title: Multimers of the bluetongue virus nonstructural protein, NS2, possess nucleotidyl phosphatase activity: similarities between NS2 and rotavirus NSP2.
abstract: The nonstructural protein, NS2, of bluetongue virus is a nonspecific single- stranded RNA-binding protein that forms large homomultimers and accumulates in viral inclusion bodies of infected cells. NS2 shares these features with the nonstructural protein, NSP2, of rotavirus, which like BTV is a member of the family Reoviridae. Recently, NSP2 was shown to have an NTPase activity and an autokinase activity that catalyzed its phosphorylation in vitro. To examine NS2 for similar enzymatic activities, the protein was expressed in bacteria with a C-terminal His-tag and purified to homogeneity. Recombinant (r)NS2 possessed nonspecific RNA-binding activity and formed 8-10S homomultimers of the same approximate size as rNSP2 homomultimers. Notably, enzymatic assays performed with rNS2 showed that the protein hydrolyzed the alpha, beta, and gamma phosphodiester bonds of all four NTPs. Therefore, rNS2 possesses a nucleotidyl phosphatase activity instead of the NTPase activity of NSP2, which only hydrolyzes the gamma phosphodiester bonds of NTPs. NS2 did not exhibit any autokinase activity in vitro, unlike NSP2. However, both NS2 and NSP2 were phosphorylated in vitro by cellular kinases. Although the nature of the enzymatic activities differs significantly, the fact that both NS2 and NSP2 hydrolyze NTPs, undergo phosphorylation, bind RNA, and assemble into multimers consisting of 6 +/- 2 subunits suggests that they are functional homologs.
authors.affiliation: Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, 7 Center Drive, Bethesda, Maryland 20892, USA.
authors.forename: Z F
authors.initials: ZF
authors.lastname: Taraporewala
authors.forename: D
authors.initials: D
authors.lastname: Chen
authors.forename: J T
authors.initials: JT
authors.lastname: Patton
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001820:Bluetongue virus / Q000201:enzymology*; D002460:Cell Line; D011232:Chemical Precipitation; D015870:Gene Expression; D006868:Hydrolysis; D008253:Macaca mulatta; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012401:Rotavirus / Q000201:enzymology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C079234:NS35 protein, rotavirus; D012313:RNA; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases