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3.2.1. Plasmids origin and sub-cloning

A codon optimised gene expressing full-length S protein of MERS-CoV (AFS88936.1) (Boheemen, Graaf, Lauber, Theo M. Bestebroer, et al., 2012), was kindly provided by Dr Davide Corti (Humabs, Biomed). Codon optimised SARS-CoV (Urbani strain) was a kind gift from Dr Graham Simmons (University of California). As previous attempts to produce viral PV particles making use of the available MERS-CoV plasmids had failed the MERS-CoV spike was sub-cloned from the phCMV vector to the pCAGGS vector. Sub-cloning was carried out as outlined in Section 2.8. with restriction enzyme sites identified from the sequence data using DNADynamo™ version 1.469. Digestions were performed using FastDigest™ enzymes (Thermo Fisher Scientific) with 400-700 ng of plasmid DNA digested in a total volume of 20 µl for each double digestion. The universal FastDigest™ buffer (Thermo Fisher Scientific, cat. n° B72) and 10 U of FastDigest® enzymes were used as per Section 2.8.3. Digestion reactions were then incubated at 37°C for 20 minutes using Mastercycler ep Gradient S therm ocycler, and subsequently, the digestion products were purified using gel electrophoresis and the Qiagen Gel Extraction Kit (Section 2.8.2). Post extraction DNA purity and concentration were assessed using a NanoDrop™ 2000 Spectrophotometer (Section 2.3.1.)

After the gel extraction step, the DNA ligation was performed, and the ligation product was transformed into DH5 cells following the protocol as outlined in Chapter 2.

3.2.2 Cell culture and cell lines

Huh-7, BHK, Caco-2, 293T, ACE2/293T/17, MDCK, Vero, QT6 and DPP- 4/HEK293T/17cells were all used as “target” cells for infection in order to determine the optimal cell line for use in further study of MERS-CoV and SARS-CoV. All cell lines were maintained

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in DMEM supplemented with10% FBS and 1% penicillin-streptomycin and cell maintenance was carried out as per Section 2.7.

3.2.3. PV production methods

PV production was carried out with a protocol as outlined in Chapter 2. However, a number of modifications were carried out there including modification of the envelope expressing plasmid concentration, the addition of various protease expression plasmids at differing concentrations and alteration of the conditions in which the cells were grown through changes in the serum concentration.

PV produced bearing a Vesicular Stomatitis Virus glycoprotein (VSV-G) was used as a positive control in assays of PV production. VSV-G PV is particularly suited to this task as the broad tropism of VSV means that PV with the VSV-G is capable of transducing a large number of cell lines (Hastie et al., 2013).

3.2.3.1. Optimisation of MERS-CoV Spike protein expressing plasmid concentration

To test the variation in yield of PV that is attributable to the concentration of envelope expression plasmid concentrations of between 200ng and 1200ng were added and the protocol described in chapter 2 adapted to a 35mm cell culture plate. Plasmids were combined in 100µl Opti-MEM® for PEI transfection and in molecular grade water for transfection with Fugene 6™.

3.2.3.2. The role of serum variation in the production of PV

Two distinct criteria were tested through the alteration of Serum content. A reduced serum concentration was used, cells were cultured 5% FBS for 48 hours prior to transfection. This experiment then followed the transfection protocol as outlined in Section 2.9 with the following exceptions:

• 48 hours prior to transfection. HEK293T/17cells were subcultured into a 100mm petri dish.

• Day of transfection. DMEM+5%FBS + 1%P/S was replaced 1 hour prior to transfection with DMEM+10%FBS + 1% P/S

After these initial steps, the protocol was carried out as per section 2.9 with 900ng of pCAGGS- MERS-CoV S added to 1000ng of p8.91 lentiviral core and 1500ng of luciferase reporter expression plasmid (pCSFLW).

3.2.3.3. Effects of protease on the production and titration of PV

Proteolytic processing of the S protein is required in order produce mature S protein that can mediate cellular fusion (Millet and Whittaker, 2014). Several studies have now

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demonstrated that MERS-CoV is capable of using different proteases to facilitate S protein maturation and subsequent cell entry (Section 1.4.3.) (Shirato et al., 2013; Simmons et al., 2013). Recent studies have also determined that the tropism of MERS-CoV is determined by this protease action (Park et al., 2016). As MERS-CoV appears to be capable of making use of several proteases at different stages of its life cycle and at different locations within hosts cells (Park et al., 2016) (Section 1.4.3.), the effects of protease on production and use of PV must be better understood. The cellular localisation of the proteases has important implications for the processing of the S protein. MERS-CoV S protein can utilise proteases expressed in endosomal compartments, to facilitate S maturation during the protein synthesis, or the S protein can interact with proteases expressed on the plasma membrane, allowing activation to occur either during the latter stages of production or during viral entry (Böttcher- Friebertshäuser, Klenk and Garten, 2013; Simmons et al., 2013; Zmora et al., 2014; Hoffmann et al., 2016).

In order to further investigate the role of proteases in the production of MERS-CoV PV, protease expression plasmids were used to transfect cells that were subsequently employed as target cells for transduction.

For both proteases expressed in the producer cells and protease expressed in target cells, a ‘no protease’ PV preparation was used as a control. No protease corresponds to a titration completed with no additional protease expression vectors added at any stage in the process.

3.2.3.4 Use of different viral cores and alternate expression vectors

To assess the potential of variation of the viral core used in PV production to affect efficiency, a murine leukaemia virus (MLV) viral core is a commonly used core in PV production (Chapter 1). In addition, the availability of multiple viral cores offers alternative research tools that may allow PV to be used in a wider variety of applications. Of particular importance is the potential for intrinsic cell restriction factors to inhibit the activity the lentiviruses (Besnier, Takeuchi and Towers, 2002; Ikeda et al., 2002), although the potential impact these factors have on PV use is not clear the availability of effective alternatives to lentiviral core PV offers a method to circumvent any limitations before they occur.

3.3.6 Production consistency in the titre of PV

A critical factor in determining the utility of a tool such as PV for research use is efficient and consistent production. In order to assess the consistency of the PV production, the protocol derived from the experiments described in this chapter was repeated with transfections labelled MERS-T194, MERS-T195, MERS-T196, MERS-T197 and MERS-T198. These transfections were carried out approximately 48 hours from each other with MERS-

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T194 being conducted first followed 48 hours later by MERS-T195, the remaining transfections were conducted sequentially following this pattern with a transfection every 48hours. Harvesting of the PV was conducted as per protocol outlined in Section 2.9. All titrations were conducted using Huh-7 cells maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin, as target cells.