title: Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus.
abstract: Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2(1)2(1)2 and diffracts up to 2.1 Å and the RdRp-Lu(3+) derivative co-crystals belong to the C222(1) space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses.
authors.affiliation: Centro Nacional de Biotecnología, CSIC, Calle Darwin No. 3, Madrid, 28049, Spain.
authors.forename: Diego
authors.initials: D
authors.lastname: Ferrero
authors.forename: Mònica
authors.initials: M
authors.lastname: Buxaderas
authors.forename: José F
authors.initials: JF
authors.lastname: Rodriguez
authors.forename: Núria
authors.initials: N
authors.lastname: Verdaguer
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D012328:RNA Viruses / Q000201:enzymology*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D012324:RNA-Dependent RNA Polymerase