Coronavirus N protein N-terminal domain (NTD) specifically binds the transcriptional regulatory sequence (TRS) and melts TRS-cTRS RNA duplexes

Journal of Molecular Biology
Nicholas E GrossoehmeDavid P Giedroc

Abstract

All coronaviruses (CoVs), including the causative agent of severe acute respiratory syndrome (SARS), encode a nucleocapsid (N) protein that harbors two independent RNA binding domains of known structure, but poorly characterized RNA binding properties. We show here that the N-terminal domain (NTD) of N protein from mouse hepatitis virus (MHV), a virus most closely related to SARS-CoV, employs aromatic amino acid-nucleobase stacking interactions with a triple adenosine motif to mediate high-affinity binding to single-stranded RNAs containing the transcriptional regulatory sequence (TRS) or its complement (cTRS). Stoichiometric NTD fully unwinds a TRS-cTRS duplex that mimics a transiently formed transcription intermediate in viral subgenomic RNA synthesis. Mutation of the solvent-exposed Y127, positioned on the beta-platform surface of our 1.75 A structure, binds the TRS far less tightly and is severely crippled in its RNA unwinding activity. In contrast, the C-terminal domain (CTD) exhibits no RNA unwinding activity. Viruses harboring Y127A N mutation are strongly selected against and Y127A N does not support an accessory function in MHV replication. We propose that the helix melting activity of the coronavirus N protein NTD pla...Continue Reading

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Citations

Apr 7, 2010·Journal of Neuroimmune Pharmacology : the Official Journal of the Society on NeuroImmune Pharmacology·Susan J Bender, Susan R Weiss
Feb 23, 2011·The Journal of Biological Chemistry·Nicholas GrossoehmeDavid P Giedroc
Jun 3, 2010·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Xiaohang TongXuemei Li
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Feb 24, 2015·PloS One·Kaja ZuwałaKrzysztof Pyrc
Mar 24, 2017·Journal of Virology·Bozena SzelazekGrzegorz Dubin
Jul 15, 2020·The Journal of Biological Chemistry·Ella HartenianBritt A Glaunsinger
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Jul 20, 2017·Scientific Reports·Yingying CongFulvio Reggiori
Nov 10, 2020·Nucleic Acids Research·Anna WackerHeidi Zetzsche
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Feb 9, 2021·Computational and Structural Biotechnology Journal·Yeu-Yang TsengWei-Li Hsu

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Methods Mentioned

BETA
immunoprecipitation
x-ray scattering
Fluorescence
isothermal titration calorimetry
FRET
in vitro runoff
PCR
ISS

Software Mentioned

TLSMD
Coot
DynaFit
MOLPROBITY
Phenix

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