Characterization of the RNA-binding domain in the Dendrolimus punctatus cytoplasmic polyhedrosis virus nonstructural protein p44.
Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV-1) belongs to the Cypovirus genus in the Reoviridae family. The ORF of genome segment 8 (S8) of DpCPV-1 was cloned into vector pMAL-c2X and used to express a 44kDa protein (p44) in E. coli, which was detected by Western blotting. The gel mobility shift assays showed that p44 had ssRNA-binding activity. Competitive assay indicated that this protein only bind to ssRNA and could not interact with DNA and dsRNA. The binding of p44 to ssRNA is sequence non-specific. To identify the domain(s) important for RNA binding of the protein, a number of deletions were made. These truncated proteins were expressed in E. coli and purified. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays and northwestern blot. The results indicated that glutamic acid-rich domain in the central region of p44 from residues 104 to 201 was the ssRNA-binding domain.
Zhao
S L
SL
Laboratory for Biological Control, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China. sdzll79@sohu.com
Liang
C Y
CY
Zhang
W J
WJ
Tang
X C
XC
Peng
H Y
HY
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D009036:Moths / Q000821:virology*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012087:Reoviridae / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly; D014779:Virus Replication
D012330:RNA, Double-Stranded; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins