Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase fromThosea asignavirus

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.

The Structure of the RNA-Dependent RNA Polymerase of a Permutotetravirus Suggests a Link between Primer-Dependent and Primer-Independent Polymerases

“…The full-length TaV ORF1 (140 kDa) fused to an N-terminal tail containing an hexa-histidine Tag (TaV rORF1; Fig 1A ) was expressed in Hi5 insect cells infected with a recombinant baculovirus, rBV-TaV ORF1. After expression, the recombinant protein was rapidly cleaved releasing a 75 kDa protein fragment ( Fig 1B ) [ 13 ]. Mass spectrometry (MALDI-TOF/TOF) showed that this polypeptide harbors the first 674 residues of the recombinant protein, including the whole RdRP domain (TaV pol from here on; Fig 1A ).…”

“…The structure of TaV pol was solved by SAD methods from Lu 3+ derivative co-crystals to 3.0 Å resolution ( Table 1 ) [ 13 ]. Native data was then used to complete and refine the model to a final resolution of 2.15 Å ( Table 1 ).…”

“…Expression and purification of TaV rORF1 and TaV pol were previously reported [ 13 ]. Briefly, a recombinant baculovirus (rBV), expressing the whole ORF1 polypeptide of Thosea asigna virus (1257 residues, 140 kDa; GenBank accession number AF282930.1) fused to a 6xhistidine tag and containing the TEV protease recognition site (hTaV ORF1), was generated, according to the Bac-to-Bac protocol (Invitrogen).…”

“…Crystals of TaV pol , apo-form and the Lutetium derivative were obtained by the sitting-drop vapor diffusion method as previously described [ 13 ]. The RNA oligonucleotide (sequences 5’-CCCAUUCGACUCCUG) was used as a template to form the binary complex in a 1:1.5 TaV pol :RNA molar ratio with 1 mg/ml TaV pol and incubated at 30°C for 30 min in a buffer containing 50 mM MES pH 6.0, 200 mM NaCl, 10% glycerol and 5 mM DTT.…”

Viruses and viral proteins

“…4). The only exception to this scheme is found in Birnavirus and Permutatetravirus polymerases, where motif C is encoded upstream of motif A (Garriga et al, 2007;Ferrero et al, 2012). Each of the seven motifs in the RNA polymerase domain adopts a specific fold that extends beyond the regions of sequence similarity into the so-called homomorphs for most RNA virus RNA polymerases (Lang et al, 2013).…”