Molecular cloning of protein coding sequences for interaction studies in the

The human IRF3 coding sequence was obtained from the pEFplink2-IRF3 (kindly provided by Prof S Goodbourn) via restriction enzyme digestion with Nco I and Xba I (Figure 4.2). As one of the host cellular proteins previously shown to interact with rotavirus NSP1, the IRF3 coding sequence had to be cloned into the mammalian two- hybrid vectors pM and pVP16 for subsequent analysis and RE sites EcoR I and Hind III have been selected for this cloning (Figure 4.3). These two RE sites were

generated in IRF3 plasmid via PCR using primers listed in table 2.1.7. The EcoR I site was generated just before the start codon of IRF3 ORF, whereas the Hind III site was created at the end of the coding sequence before the stop codon, in which case the gene will be using the stop codon from the vector (Figure 4.4). Positive colonies were selected via Blue/White screening and the DNA extracted was subjected in restriction enzyme double digestion followed by sequencing (Figure 4.5A). However this analysis revealed that the original design of the primer used for IRF3 cDNA amplification had made IRF3 protein to be out of frame with the upstream DNA binding and transcription activation domains of the fusion protein. To overcome this oversight the fusion construct plasmid was digested with EcoR I, the staggered ends of the EcoR I site were filled in using Klenow DNA polymerase I and the plasmid was re-circularised to give a construct in which the two halves of the fusion protein were joined to each other in frame (Figure 4.5B).

Figure 4.2 IRF3 obtained from vector pEFplink2.

IRF3 expression plasmid was obtained by double digestion with restriction enzymes Nco I and Xba I from the pEFplink2-IRF3 plasmid, which is ready for the

subsequence re-amplification with PCR to add other restriction enzyme sites for cloning into the mammalian two-hybrid vectors.

Figure 4.3 Experimental strategies for re-amplifying IRF3 coding sequence into the mammalian two-hybrid vectors.

EcoR I and Hind III sites are generated using PCR with appropriate primers at both the 5’ and 3’ end of the IRF3 coding sequence for re-amplifying IRF3 coding sequence into the mammalian two-hybrid vectors.

A.

Figure 4.4 Schematic diagrams of the mammalian two-hybrid system cloning vectors pM and pVP16, showing the multi-cloning site sequence and unique restriction enzyme sites.

Schematic diagrams of the main features of vectors pM (panel A) and pVP16 (panel B) are shown. pM expresses the DNA binding domain (amino acids 1-147) of the GAL4 protein. pVP16 generates the transcriptional activation domain (amino acids 446-490) of the HSV VP16 protein. These plasmids have unique RE sites located in the multi-cloning site (MCS) region at the 3’ end of the open reading frame for either the GAL4 DNA binding domain or the VP16 activation domain. The gene encoding the protein of interest is ligated into the MCS in the correct orientation and reading frame, such that fusion proteins with the GAL4 or VP16 domains are expressed. The unique restriction sites that chosen to be used for cloning both IRF3 (EcoR I and Hind III) and β-TrCP (BamH I and Xba I) in the MCS of these vectors are indicated below each plasmid diagram (This figure is adapted from the Clontech Matchmaker Mammalian Assay Kit User Manual).

A

Figure 4.5 Confirmation of the constructs pM-IRF3 and pVP16-IRF3.

(A) DNAs of the pM-IRF3 and pVP16-IRF3 constructs were amplified in E.coli and purified by maxi-prep as described in Materials and Methods. Plasmid DNA was then double digested with EcoR I and Hind III and fractionated on a 1% agarose gel as described in Materials and Methods. The position of the expected 1300bp IRF3 insert band is indicated down the right hand side of each gel. Lanes labelled D indicate the samples after double digestion and lanes labelled U indicate the

undigested samples, lanes labelled M indicate the 1kb DNA marker and the sizes of the markers are given in base pairs.

(B) Sequencing analysis was performed to confirm the correct orientation and open reading frame of IRF3 at the junction with the Gal4 domain in the pM vector, or the VP16 domain in the pVP16 vector.

