Date of Award
Fall 2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular, Cellular, and Developmental Biology
First Advisor
Pyle, Anna Marie
Abstract
RIG-I is our first line of defense against RNA viruses, serving as a patternrecognition receptor (PRR) that identifies molecular features common among dsRNA and ssRNA viral pathogens. RIG-I is maintained in an inactive conformation as it samples the cellular space for pathogenic RNAs. Upon encounter with the triphosphorylated terminus of blunt-ended viral RNA duplexes, the receptor changes conformation and releases a pair of signaling domains (CARDs) that are selectively modified and interact with an adapter protein (MAVS), thereby triggering a signaling cascade that stimulates transcription of type I interferon. While the molecular mechanisms of RIG-I recognition of viral pathogen-associated molecular patterns (PAMPs) is well understood, our understanding of RIG-I’s surveillance of the cell remains incomplete. In particular, direct observation of RIG-I encountering viral RNA PAMPs within the cell have been scarcely reported, and prior observations of RIG-I signaling at MAVS are largely not aligned with the actual dynamics of RIG-I signaling. In my thesis work, I have focused on more fully understanding the biology of RIG-I signaling within the cell. To that end, I stimulated endogenous RIG-I with both a small, synthetic double stranded RNA and a negative strand virus, and determined the level of total signaling induced via RT-qPCR. From these experiments, I determined that upon encountering viral RNA, an active RIG-I signal is rapidly transmitted to enable a swift response. Furthermore, careful titration of RNA PAMPs determined that an average of about 10 RNAs/cell are required to initiate an antiviral response. Next, I employed high-resolution confocal microscopy to track the distribution of fluorescently-labeled RIG-I before and after stimulation with short double-stranded RNA (dsRNA) PAMPs, and observed that throughout the RIG-I signaling response, no increase in RIG-I concentration at the mitochondria could be observed. Instead, RIG-I aggregations could be observed colocalizing with labeled dsRNA within the cell. These RIG-I:dsRNA RNP bodies form regardless of whether the dsRNA can stimulate signaling, suggesting that these bodies are not directly connected to signalling. Taken together, these findings indicate that an active RIG-I signal is quickly transmitted by a small number of receptors, and that even when numerous receptors are active, it does not accumulate at the mitochondria network where MAVS resides. All of these findings recommend a significant revision in how RIG-I signaling is viewed within the cell, from the slow accumulation of massive oligomeric signaling complexes to a much smaller and transient signaling complex.
Recommended Citation
Thoresen, Daniel, "A Rapid RIG-I Signaling Relay Mediates an Efficient Antiviral Response" (2022). Yale Graduate School of Arts and Sciences Dissertations. 736.
https://elischolar.library.yale.edu/gsas_dissertations/736
