the basis for designing experiments and developing hypotheses that will lead to new molecular-level insights into how RNA structure and RNA-protein interactions regulate coronavirus replication. This application focuses on three aspects of our model. In the first aim we will investigate our hypothesis that SL1 must be a dynamic structure to allow interactions with other RNA sequences and/or replication accessory proteins that ultimately mediate an interaction between the 5'and 3'UTRs in genome circularization. Biochemical, biophysical (NMR, thermodynamic studies), and reverse genetic approaches will be utilized to determine the stability of SL1, and the requirement for a dynamic SL1 in viral replication. Biochemical studies will be performed to identify proteins that bind to SL1 and their role in circularization of the coronavirus genome. In the second aim we will investigate SL2, a stem-loop that our preliminary studies have indicated adopts an unusual U-turn like structure. We propose to solve the solution structure of SL2 at high resolution using NMR methods, and perform a series of reverse genetic studies to test predictions of our structural model in the context of the MHV genome and determine their effects on viral replication. In the third aim we will employ a combination of biophysical and reverse genetic experiments to investigate the interaction of the coronavirus nucleocapsid protein with the transcriptional regulatory sequences in the 5'leader (TRS-L) RNA. We will determine the solution structure of nucleocapsid protein:TRS-L RNA complexes by NMR spectroscopy and biophysical methods. Informed by the nucleocapsid protein:TRS-L RNA structure, we will perform a series of reverse genetic studies to determine if the TRS:nucleocapsid interaction plays a role in RNA replication, subgenomic RNA synthesis and/or translation. The proposed studies will significantly advance our understanding of the detailed structure and function of critical coronavirus cis- acting sequences and interacting proteins, and provide mechanistic insights into coronavirus replication. Project Description Page 6
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