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generated were able to produce viral proteins and Renilla luciferase protein, the constructs were then examined in cell culture using two different reverse genetics systems. The MNV replication permissive cells, BSR-T7, which are used in both systems, allows viral protein expression and a single cycle of MNV replication (Chaudhry et al., 2007; Yunus et al., 2010). However they lack a suitable viral receptor to enable reinfection and multi-cycle virus replication. In the cDNA based

(a) (b)

(c)

Figure 6.2 In vitro translation of Renilla luciferase gene in MNV genome from full length genomic and subgenomic of in vitro transcribed capped reporter tagged MNV RNAs. (a) In vitro translation of full length MNV genomic (G) (WT and FS) and Renilla luciferase-FMDV 2A-VP2 fusion (RL2A-VP2^WT and RL2A-VP2^FS) capped RNA. (b) In vitro translation of WT and RL2A-VP2 ^WT capped SG RNA. In both cases (a and b), the enzymatically capped in vitro transcribed RNA were prepared and used in in vitro translation using rabbit reticulocyte lysates as described in Section 2.12. The translation products were resolved on 15% SDS PAGE, dried and exposed to phosphorimager for visualization. (c) Luciferase activity quantification of reporter RNAs translation products.

The in vitro translation products from full length G RNA (G – WT, G – Pol^FS) and SG RNA of reporter construct were diluted in reporter lysis buffer (RLB) and subjected to luciferase assay as detailed in Section 2.21. The luciferase activity was measured as relative luminescene unit (RLU). The error bars represent standard deviation.

recovery system, the reporter tagged MNV cDNA was transfected into FPV-T7 infected BSR-T7 cells and protein samples analysed for luciferase activity. Western blot analysis was used to detect viral protein expression and virus production was assayed by TCID50 at 48h post transfection. The luciferase activity analysis in Figure 6.3a shows the presence of active Renilla luciferase in the lysates from the transfected cells confirming the expression of reporter tagged MNV cDNA in cell culture. The detection of equal levels of MNV NS7 and NS3 proteins in the western blot analysis of all the protein samples confirms that the BSR-T7 cells were transfected at the same transfection efficiency (Figure 6.3b). Given the protein levels were the same in the POL^FS and POL^- mutants, we can conclude that the protein level observed in this assay were only a reflection of viral RNA transfection rate rather than replication competency. Therefore, the increased luciferase activity obtained from the WT replicon compared to the POL^FS and POL^- was presumably due to replication of the viral genome in the transfected cell. By comparing the Renilla luciferase signal obtained from POL^- and POL^FS, the high signal from POL -could be due to the internal reinitiation of translation. This event is less likely to occur in POL^FS becausethe stop codon introduced in NS7 in this construct (Chaudhry et al., 2007) is far upstream from the start of the ORF2 and presumably the ribosome dissociates at an earlier point. Even though the reporter tagged constructs were able to translate efficiently, there were no infectious virus particles detected by TCID50 analysis, except for those samples from the full-length MNV infectious cDNA clone transfected cells (Figure 6.3c).

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Figure 6.3 Translation of Renilla luciferase from reporter tagged MNV constructs in tissue culture by cDNA based reverse genetics system. (a) Renilla luciferase activity of protein lysates obtained from reporter plasmids transfected BSR-T7 cells previously infected with recombinant FPV-T7. The cDNA based reverse genetics system was carried out as described in Section 2.13.1 by transfecting full-length WT, NS7 frame shift (FS) and active site mutant NS7 (POL^-) of pT7MNV-RL2A-VP2-3’Rz constructs. Transfection of full-length MNV infectious cDNA clone was carried out as a control. At 48h post transfection, the transfected cells were harvested and subjected to lysis in reporter lysis buffer (Promega) before a luciferase assay was carried out as described in Section 2.21.

The error bars represent standard deviations and the luciferase activity was measured as relative luminescene unit (RLU). Statistical differences compare to the WT (calculated by one way ANOVA with Tukey’s post test) are represented by the P-values P < 0.05 (*) and P < 0.01 (**). (b) Western blot analysis of reporter tagged construct transfected BSRT7 cells. The same protein samples obtained in (a) were used in western blot using antisera to the viral NS7 and NS3 and GAPDH as a loading control. (c) TCID50 of the recoveries from reporter tagged construct transfected cells and full-length infectious MNV cDNA clone. Samples were harverted at 48h post transfection by one freeze thaw cycle and the cleared supernatants were titrated onto RAW264.7 cells. The TCID50 was carried out in triplicate with the titre values represented being the mean virus titre observed (asterisk symbol in figure). Standard deviation around the mean is also given in brackets.

In addition to the cDNA based recovery system, the RNA based reverse genetics system was also performed to further examine the translation and replication of length reporter tagged replicon MNV RNA in tissue culture. The full-length in vitro transcribed and enzymatically capped MNV reporter tagged RNA was again transfected into BSR-T7 cells. As a control for virus recovery, the full-length in vitro transcribed and enzymatically capped RNA from the infectious cDNA clone of MNV (Yunus et al., 2010) was also included in the recovery assay. At 24h post transfection, protein samples were prepared and subjected to luciferase assay and western blot analysis. Comparative transfection efficiencies were achieved in all the

WT POL^FS POL

transfected RNA samples, as indicated by western blot analysis in Figure 6.4b.

Active Renilla luciferase protein was detected in protein samples from all reporter tagged RNA transfected cells as indicated in the luciferase assay in Figure 6.4a.

Again, the WT reporter tagged RNA transfected BSR-T7 cells produced the highest luciferase signal compared to its POL^FS and POL^- derivatives. This observation suggested that the transfected WT reporter tagged RNA of MNV from in vitro transcription was able to replicate in BSR-T7 cells while transfection of replication defective reporter tagged in vitro transcribed RNA (POL^FS and POL^-) resulted in no replication. The lower luciferase signal obtained from the transfection of these replication defective reporter tagged RNA was presumably as a result of translation reinitiation at the ORF2 in the viral genome of the input capped RNA transcript in the transfection reactions. This obervation will be further discussed in section 6.2.4.1 of this chapter. As observed in the cDNA based reverse genetics system previously, recoveries from RNA based reverse genetics also failed to produce any detectable level of recombinant reporter virus, as shown by TCID50 analysis (Figure 6.4b).

(a) (b)

Figure 6.4 Translation of Renilla luciferase from in vitro transcribed and enzymatically capped reporter tagged MNV RNA in tissue culture by RNA based reverse genetics system. (a) Renilla luciferase activity measurement and western blot analysis of protein samples (targeting NS7) from reporter tagged MNV RNA transfected BSR-T7 cells harvested at 24h post transfection. The protein samples for luciferase assay were prepared in RLB as described in Section 2.21, while the protein samples for western blot analysis were prepared in RIPA buffer as described in Section 2.20. The error bars represent standard deviation and the luciferase activity was measured as relative luminescene unit (RLU). Statistical differences compared to the WT (calculated by one way ANOVA with Tukey’s post test) are represented by the P-values of P < 0.05 (*) and P < 0.01 (**). (b) TCID50 of the recoveries from the in vitro transcribed and enzymatically capped reporter tagged RNA and full length infectious MNV RNA transfected into the BSR-T7 cells. Virus samples were harverted at 24h post transfection and titrated using RAW264.7 cells. The TCID50 was carried out as described in Section 2.15 in triplicate with the mean titre values presented here (asterisk symbol in figure) and standard deviations shown in brackets.