Size-independent and noncooperative recognition of dsRNA by the Rice stripe virus RNA silencing suppressor NS3.
Plant and animal viruses employ diverse suppressor proteins to thwart the host antiviral reaction of RNA silencing. Many suppressors bind dsRNA with different size specificity. Here, we examine the dsRNA recognition mechanism of the Rice stripe virus NS3 suppressor using quantitative biochemical approaches, as well as mutagenesis and suppression activity analyses in plants. We show that dimeric NS3 is a size-independent, rather than small interfering RNA-specific, dsRNA-binding protein that recognizes a minimum of 9 bp and can bind to long dsRNA with two or more copies. Global analysis using a combinatorial approach reveals that NS3 dimer has an occluded site size of ∼ 13 bp on dsRNA, an intrinsic binding constant of 1 × 10(8) M(-1), and virtually no binding cooperativity. This lack of cooperativity suggests that NS3 is not geared to target long dsRNA. The larger site size of NS3, compared with its interacting size, indicates that the NS3 structure has a border region that has no direct contact with dsRNA but occludes a ∼ 4-bp region from binding. We also develop a method to correct the border effect of ligand by extending the lattice length. In addition, we find that NS3 recognizes the helical structure and 2'-hydroxyl group of dsRNA with moderate specificity. Analysis of dsRNA-binding mutants suggests that silencing of the suppression activity of NS3 is mechanistically related to its dsRNA binding ability.
Graduate Program at Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
Mei
M
Shen
Yi
Y
Xu
Ru
R
Jia
Xueping
X
Zhou
Keqiong
K
Ye
D000818:Animals; D054884:Host-Pathogen Interactions; D007700:Kinetics; D011485:Protein Binding; D055503:Protein Multimerization; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D029364:Tenuivirus / Q000201:enzymology / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins