Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Charles M. Rice
Abstract
Positive-sense RNA viruses, such as those in the families Flaviviridae and Coronaviridae, exploit host cell machinery for replication. The ER-resident transmembrane protein TMEM41B has emerged from independent genome-wide CRISPR knockout screens as a convergent pan- viral host factor. Yet, TMEM41B also performs essential cellular functions required for autophagosome biogenesis, lipid droplet homeostasis, phospholipid scramblase activity, and ER Ca²⁺ release. This functional pleiotropy renders pharmacological inhibition therapeutically untenable and demands a strategy capable of targeting its proviral role with single-residue precision. This thesis investigates whether specific residues exist where TMEM41B’s proviral function can be disrupted without abolishing its essential cellular activities. To quantitatively define TMEM41B’s functional landscape, I first established four orthogonal phenotypic assays spanning viral susceptibility, lipid mobilization, autophagic flux, and scramblase activity. I then deployed two conventional deep mutational scanning (DMS) platforms, lentiviral cDNA overexpression and cytidine base editing at the endogenous locus, that identified structural features such as transmembrane (TM) helices as the most constrained regions. These conventional DMS approaches mapped TMEM41B’s mutational profile but failed to resolve intermediate phenotypes because loss-of-expression variants (e.g. early stop codons) overwhelmed the surviving population. To overcome this limitation, I engineered a saturation genome editing (SGE) platform with a novel mNeonGreen break-and-rescue system that filters for stably expressed variants before viral challenge, isolating functional phenotypes from the dominant loss-of-expression background. Deployment of this platform in an Exon 3 screen (positions 80–122) identified two functional clusters in the structured loop between TM helices 1 and 2, unified by steric sensitivity at the predicted lipid translocation groove. At groove-lining positions (A95, G99), and the adjacent Ca²⁺-coordination triad (D91, D93, D94), loss of proviral function correlates with the steric footprint of the substitution; consistent with physical occlusion of the translocation pathway. A distinct, electrostatic mechanism operates at groove-rim positions (M86, V88), where only negatively charged substitutions disrupt proviral function. Four priority candidates spanning both mechanisms—A95W, G99L, D94P/W, and V88D—emerged from this screen. All four variants produce stably expressed protein yet specifically fail to support viral replication and represent candidates for validation against the cellular assay panel to determine whether true separation of function has been achieved. This work establishes the experimental infrastructure necessary to determine whether TMEM41B’s proviral function can be selectively targeted and map the structure–function relationships that govern its pleiotropic biology. The break-and-rescue SGE–mNeonGreen platform offers a generalizable approach for studies of proviral host factors amenable to endogenous tagging, where pleiotropy complicates therapeutic intervention and sensitive multiplexed variant interpretation is required.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Grodus, Michael, "Decoupling TMEM41B'S Proviral and Cellular Functions via Saturation Genome Editing" (2026). Student Theses and Dissertations. 858.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/858
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy