Methods:
For the full methods see Qi et al 2010. Only the key methods referred to in this blog are detailed:
Structure Determination:
1. Protein Expression and Purification:
The full length LASV NP gene was cloned into a pMAL-c2X derived pLou3 plasmid. This plasmid is designed to produce N terminal MBP (maltose binding protein) tagged fusion proteins. This plasmid was transformed into Rosetta cells (supplied by Novagen). IPTG was used to induce protein expression. An amylose column was used to extract and purify the target protein by affinity chromatography. Trypsin digestion and mass spectrometry were used to ensure the purified LASV NP was homogenous.
2. Crystallization and data collection:
Native crystals of the LASV NP were obtained in 0.2 M LiCl2 and 20% PEG3350 in 1 week (20oC). The Nucleoprotein was co-crystallised with m7GpppG, m7GTP or m7GDP (In the N terminal domain binding pocket) by incubation with compounds at 2mM for 30 minutes. These crystals grew in 0.2M KCl and 14-22% PEG3350. Crystals complexed with Mn ions (C terminal active site) were obtained by incubation with 0.2 M MnCl2. The structure was resolved to 1.80 Å
Activity Assays:
3. In vitro 3’-5’ exoribonuclease assay:
The in vitro 3′–5′ exoribonuclease assays were carried out in 10 μl of the reaction solution (0.3 M NaCl, 10% glycerol, 20 mM Tris pH 7.5, 10 mM MnCl2). 7 μg of either wild-type or mutant NP proteins and 8 units of the RNaseIN inhibitor (Promega) were added.The exoribonucleasse activity was analysed on arious ribonucleotide substrates. The control reactions were run without MnCl2 (20mM EDTA was added). The reactions were stopped simultaneously by the addition of EDTA. The samples were mixed with RNA loading buffer (supplied by Ambion), and heated at 95 °C for 3 mins. These were then cooled on ice for 5 min, and separated in 15% or 6% urea-polyacrylamide gel, or 2% agarose gel. The gels were stained in ethidium bromide for 25 min and visualized using the 2UV transilluminator (UVP).
4. LASV minigenome (MG) transcription assay:
The full-length LASV L and NP genes (Josiah strain) were cloned into the pCAGGS vector for expression in mammalian cells. The LASV MG construct contains the T7 promoter-directed LASV S-segment-like sequences that include all the cis-acting elements required for viral RNA synthesis (5′ UTR, intergenic region and 3′ UTR) and encode a Renilla luciferase (RLuc) gene in place of the viral NP coding sequence. This LASV-based LUC-encoding minigenome (MG) RNA was transcribed in vitro by the T7 MEGAScript kit (supplied by Ambion) and transfected into 293T cells, together with the LASV L expression plasmid, and wild-type or mutant NP expression plasmid. LUC activity was determined at 24 h after transfection and shown as fold increase over a control sample that lacked the L expression plasmid.