Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells.
The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a right hand with a flexible, positively charged beta-hairpin and a hydrophobic palm. This domain was shown to interact with the genomic RNA for coronavirus infectious bronchitis virus (IBV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Based on its 3D structure, we used site-directed mutagenesis to identify residues essential for the RNA-binding activity of the IBV N protein and viral infectivity. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells. In contrast, mutation of amino acid Gln-74 to an alanine, which does not affect the binding activity of the N-terminal domain, showed minimal, if any, detrimental effect on the infectivity of IBV. This study thus identifies residues critical for RNA binding on the nucleocapsid surface, and presents biochemical and genetic evidence that directly links the RNA binding capacity of the coronavirus N protein to the viral infectivity in cultured cells. This information would be useful in development of preventive and treatment approaches against coronavirus infection.
Tan
Yong Wah
YW
Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.
Fang
Shouguo
S
Fan
Hui
H
Lescar
Julien
J
Liu
D X
DX
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids; D000818:Animals; D002522:Chlorocebus aethiops; D000086462:Coronavirus Nucleocapsid Proteins; D004252:DNA Mutational Analysis; D001351:Infectious bronchitis virus / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014709:Vero Cells
D000596:Amino Acids; D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins