The long-term goal of this project is to obtain a detailed understanding of the high resolution RNA structures of these critical cis-acting 5'and 3'UTRs and of protein-RNA interactions that direct the replication and propagation of SARS-coronavirus (SARS-CoV) and the closely related group 2 coronavirus, mouse hepatitis virus (MHV). Such information is integral to the eventual development of specific antiviral agents that could be used to abrogate coronavirus replication. Towards that end we have developed a covariation-based model of the secondary structure of the coronavirus 5'UTR that contains three conserved stem-loop structures, SL1, SL2, and SL4. This model has provided 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. Coronaviruses are important human and veterinary pathogens, with the SARS-coronavirus being the most serious human pathogen. Although the original outbreak of SARS was brought under control, this virus is a threat to re-emerge from its zoonotic source. The research proposed in this application will advance our detailed knowledge of coronavirus replication, and by doing so lay the basis for future development of additional anti-virals directed against this group of viruses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI067416-03
Application #
7753657
Study Section
Virology - A Study Section (VIRA)
Program Officer
Salomon, Rachelle
Project Start
2008-01-01
Project End
2012-12-31
Budget Start
2010-01-01
Budget End
2010-12-31
Support Year
3
Fiscal Year
2010
Total Cost
$359,003
Indirect Cost
Name
Indiana University Bloomington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
Yang, Dong; Leibowitz, Julian L (2015) The structure and functions of coronavirus genomic 3' and 5' ends. Virus Res 206:120-33
Yang, Dong; Liu, Pinghua; Wudeck, Elyse V et al. (2015) SHAPE analysis of the RNA secondary structure of the Mouse Hepatitis Virus 5' untranslated region and N-terminal nsp1 coding sequences. Virology 475:15-27
Keane, Sarah C; Giedroc, David P (2014) ¹H, ¹³C, ¹?N resonance assignments of murine hepatitis virus nonstructural protein 3a. Biomol NMR Assign 8:15-7
Keane, Sarah C; Giedroc, David P (2013) Solution structure of mouse hepatitis virus (MHV) nsp3a and determinants of the interaction with MHV nucleocapsid (N) protein. J Virol 87:3502-15
Liu, Pinghua; Yang, Dong; Carter, Kristen et al. (2013) Functional analysis of the stem loop S3 and S4 structures in the coronavirus 3'UTR. Virology 443:40-7
Bhardwaj, Kanchan; Liu, Pinghua; Leibowitz, Julian L et al. (2012) The coronavirus endoribonuclease Nsp15 interacts with retinoblastoma tumor suppressor protein. J Virol 86:4294-304
Keane, Sarah C; Liu, Pinghua; Leibowitz, Julian L et al. (2012) Functional transcriptional regulatory sequence (TRS) RNA binding and helix destabilizing determinants of murine hepatitis virus (MHV) nucleocapsid (N) protein. J Biol Chem 287:7063-73
Leibowitz, Julian; Kaufman, Gili; Liu, Pinghua (2011) Coronaviruses: propagation, quantification, storage, and construction of recombinant mouse hepatitis virus. Curr Protoc Microbiol Chapter 15:Unit 15E.1
Stammler, Suzanne N; Cao, Song; Chen, Shi-Jie et al. (2011) A conserved RNA pseudoknot in a putative molecular switch domain of the 3'-untranslated region of coronaviruses is only marginally stable. RNA 17:1747-59
Lee, Chul Won; Li, Lichun; Giedroc, David P (2011) The solution structure of coronaviral stem-loop 2 (SL2) reveals a canonical CUYG tetraloop fold. FEBS Lett 585:1049-53

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