title: Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1.
abstract: Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity. The purified protein is enzymatically active and contains both MTase and GTase activity indicating that SINV nsP1 does not require membrane association for its enzymatic function. Biochemical analysis shows that detergents abolish nsP1 GTase activity, whereas nonionic detergents do not affect MTase activity. Furthermore, SINV nsP1 contains the metal-ion dependent GTase, whereas MTase does not require a metal ion. Circular dichroism spectroscopic analysis of purified protein indicate that nsP1 has a mixed α/β structure and is in the folded native conformation.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, India. shailfbt@iitr.ernet.in
authors.forename: Shailly
authors.initials: S
authors.lastname: Tomar
authors.forename: Manju
authors.initials: M
authors.lastname: Narwal
authors.forename: Etti
authors.initials: E
authors.lastname: Harms
authors.forename: Janet L
authors.initials: JL
authors.lastname: Smith
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kuhn
mesh_terms: D018354:Alphavirus Infections / Q000821:virology; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012845:Sindbis Virus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014169:Transformation, Bacterial; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000098556:mRNA Guanylyltransferases
chemical_list: D003902:Detergents; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C104211:nsP1 protein, Sindbis virus; D006639:Histidine; D006160:Guanosine Triphosphate; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases