The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA.
Binding of coat protein (CP) to the 3' nontranslated region (3'-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo- and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3'-NTR of its genomic RNA using purified E. coli- expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3'-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3'-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg(2+), lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3'-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3'-NTR level in Alfamo- and Ilarvirus genera.
Aparicio
Frederic
F
Instituto de Biologia Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Avenida de los Naranjos s/n, E-46022 Valencia, Spain.
Vilar
Marçal
M
Perez-Payá
Enrique
E
Pallás
Vicente
V
D020413:3' Untranslated Regions / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D024202:Electrophoretic Mobility Shift Assay; D019179:Ilarvirus / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D027861:Prunus / Q000821:virology*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020413:3' Untranslated Regions; D036022:Capsid Proteins; D012367:RNA, Viral; D008274:Magnesium