Similarly, β-TrCP, the other possible interaction partner for rotavirus NSP1, was also cloned into pM and pVP16 vectors. The β-TrCP coding region from a plasmid carrying HA-tagged human β-TrCP (TRcP 1a/Fbw 1a) (Kindly provided by Dr A Stephanou) was amplified with primers listed in table 2.1.7 and cloned into the mammalian two-hybrid vectors between RE sites BamH I and Xba I for pM or only BamH I in the case of pVP16 (Figure 4.3). BamH I and Xba I sites were created at respectively the 5’ end of the β-TrCP coding sequence upstream of the start codon and the 3’ end of the ORF following the stop codon for cloning into the pM vector. For cloning into pVP16 activation domain vector, BamH I sites were generated at both ends of the β-TrCP ORF. Positive colonies were selected via Blue/White screening, and plasmid DNA extracted was subjected to restriction enzyme double digestion (Figure 4.6A). Selected clones were then sequenced to confirm the correct joining points in both vectors (Figure 4.6B).

A

Figure 4.6 Confirmation of the constructs pM-β-TrCP and pVP16-β-TrCP. (A) DNAs of the pM- β-TrCP and pVP16- β-TrCP constructs were amplified in E.coli and purified by maxi-prep as described in Materials and Methods. Plasmid DNA was then double digested with BamH I and Xba I or digested with BamH I alone, and fractionated on a 1% agarose gel as described in Materials and Methods. The position of the expected 1800bp β-TrCP insert band is indicated down the right hand side of each gel. Lanes labelled D indicate the samples after digestion and lanes labelled U indicate the undigested samples, lanes labelled M indicate the 1kb DNA marker and the sizes of the markers are given in base pairs.

(B) Sequencing analysis was performed to confirm the correct orientation and open reading frame of β-TrCP at the junction with the Gal4 domain in the pM vector, or the VP16 domain in the pVP16 vector.

The parental UKtcNSP1 and OSUNSP1 cDNAs were also cloned into the two mammalian two-hybrid vectors. The UKtcNSP1 coding sequence was cloned in between EcoR I and Hind III restriction enzyme sites using the same strategy as that employed for IRF3. The OSUNSP1 coding sequence was cloned in between BamH I and Hind III restriction enzyme sites again using the strategy described earlier for their putative interaction partners, i.e: IRF3 and β-TrCP. The expected insert bands were detected as indicated (Figure 4.7A, Figure 4.8A) and the sequencing results of the corresponding junctions in all constructs confirmed the correct construction of these plasmids (Figure 4.7B, Figure 4.8B).

A

Figure 4.7 Cloning of UKtcNSP1 into mammalian two-hybrid vectors pM and pVP16.

(A) Around 500ng of DNA sample were digested with EcoR I and Hind III and correct sized bands were released indicating the correct insert of the UKtcNSP1. U indicates the undigested product and D indicates the products after digestion, 1kb DNA marker on the left hand side indicates the sizes of the bands.

(B) Selected clones were sequenced using pM and pVP16 sequencing primers (Table 2.1.7) and the junction regions are illustrated. Sequencing analysis confirmed that the UKtcNSP1 gene was inserted correctly with correct orientation in both pM and pVP16 vectors.

A

Figure 4.8 Cloning of OSUNSP1 into mammalian two-hybrid vectors pM and pVP16.

(A). Around 500ng of DNA sample were digested with BamH I and Hind III, and correct sized bands were released indicating the correct insert of the NSP1. U indicates the undigested product and D indicates the products after digestion, 1kb DNA marker on the left hand side indicates the sizes of the bands.

(B). Selected clones were sequenced using pM and pVP16 sequencing primers (Table 2.1.7) and the junction regions are illustrated. Sequencing analysis confirmed that the OSUNSP1 gene was inserted correctly with correct orientation in both pM and pVP16 vectors.

4.3 Protein-Protein interaction studies using the mammalian two-hybrid