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With the unsuccessful outcome of the TAP system in the RAW264.7 cell line, another attempt to identify potential NS7 binding partners was carried out by employing two different TAP NS7 constructs, the N-terminally and C-terminally TAP fusion NS7 (NTAP and CTAP NS7) using the same tetracycline inducible system but in a different cell type (Figure 3.4a). In this attempt, stable HEK 293T TRex cell lines

expressing either NTAP or CTAP NS7 under the control of a doxycycline inducible promoter were generated (see Section 2.8.2). A number of advantages have contributed to the selection of this cell line. First of all, HEK 293T TREX cells are well known for their high transfection efficiency. These cells also demonstrate good expression of exogenous proteins if transfected with a cytomegalovirus (CMV) promoter driven expression plasmid. HEK 293T cells cannot be infected with MNV, although these cells support MNV genome replication. The purpose of generating these stable cell lines was purely to identify any potential host cell proteins, which might potentially bind to NS7. Successful clones from each stable cell line were selected after analysing the protein expression levels from doxycycline induced (+) and uninduced (-) cells of each clone (Figure 3.4b). In this case, the best inducible clones with the most tightly regulated expression compared to their uninduced counterpart sample in western blot analysis were preferred to be used in subsequent TAP purification assays. The clones that were induced with doxycycline displayed a stronger NS7 signal on western blot analysis compared to their uninduced counterpart. However, all the clones also displayed a high background level of TAP NS7 expression in all of the uninduced cells as seen previously in the RAW264.7 cells (Figure 3.4b). Regardless of this issue, Clone 1 from each N-TAP and C-TAP MNV NS7 was selected to proceed with tandem affinity purification based on the protein expression levels and the growth characteristics of the cells (Figure 3.4b).

A large-scale tandem affinity purification was carried out by inducing cell lines expressing N-TAP and C-TAP MNV NS7 (Clone 1) with 1µg/ml doxycycline. Induced cells were lysed 24 hours post induction and cell lysates of N-TAP and C-TAP MNV NS7 were purified via TAP purification assay (see Section 2.8.1 and 2.8.2). The final elution products were examined on SDS PAGE using silver staining (Figure 3.5).

Unlike the previous approach using RAW264.7 cells, several potential binding partners from host cells were successfully co-precipitated (Figure 3.5). Interestingly, more potential cellular binding partners were eluted from the NTAP-NS7 than the CTAP-NS7 as observed in the Figure 3.5. This may have been due to the fact that the N-TAP tagged protein appeared to purify better and resulted in a greater overall final yield.

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Figure 3.4 Generation of stable HEK 293T TREX cell lines expressing NTAP and CTAP-NS7. (a) Schematic representation of the plasmids pcDNA4/TO with N-terminal and C-terminal fusion of MNV NS7 gene used to generate stable 293T cell lines expressing the tetracycline repressor. Expression of both NTAP and CTAP NS7 were under the control of a tetracycline inducible CMV promoter. Stably TAP-NS7 expressing cell lines were generated by transfection of NTAP-NS7 or CTAP-NS7 plasmid into TetR expressing HEK 293T cells These stable cell lines were maintained by selection with zeocin and blasticidine (see Section 2.8.2) (b) Western blot analysis of the induced and un-induced TAP-tagged NS7 expressed from the HEK 293T TREX cells. 5 clones were selected, induced with doxycycline and lysed after 16 hour post induction. All the proteins concentration in the cell lysates were normalised before being resolved in SDS-PAGE gel.

MNV NS7 antisera was used as a probe for western blot analysis where the TAP-NS7 produced signal at ~70 kDa. Clone 1 (red circled) from each TAP-NS7 were selected for a large scale TAP purification.

Based on these initial observations, the final elution samples were further concentrated and resolved in gradient SDS PAGE gel and were stained with colloidal coomassie blue. Selected protein bands were excised from the gel and were sent to Proteomics Services at McGill University and Genome Quebec Innovation Centre in Quebec, Canada for mass spectrometry analysis. The obtained mass spectrometry data were then analysed against a custom human and MNV protein database. The final data was then analysed using proteomic software Scaffold 2. Based on the final data, the identified proteins with high peptide hits and high probability were correlated to the original protein bands identified [Figure 3.6 (C-TAP NS7) and Figure 3.7 (N-TAP NS7)]. The identified proteins were also screened against the list of proteins identified from the lysates of the ‘TAP only’ pulldown as a control.

Figure 3.5 Analysis of final elution products from tandem affinity purification of MNV N-TAP and C-TAP NS7 doxycycline induced HEK 293T TREX cell lines by silver staining of gradient SDS PAGE gel. Cells were harvested and lysed 24 hours post doxycycline induction followed TAP purification. The final elution samples were resolved on gradient PAGE and visualised by silver staining as described in Section 2.8.2. NS7 proteins were highlighted with asterisk and lysate from a purification performed on a cell line expressing only the TAP tag alone was run on the gel as a control.

All the identified proteins that were co-precipitated from the C-TAP MNV NS7 were determined to be non-specifically binding proteins to NS7 since these proteins were previously identified in the empty TAP purification (TAP only) (previous observations within the laboratory). Therefore the interaction of these proteins with the C-TAP MNV NS7 protein was determined to be non-specific. Human General transcription factor II (GTF2), which had a high peptide score (Figure 3.6b and Figure 3.7b) was identified in the TAP only pull down as a control. GTF2 has been pulled down non-specifically in a previously published TAP purification assay (Burckstummer et al., 2006). It is a multifunctional nuclear protein, which plays roles in RNA Pol II mediated transcription as well as signal transduction. There are several isotypes of GTFs and together these factors are responsible for promoter recognition and the formation of transcription preinitiation complex with RNA polymerase II.

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Figure 3.6 Identification of co-precipitated proteins from TAP purification of the C-TAP MNV NS7 by mass spectrometry analysis. (a) Analysis of final elution product from the C-TAP MNV NS7 TAP purification by SDS PAGE gel and colloidal coomassie blue staining. The particular protein bands were excised for mass spectrometry analysis. (b) Summary of the identified proteins for the C-TAP MNV NS7 co-precipitated proteins from TAP purification by mass spectrometry based on the relatively high peptide hits with the highest probability.

Although this protein plays a role in cells RNA transcription, it is DNA-dependent and interacts with RNA polymerase II, a multisubunit polymerase protein and catalyses the transcription process using DNA as a template. The association between RdRp with this factor is highly unlikely since all the RdRp’s are RNA-dependent polymerases. Furthermore, since the GTFs are nuclear protein, it would be an indication that they are not involved in MNV replication.

Tubulin, used as the building block of microtubules, is highly expressed and in this case, it may simply be co-purified due to the high levels of this protein present in cells. In addition, the peptide hit obtained from mass spectrometry was also relatively low (Figure 3.6b) and it has been co-purified in the TAP only control as well.

Microtubules are involved in several basic cellular processes, such as segregation of genetic material, intracellular transport, maintenance of cell shape and extracellular transport by means of cilia. Interestingly, in vitro mRNA synthesis for Measles virus and Sendai virus (a negative strand RNA viruses) is stimulated by tubulin and this process has been demonstrated to be inhibited by anti-β-tubulin antibodies (Houben et al., 2007). This observation lead to the hypothesis that tubulin might serve as an anchoring site on the cytoskeleton for the viral RNA polymerase. Studies on influenza virus polymerase supported this hypothesis as it has been shown that the incoming viral ribonucleoproteins associate with the cytoskeletal network where they are active in RNA synthesis (Klumpp et al., 1997). Assembling the RNA polymerase on the cytoskeleton provides focal concentration of the replication components and as a result, it increases the rates or efficiency of viral genome replication in infected cells.

Compared to the C-TAP NS7, the N-TAP NS7 final elution product from the TAP purification process contained a higher number of pulled down proteins (Figure 3.7). As discussed above, the GTF2 protein was identified in this purification as a non-specific binding protein (Figure 3.7b). Furthermore, identification of other proteins such as, 60S ribosomal protein, beta globin chain, and proteosomal components were considered to be irrelevant since these proteins had been identified in the TAP only control purification as well. Therefore these proteins were classified as non-specific products that been co-purified during the TAP purification process.

Intriguingly, additional proteins identified by mass spectrometry analysis with a relatively high peptide score were the human guanosine monophosphate reductase 1 and 2 (GMPR) and N(2)-dimethylguanosine tRNA methyltransferase. (Figure 3.7b).

Both of these proteins have previously not been identified in the TAP alone control.

Therefore, it is feasible that these proteins are directly interacting with NS7. The human GMPR protein catalyses the irreversible NADPH-dependent deamination of

GMP (guanine nucleotide) to IMP (adenine nucleotide), maintaining the intracellular balance of nucleotides A and G during cellular differentiation. It plays a role in modulating cellular differentiation where overexpression of GMPR2 has been shown to promote the monocytic differentation of HL-60 leukemia cells a cancer cell-line (Zhang et al., 2003). The N(2)-dimethylguanosine tRNA methyltransferase is involved in tRNA processing as it dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl-L-methionine as donor of the methyl group. These two identified proteins also may have other roles that have yet to be determined. The interaction between these proteins and MNV NS7 warrants further investigation to validate and confirm this observation.

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Figure 3.7 Identification of co-precipitated proteins from TAP purification of the N-TAP MNV NS7 by mass spectrometry analysis. (a) Analysis of final elution product from the N-TAP MNV NS7 purification by SDS PAGE gel and colloidal coomassie blue staining. The particular protein bands in bracket were excised out for mass spectrometry analysis. (b) Summary of the identified proteins for the N-TAP MNV NS7 from TAP purification by mass spectrometry based on the relatively high peptide hits with the highest probability.