3University of Leeds, School of Chemistry, The Astbury Centre for Structural Molecular Biology, Leeds LS2 9JT, UK
The fusogenic avian Orthoreoviruses of Reoviridae family are important pathogens of birds that can cause considerable economic losses in the poultry industry. Avian reoviruses have been associated with a variety of disease conditions in poultry, including enteric and respiratory diseases, myocarditis, hepatitis, arthritis syndrome and the so-called stunting/malabsorption syndrome [1]. The avian reovirions are non-enveloped icosahedral particles of 85 nm external diameter with 10 dsRNA genomic segments (23.5 kb) encased within two concentric protein shells, forming the outer capsid and the core [2]. The RNA replication and morphogenesis of reoviruses occurs exclusively within cytoplasmic inclusion bodies, also known as viral factories, or ‘viroplasms’. The viroplasms are formed by non-structural protein μNS in association with non-structural protein σNS [3]. The σNS acts as RNA chaperone and destabilizes helical regions of RNAs. The structure is not known yet. The σNS protein was constructed in order to study the process of the viroplasm formation in details. The σNS is a non-structural protein approximately 41 kDa large and is composed of 367 amino acids. The homology modelling by Phyre2 prediction server estimated a high α-helical structure [4]. The SAXS experiments revealed the elongated pear-shaped structure. The σNS protein is homodimer as a biological unit with high probable further hexamerisation. It forms likely octamers in the presence of bound ssRNA in solution by hydrophobic interactions. The σNS rapidly binds ssRNA in a sequence-independent manner and then form large nucleoprotein complex [5].
The σNS gene was amplified by Q5® Polymerase (New England Biolabs, UK) and cloned into the pET SUMO expression vector. Recombinant σNS was produced in E. coli BL21 (DE3) cells at a constant temperature of 37°C and 220rpm for 4 hours. Cells were subsequently harvested by centrifugation, lysed using French press and the cell lysate was clarified by ultracentrifugation at 25000rpm for 1 hour. Recombinant σNS was purified from collected supernatant by various chromatography methods including affinity chromatography, anion exchange chromatography size exclusion chromatography. The purity and homogeneity of sNS protein was analyzed via SDS-PAGE and MALS analysis, respectively. The electron microscopy (EM) negative staining was used to clarify the oligomeric state of the purified samples. The purified samples were used for the crystallization experiment by the sitting-drop vapor-diffusion procedure. For the initial screening several commercial precipitant kits were applied to grow 3D crystals suitable for the diffraction measurement (Morpheus II, Molecular Dimensions, UK; PEG/Ion, Hampton Research, USA).
Here we report the results of the expression, purification and further crystallization experiments of the σNS protein. Subsequent monitoring of purified samples revealed that protein is quite stable and in presence of ssRNAs forms octamers. These afterwards forms pseudocapsids that were observed by EM negative staining.