Rig-1-dependent innate immune signaling by hepatitis C virus RNA

The cytosolic pattern recognition receptor RIG-I recognizes viral RNAs and initiates downstream signaling resulting in expression of interferon (IFN)-α/β and establishment of an antiviral state in infected cells. The goal of this dissertation is to provide a more detailed understanding of how the RIG-I/viral RNA interaction activates RIG-I-dependent innate immune signaling. Many different RIG-I activator RNAs have been identified. In Chapter 2, I demonstrate that an RNA 5' triphosphate (5'ppp) is insufficient for robust activation of RIG-I signaling. 5'ppp-containing single-stranded RNAs with uridine- or adenine-rich motifs, such as the HCV polyU/UC and polyAG/A RNAs, are potent RIG-I stimulators. I show that polyU/UC RNAs transcribed in vitro using 2'fluoro-modified (2'F-dU) or pseudouridine-modified ribonucleotides lack signaling activity, but retain the ability to bind RIG-I. In Chapter 3, I examine RIG-I binding affinities of 2'F-dU- and pseudouridine-modified polyU/UC RNAs, and show that they have slightly enhanced RIG-I binding affinities compared to unmodified polyU/UC RNA. In addition, RIG-I is ubiquitinated in the presence of unmodified or 2'F-dU-modified polyU/UC RNA. Interestingly, an interaction between RIG-I and its downstream signaling partner MAVS is detected in the presence of 2'F-dU-modified polyU/UC RNA but not unmodified polyU/UC RNA, suggesting that unmodified polyU/UC RNA induces a transient but functional RIG-I/MAVS complex, whereas 2'F-dU-modified polyU/UC RNA induces a stable, signaling-defective RIG-I/MANS complex. In Chapter 4, I describe collaborative studies that test whether potent RIG-I activator RNAs, such as polyU/UC and polyAG/A RNAs, can be delivered efficiently to both an in vitro human liver model and in vivo to mice to induce innate immune responses. I show that polyU/UC RNA can be delivered into primary human hepatocytes using a novel lipid-like delivery reagent and that it induces robust IFN-β production. In addition, I provide evidence that a rapid, robust TLR3-independent innate immune response can be stimulated by polyAG/A RNA in vivo in mice. In summary, this dissertation identifies 5'ppp-containing polyU/UC and polyAG/A RNAs as potent RIG-I activators, provides insight into how RIG-I binding and signaling are uncoupled with modified RNAs, and demonstrates potential for RIG-I activator RNAs as agonists for therapeutic applications.