Coronavirus N protein N-terminal domain (NTD) specifically binds the transcriptional regulatory sequence (TRS) and melts TRS-cTRS RNA duplexes.
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 plays a critical accessory role in subgenomic RNA synthesis and other processes requiring RNA remodeling.
Department of Chemistry, Indiana University, Bloomington, IN 47405-7102, USA.
Nicholas E
NE
Grossoehme
Lichun
L
Li
Sarah C
SC
Keane
Pinghua
P
Liu
Charles E
CE
Dann
Julian L
JL
Leibowitz
David P
DP
Giedroc
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000086462:Coronavirus Nucleocapsid Proteins; D018360:Crystallography, X-Ray; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013816:Thermodynamics
D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins