Localization of the single-stranded RNA-binding domains of bluetongue virus nonstructural protein NS2.
The S2 gene of bluetongue virus, serotype 17, has been cloned, and the nonstructural protein NS2 has been expressed. Synthetic peptides matching regions within the amino acid sequence of NS2 were used to map three single-stranded RNA (ssRNA)-binding regions within the protein. A prokaryotic expression system was then used to generate a series of deletion mutants with the ssRNA-binding domains of NS2 removed, singly and in different combinations. These truncated proteins were expressed on a large scale and purified to near homogeneity. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays. As a result, the three ssRNA-binding domains of BTV nonstructural protein NS2 have been conclusively localized, and removal of these three domains completely abrogates the ability of NS2 to bind to ssRNA.
Fillmore
G C
GC
Department of Biology, Utah State University, Logan, Utah 84322-5305, USA.
Lin
H
H
Li
J K K
JK
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D001820:Bluetongue virus / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins