Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Immunobiology

First Advisor

MacMicking, John

Abstract

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to a range of clinical outcomes, varying from asymptomatic to severe disease. One of our body’s early immune responses against viruses involves the activation of the interferon (IFN) signaling pathway, which mobilizes a diverse set of proteins that defends the host against the invading pathogen. The importance of the IFN response to combatting SARS-CoV-2 infection has been demonstrated by studies showing that patients with genetic deficiencies interfering with the IFN pathway or neutralizing autoantibodies against type I IFNs are predisposed to developing severe COVID-19 disease. Additional studies have shown that prophylactic treatment with type I or III IFN can help control SARS-CoV-2 infection. Together, these findings suggest that our innate immune system plays an important role in defense against SARS-CoV-2, likely through the deployment of IFNs and subsequent activation of antiviral effector proteins. However, the exact host restriction factors involved in IFN-mediated control of SARS-CoV-2 remain largely uncharacterized. The overall goal of this thesis is to identify IFN-stimulated genes (ISGs) that act as antiviral effectors against SARS-CoV-2 and investigate their potential mechanisms of action. First, we performed parallel genome-wide loss-of-function screens in human lung epithelial and hepatoma cells, from which we identified phospholipid scramblase 1 (PLSCR1) as a significantly enriched hit in both screens (Chapter 2). Next, we validated the antiviral activities of PLSCR1 against SARS-CoV-2 across multiple cell types and species of organisms, revealing its evolutionary importance as an anti-SARS-CoV-2 host defense factor (Chapter 3). Subsequently, we interrogated the SARS-CoV-2 infectious life cycle and narrowed down the antiviral activities of PLSCR1 to the viral entry stage. After further dissection of specific steps during the entry process, we found that PLSCR1 antagonizes SARS-CoV-2 infection by interfering with virus-cell fusion (Chapter 4). Lastly, we began to delineate potential mechanisms by which PLSCR1 inhibits SARS-CoV-2 infection. Through fluorescence microscopy and colocalization studies, we discovered that PLSCR1 directly targets to SARS-CoV-2-containing vesicles and relocates to endolysosomal compartments where viral membrane fusion may occur (Chapter 5). Collectively, these results showed that PLSCR1 is a bona-fide IFN-induced host defense protein that restricts the viral entry of SARS-CoV-2 by blocking virus-cell fusion.

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