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2.10.1 Cloning

Expression plasmids already generated in the lab (namely pET26Ub:MNV NS7 His and pET26Ub:MNV VPg His), containing either NS7 or VPg as C-terminal histidine were used as vectors. Both the NS7 and VPg coding region were amplified using expression primers as shown in the Table 2.1 using full-length MNV-1 cDNA clone (pT7MNV3’Rz) as a template. A stop codon was introduced immediately after the last amino acid residue of both the C-terminally tagged NS7 and VPg coding regions in order to produce the non-his tagged version of recombinant protein. The cloning of both genes was performed by employing the Sac II and Bam HI sites.

2.10.2 MNV NS7 (non-His tagged)

The expression plasmid (pET26Ub:MNV NS7 non His ) was transformed into BL21 DE3 + pCG1 bacterial cells (Gohara et al., 1999). The expression plasmid was designed to fuse the ubiquitin to the N-terminus of the desired protein in order to increase the expression and the solubility. Co-expression with pCG1 in BL21DE3 bacterial cells results in the specific removal of the ubiquitin domain, producing a

protein with the precise N-terminus as seen in the virus. For induction purposes, the transformants were grown for 24 hours in Overnight Express^TM Instant TB medium at 37^°C in the presence of kanamycin and chloramphenicol. After 24 hours, bacterial cells were harvested by centrifugation at 3000 x g and pellets were lysed in buffer A (500 mM NaCl, 50 mM Tris-HCl pH 8.0, 5% glycerol, 10 mM 2-mercaptoethanol, 0.1% NP-40) containing protease inhibitor and lysozyme by single freeze-thaw cycle at -80^°C and sonication for 2 minutes total. A 0.25% final concentration of polyethylenimine (PEI) was added slowly to the lysate and the precipitated nucleic acid was removed by centrifugation at 11,000 x g for 30 minutes at 4°C. Ammonium sulphate was then added slowly to the PEI supernatant until 40% to 60% saturation was reached before being centrifuge. The precipitated pellet was then re-suspended in 100 mM NaCl buffer A and dialysed against the same buffer overnight at 4°C. Thereafter, column chromatography was performed by using AKTAprime™

Automated Liquid Chromatography System from GE Healthcare according to the manufacturer’s manual. Phosphocellulose (PC), heparin and SP Sepharose columns were used in a series of purifications. After the removal of nucleic acid, an initial ammonium sulphate mediated precipitation and dialysis, the sample of non-his NS7 in Buffer A+100mM NaCl was first loaded onto a PC column, equilibrated in the same buffer and gradient eluted with 100 mM to 1M NaCl Buffer A. SDS-PAGE indicated that the MNV NS7 protein did not bind to the PC column under these conditions and was found exclusively in the pass through. The collected pass through from the PC column was further loaded onto a heparin column and the non-his NS7 was eluted around 400 mM NaCl. The cleanest fractions were then pooled, diluted in Buffer A to the desired final concentration of NaCl (typically 40-100mM) and subjected to further purification by loading onto a SP Sepharose column. The untagged NS7 eluted from the SP resin at around 40 mM NaCl. The cleanest fractions were pooled together again, diluted with Buffer A and loaded onto heparin column again. The non-his NS7 was eluted from the column at between 300 and 400 mM NaCl. The cleanest extinction coefficient of 80,780 units. The protein sample was aliquoted and frozen at -80°C.

2.10.3 MNV VPg (non-His tagged)

The MNV VPg (non His-tagged) expression plasmid (pET26Ub:MNV VPg non His) was transformed into BL21 DE3 + pCG1 bacterial cells (Gohara et al., 1999), induced, lysed and prepared as described for MNV NS7 above. The ammonium sulphate precipitated pellet was suspended in 250 mM NaCl Buffer A and dialysed overnight against the same buffer. PC and SP Sepharose columns were used for MNV VPg purification. The dialysed sample was first loaded onto a PC column previously equilibrated with 250 mM NaCl Buffer A. Gradient elution was performed with 250 mM to 1.5 M NaCl buffer A and VPg was eluted between 400 to 500 mM NaCl. The cleanest fractions were pooled together and diluted with Buffer A to a final concentration of around 100 mM NaCl. A SP Sepharose column was then employed as a further purification step. The purified VPg was eluted using at gradient at around 600 mM NaCl in Buffer A. The cleanest fractions containing VPg protein were pooled and dialysed overnight against 300 mM buffer A without NP-40. The VPg protein concentration was determined as described for NS7 but using a calculated molar extinction coefficient of 16,500 units.

2.11 T7 in vitro RNA transcription, purification and enzymatically capping of RNA transcripts

In vitro transcription reactions using recombinant T7 RNA polymerase were carried out to produce RNA transcripts of full-length genomic and sub-genomic RNAs from various cDNA constructs based on pT7MNV3’Rz. For the production of full-length genomic viral RNA transcripts, the cDNA clones (WT, mutants or reporter tagged) were linearised using the Nhe I site which is situated at the last adenine nucleotide of the 26 poly A tail in the cDNA clone, purified and used as a template in reactions. For the production of sub-genomic RNA transcripts, purified PCR products representing the subgenomic region (with an introduced T7 promoter sequence at the 5’ end of sub-genomic region) were used as templates for the reactions.

Typically, the transcription reactions contained 200 mM HEPES pH7.5, 32 mM magnesium acetate, 40 mM DTT, 2 mM spermidine, 7.5 mM of each NTP (ATP, UTP, GTP and CTP), 40 unit of RNAse inhibitor (Promega), 250 ng of DNA template and 50 µg/ml of T7 RNA polymerase. Reactions were incubated at 37°C for 2-7 h and then treated with DNase I at a final concentration of 0.1 unit/µl (New England Biolabs) at 37°C for 30 minutes. A subsequent DNA purification step was carried out by precipitation using lithium chloride at final concentration of 2.5M and incubation at -20°C for 30 minutes. The precipitated RNA were then washed in 70% ethanol and

resuspended in RNA storage solution (Ambion). The final concentration of the RNA