 cells. These knockout A549 cell lines will be single cell cloned and then infected with SARS-CoV-2 to test for key responses including pyroptotic cell death via LDH release, ELISA for cytokine release, qPCR for host and viral gene expression, and ASC speck formation. These results will be compared between the gene-specific knockout, wild-type, and ASC-knock out lines to determine the contribution of each inflammasome component in the immune response in the SARS-CoV-2 infection. Ultimately, this work will clarify the molecular mechanisms connecting virus-induced cell death to the host inflammatory response, contributing to the development of targeted therapies for coronavirus infections in the future.
