Grant: Discovery Grants Program - Individual

Overview: A key initiator of the innate immune response to viral infection is foreign double-stranded RNA (dsRNA) derived from viral genomes. Central to this response are interferon-stimulated proteins that are tuned to bind and respond to viral dsRNA. We currently study two such cytosolic protein systems: the family of 2'-5'-oligoadenylate synthetases (OAS) and the RNA-dependent protein kinase (PKR). The recent surge in industrially important RNA biotechnology applications require evasion of the OAS/PKR immune response. Therefore, the continued goal of the proposed research program is to characterize the molecular recognition of dsRNA molecules by OAS and PKR enzymes. Research Program: Understanding the recognition of highly structured viral RNA targets by OAS/PKR is the first objective of the program. We will focus our attention on the 5'- and 3'- terminal regions of multiple flavivirus RNA genomes, as these regions share highly conserved secondary structure elements required for viral fitness. OAS and PKR enzymes have an established role in attenuating flavivirus infection and therefore understanding the core structural features required for direct interaction will shed light on innate immune function. Using purified RNA and protein, we will examine binding affinity, enzyme activation, and determine structures of complexes. Identifying host cell RNA and protein regulators of the OAS/PKR pathways is the second short-term objective of the program. While originally thought to only function as part of the interferon response to viral infection, recent reports strongly suggest that cytosolic OAS/PKR respond to endogenous RNA and protein cofactors during both cellular stress conditions and at various points in the normal cell cycle. Immunoprecipitation of OAS/PKR in cells subjected to stressors will enable isolation and identification of novel host cell binding partners. Understanding potential chemical modifications that prevent recognition by OAS/PKR is the third objective of the program. In vitro transcribed or synthesized RNA have emerged as key players in biotechnologies and biologics. Extensive research efforts are currently focused on optimizing RNA constructs to overcome stability, delivery, and immunogenicity through sequence optimization, chemical engineering of nucleotides and 5' cap, and optimization of in vitro transcription. We will test the impact of multiple chemical modifications and structural features in RNA in an attempt to evade detection by the OAS/PKR systems. Results will inform the long-term goal of the research program in understanding the structural/chemical features of RNA biomolecules required to enzymatically activate cytosolic RNA sensor proteins. Impact: The research program addresses fundamental properties of the stress response to dsRNA by sensor proteins. Beyond the characterization of these important proteins, the immediate impact will be felt by researchers and industries using RNA delivery approaches.