Cloning of rIBV donor plasmids

4.2.13.1 Cloning of rIBV HPS donor plasmid via unique restriction sites

To obtain the rIBV HPS donor plasmid, the unique restriction sites of Tth111I in the 1b region upstream of the spike protein gene and ClaI in the 3a region downstream of the spike protein gene were targeted to substitute the H52 mIBV spike protein of the H52 mIBV donor plasmid (Figure 6) with the IB66HP spike protein. A construct was designed with the complete IB66HP spike protein and flanked by Tth111I and ClaI the restriction sites. The designed sequence was synthesized by GenScript in the pUC57 simple plasmid (Figure 7) and digested with Tth111I, ClaI and ScaI. ScaI was used to cut the plasmid backbone and thus minimize possible re-isolation of the original plasmid. The H52 mIBV donor plasmid was digested with Tth111I and ClaI. Each of the restriction

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endonucleases recognition sequence is also present in the MHV spike ectodomain (Figure 6); therefore the probability for contamination from the template plasmid is automatically reduced.

Figure 6: H52 mIBV donor plasmid with marked restriction sites ClaI and Tth111I

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Figure 7: IB66HP with H52 flanks in pUC57 simple plasmid with the marked restriction sites ClaI and Tth111I as well as ScaI which was used to cut the plasmid backbone.

4.2.13.2 Cloning of rIBV donor plasmids via Gibson assembly®

To obtain rIBV BS, rIBV HPS1-H52S2, rIBV H52S1-HPS2, rIBV BS1-H52S2 and rIBV H52S1-BS2, Gibson assembly® was conducted.

Gibson assembly® is an isothermal DNA-amplification method in which fragments are flanked with overlapping regions into each other. This allows cloning of a construct without the need of unique restriction sites. The fragments are obtained with primers that have ~15-25 nt overlap to the respective neighboring fragment (Figure 8).

Primers were designed with the NEBuilder® Assembly Tool (http://nebuilder.neb.com/).

The IBV H52 S1 subunits was amplified from the H52 rIBV wt donor plasmid PB485 [81]

using primers PO1967 & PO1769 and the IBV H52 S2 subunits with PO1772 & PO2045 from the pcDNA3.1 Beaudette S expression plasmid (PB572, synthesized by GenScript) using the Q5® HF DNA Polymerase PCR (see 4.2.9.1). The Beaudette S1 subunit was amplified using PO1967 & PO1769 from PB572; the Beaudette S2 subunit was amplified with PO1770 & PO2045 from the same plasmid. The IB66HP S1 and S2 subunits were amplified using the same primers that were used for the IBV H52 spike protein subunits using the rIBV HPS donor plasmid. For rIBV BS donor plasmid, which comprises the full Beaudette spike protein in the H52 background, primers PO1765 &

PO1766 were used with PB572 as the template. The spike protein PCR fragments were

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separated via gel electrophoresis (see 4.2.11.2) and purified from the gel (see 4.2.11.3).

The plasmid backbone for the constructs was amplified using PB485 as template with the primers PO2043 & PO2044 and the PCR-linearized plasmid treated with 5 µl DpnI for 90 min at 37 °C to remove template plasmid.

Subsequently, concentration of the PCR products was determined. For 2-3 fragment assembly, the NEBuilder® HiFi Assembly manual suggests to use 50-100 ng of the linearized vector and double amount of the inserts, so 1 µl of the vector was used (~70 ng) and ~140 ng of the respective inserts. Each rIBV donor plasmid assembly mix was then filled up to 10 µl with H2O and 10 µl NEBuilder® HiFi DNA Assembly Master Mix added and the mixture incubated for 15 min at 50 °C. Subsequently transformation of the assembled rIBV donor plasmid was performed as in 4.2.10.1.

Figure 8: Schematic assembly of rIBV donor plasmids with heterologous spike protein subunits utilizing Gibson assembly®. The whole donor plasmid is assembled from PCR amplified fragments with overlapping primers. Grey-colored: linearized vector amplified with primer set 3. Light red-colored: S1 subunit amplified with primer set 1. Blue-colored: S2 subunit amplified primer set 2.

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4.2.13.3 Substitution of targeted amino acids in rIBV donor plasmids via SDM

To substitute cysteine on AA position 267 of the spike protein (see 5.3.2), Q5® Site-Directed Mutagenesis Kit with the primers PO1942 & PO1943 was used in order to mutate the thymidine at position 800 in the IBV H52 spike protein sequence to guanidine, which results in the substitution of phenylalanine to cysteine of amino acid 267 of the spike protein resulting in the donor plasmid for rIBV H52-F267C

Furthermore, to analyze, if the cysteine to phenylalanine mutation on position 267 would disrupt the extended cell culture tropism on EB66® cells, the opposite was conducted by mutating the cysteine at position 267 of the donor plasmids rIBV HPS and rIBV HPS1-H52S2 into a phenylalanine, using the primers PO1941 & PO1942. Thus, in both donor plasmids, the reverse point mutation at position 800 of the IBV spike protein from guanidine to thymidine was conducted. The rescued rIBVs with the phenylalanine substitution on position 267 were named rIBV HPS-C267F and rIBV HPS1-H52S2-C267F

To determine whether the cysteine substitution could also extend the cell culture tropism in a 4/91 type IBV, the position homologue, a leucine at AA position 269 in the spike protein sequence of the CR88 rIBV donor plasmid (provided by BI) was mutated to a cysteine using PO1886 changing bp 806 and bp 807 of the CR88 spike protein from thymidine and adenine to guanidine and thymidine. This step was performed together with other primers substituting additional residues (schematically shown as primer y in Figure 9) with the multi SDM kit (4.2.9.5) and subsequent transformation (4.2.10.2). The resulting clones had a random distribution of substitutions from the used mutagenic primers. Upon sequencing, the clone with the plasmid harboring only the L>C269

substitution was further used for this work, obtaining the rIBV CR88-L269C donor plasmid.

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Figure 9: Site-directed mutagenesis (SDM) with single mutation present in forward and reverse primer for H52 rIBV SDM (left) and with multiple SDM kit for CR88 rIBV SDM (right). From the CR88 SDM only a clone with the desired substitution L269C was pursued for this work.