Coronavirus Replication: RNA Structure and Protein-RNA Interactions

In this project, initiated as a collaborative project at Texas A&M University, we used the tools of biophysical chemistry, SHAPE and NMR spectroscopy to understand the structure and biological function of the very “tips” of the coronavirus genome, the 5′ and 3′ untranslated regions (UTRs) that direct the replication, subgenomic RNA (sgRNA) transcription, and propagation of SARS-CoV and closely related group 2 CoVs, including SARS-CoV-2. In this project, we also investigated the structure and function of the CoV nucleocapsid (N) protein as a regulator of the switch from sgRNA transcription early in infection to viral RNA synthesis late in infection. Quantitative RNA binding and unwinding experiments coupled with NMR studies were central to this work.

Publications

SHAPE analysis of the RNA seconday structure of the Mouse Hepatitis Virus 5′ untranslated region and N-terminal nsp1 coding sequences
Yang, D., Liu, P., Wudeck, E.V., LEIBOWITZ, J.L., & GIEDROC, D. P. (2015)
Virology. 475, 15-27 (doi: 10.1016/j.virol.2014.11.001)
View: PubMed

Solution structure of mouse hepatitis virus (MHV) nsp3a and determinants of the interaction with MHV nucleocapsid (N) protein
Keane, S. C. & GIEDROC, D. P. (2013) (doi: 10.1128/JVI.03112-12)
J. Virol. 87, 3502-3515
View: PubMed

(1)H, (13)C, (15)N resonance assignments of murine hepatitis virus nonstructural protein 3a.
Keane, S. C. & GIEDROC, D. P. (2012)
Biomol. NMR Assign.8, 15-17 (doi: 10.1007/s12104-012-9443-5)
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Functional transcriptional regulatory sequence (TRS) RNA binding and helix destabilizing determinants of the murine hepatitis virus (MHV) nucleocapsid (N) protein.
Keane, S. C., Liu, P., Leibowitz, J. L. & GIEDROC, D. P. (2012)
J. Biol. Chem. 287, 7063-7073.
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A conserved RNA pseudoknot in a putative molecular switch domain of the 3′-untranslated region of coronaviruses is only marginally stable.
Stammler, S. N., Cao, S., Chen, S. J., & GIEDROC, D. P. (2011)
RNA 17, 1747-1759.
View: PubMed

Mouse hepatitis virus stem-loop 4 functions as a spacer element required to drive subgenomic RNA synthesis.
Yang, D., Liu, P., GIEDROC, D. P., & Leibowitz, J. L. (2011)
J. Virol. 85, 9199-9209.
View: PubMed

The solution structure of coronaviral stem-loop 2 (SL2) reveals a canonical CUYG tetraloop fold.
Lee, C. W., Li, L.,& GIEDROC, D. P. (2011)
FEBS Lett. 585, 1049-1053.
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Mouse hepatitis virus stem-loop 2 adopts an uYNMG(U)a-like tetraloop structure that is highly functionally tolerant of base substitutions.
Lui, P., Li, L., Keane, S. C., Yang, D., Leibowitz, J. L. & GIEDROC, D. P. (2009)
J. Virol. 83, 12084-12093.
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Coronavirus N protein N-terminal domain (NTD) specifically binds the transcriptional regulatory sequence (TRS) and melts TRS-cTRS RNA duplexes.
Grossoehme, N. E., Li, L., Keane, S. C., Liu, P., Dann, C. E., III, Leibowitz, J. L., & GIEDROC, D. P.  (2009)
J. Mol. Biol. 394, 544-557.
View: PubMed

Structural lability in stem-loop 1 drives a 5′ UTR-3′ UTR interaction in coronavirus replication.
Li, L., Kang, H., Liu, P., Makkinje, N., Williamson, S. T., Leibowitz, J. L., and Giedroc, D. P. (2008)
J. Mol. Biol. 377, 790-803.
View: PubMed

A U-turn motif-containing stem-loop in the coronavirus 5′ untranslated region (UTR) plays a functional role in replication.
Liu, P., Li, L., Millership, J. J., Leibowitz, J. L., & Giedroc, D. P. (2007)
RNA 13, 763-780.
View: PubMed

Cis-acting stem-loops in the 5′ untranslated region of the severe acute respiratory syndrome coronavirus can substitute for their MHV counterparts.
Kang, H., Feng, M., Schroeder, M. E., Giedroc, D. P., & Leibowitz, J. L. (2006)
J. Virol. 80, 10600-10614.
View: PubMed