title: Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro.
abstract: The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3' end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3' CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA.
authors.affiliation: Department of Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Jon K
authors.initials: JK
authors.lastname: Rubach
authors.forename: Brian R
authors.initials: BR
authors.lastname: Wasik
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Rupp
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kuhn
authors.forename: Richard W
authors.initials: RW
authors.lastname: Hardy
authors.forename: Janet L
authors.initials: JL
authors.lastname: Smith
mesh_terms: D002413:Cations, Divalent / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012845:Sindbis Virus / Q000201:enzymology*; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase; D003546:Cysteine Endopeptidases; D008274:Magnesium