Mapping of the RNA-binding domain of the cucumber mosaic virus movement protein.
A series of in-frame deletion mutants was used to identify a domain within the 3a protein of cucumber mosaic virus (CMV) that is required for RNA-binding activity. Deletions in the 3a gene were generated by PCR and restriction digestion, and the resulting mutated 3a sequences were cloned either in pT7-7 or in pGEX-5X3 expression vectors. The mutated 3a proteins or fusions with glutathione S-transferase (GST) were expressed in E. coli, purified, and their nucleic acid-binding activities analysed by photochemical UV cross-linking assays using digoxigenin-UTP-labelled RNA probes. Comparative analyses of seven mutated 3a proteins obtained from inclusion bodies and eight GST fusion proteins revealed that there is an RNA-binding domain located between amino acids 174 and 233. This RNA-binding domain is able to bind single-stranded RNA out of the context of the complete 3a movement protein and is highly conserved within both subgroups of CMV.
Vaquero
C
C
Institut fur Biologie I (Molekulargenetik/Botanik), RWTH Aachen, Germany.
Liao
Y C
YC
Nähring
J
J
Fischer
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D017799:Cucumovirus / Q000378:metabolism*; D005982:Glutathione Transferase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D053830:Plant Viral Movement Proteins; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D053830:Plant Viral Movement Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005982:Glutathione Transferase