Genome-wide and Targeted Screening Methods Reveal Host Antiviral Mechanisms to Limit Norovirus Infection
Date of Award
Doctor of Philosophy (PhD)
The goal of this thesis was to identify and characterize novel endogenous antiviral genes and to elucidate their mechanisms within the context of murine norovirus (MNoV) infection using cutting-edge techniques. Our studies provide a resource for understanding enteric viral pathogenesis, demonstrate robust methodologies for broadly identifying novel endogenous antiviral molecules, and potentially pave the way for future therapeutics.
Here, we describe genome-wide CRISPR activation (CRISPRa) screens performed in both naturally permissive murine BV2 cells and MNoV-permissive engineered HeLa cells which revealed high-scoring gene candidates that inhibit MNoV infection when overexpressed at the endogenous locus. TRIM7, an E3 ubiquitin ligase, emerged as the strongest hit in both screens, emphasizing its potent activity against MNoV regardless of cellular context. We further characterized the molecular mechanisms underlying its antiviral effect and have found TRIM7 to act early in the viral life cycle to limit viral nonstructural protein levels by interacting directly with the viral protease.
We also performed an arrayed interferon stimulated gene (ISG) overexpression screen with the goal of uncovering novel anti-MNoV ISGs. This screen was performed in STAT1-deficient MNoV-permissive human fibroblasts. Cells overexpressing a single ISG were challenged with MNoVCW3, and the effect of the ISG on viral infection was quantified by flow cytometry. Our screen identified multiple antiviral ISGs including KCTD14, thought to potentially mediate the activity of the CUL-3 ubiquitin ligase. We found that that KCTD14 inhibits MNoV replication after viral entry, likely by disrupting formation of the replication complex via interaction with viral protein NS3.
Data produced via these varied screening methods provide an entirely new lens by which to understand norovirus biology – via both genome-wide and targeted ISG interrogation - leading to the discovery of novel anti-NoV genes that may be leveraged in the future to limit NoV infections.
Chair and Committee
Megan T. Baldridge
Daniel E. Goldberg
Sullender, Meagan E., "Genome-wide and Targeted Screening Methods Reveal Host Antiviral Mechanisms to Limit Norovirus Infection" (2022). Arts & Sciences Electronic Theses and Dissertations. 2661.
Available for download on Monday, May 20, 2030