Descripción del proceso: 3 El proceso con los alumnos:

PML-II is known to facilitate a robust IFN response and at the same time it is upregulated significantly from basal level with stimulation by IFN-α or β (Der et al., 1998, Chen et al., 2015). Hence, the straightforward mechanism that could be proposed here is that the reduced level of IFN response in PML-II Kd cells allows Ad5 to grow better, even though Ad5 expresses E4Orf3 that presumably inhibits this factor. To test this, the idea was to block the interferon response before Ad5 infection in both PML-II Kd and EV cells by another means, then to compare the outcome of Ad5 infection. IRF3 is a transcriptional factor that is retained in an inactive form in the cytoplasm due to a constitutive nuclear export signal. When a cell senses a pathogen, IRF3 becomes activated and is transported to the nucleus where it starts its transcription function in triggering the type I interferon response (Yoneyama et al., 1998). Adenovirus infection in particular induces interferon type I in an IRF3-dependent manner soon after infection which results in a direct stimulation of ISG56 and other ISGs (Stein and Falck-Pedersen, 2012).

IRF3 siRNA treatment was optimised by using a series of concentrations: 31.25, 62.5 and 125 pmol (data not shown); the best concentration was 62.5 pmol which showed about 50% reduction in ISG56 mRNA (Fig4.6A) which was used later as the standard for IRF3 knockdown experiments.

To confirm the functional effect of IRF3 knockdown EV cells were treated with control or IRF3 siRNA, stimulated with poly I:C and ISG56 mRNA was quantified as one of the directly IRF3-stimulated genes (Grandvaux et al., 2002). In both poly I:C stimulated and un-stimulated cells, IRF3 knockdown caused the IFN response to decline to about 50 and 40% respectively (Fig.4.6B). IRF3 knockdown did not block the response completely, either because IRF3 loss was not complete or because there is another transcription factor, NF-kB, that is also activated and contributes to the IFN response within this same classical induction pathway (Haller et al., 2006)

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Next, the effect of IRF3 knockdown on Ad5 infection was tested. PML-II Kd and EV cells were transfected with IRF3 siRNA or siC for 48 hours, then infected with Ad5wt300 for 20 hours. Late viral proteins was analysed and compared in both cell lines (Fig.4.6C). The level of late viral proteins was considerably greater inPML-II Kd cells than in EV cells when treated with siC, which is consistent with results that were obtained in these cells without siRNA treatment. IRF3 knockdown in PML-II Kd cells had no effect on the already high level of hexon being expressed. This reflects the fact that the interferon response is already strongly inhibited in these cells through the absence of PML-II.

In EV cells the effect of IRF3 knockdown was to increase hexon expression somewhat compared to the control. Significantly though, this hexon expression was still substantially lower than was observed in PML-II kd cells (Fig 4.6C). Thus, blocking of type I interferon responses by IRF3 knockdown in EV cells does not make them equivalent to PML-II Kd cells in terms of their hexon expression. This sort of effect suggests that there might be other mechanisms in addition to loss of the IFN response that contribute to increased viral gene expression in PML-II Kd cells.

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Fig.4.6. Ad5 late gene expression in PML-II Kd and EV cell lines with or without IRF3 knockdown. PML-II Kd and EV cells were seeded at a density of 1×105 /ml for 24 hours in 24-well cultures. A. IRF3 expression in PML-II Kd and EV cells. Cells were transfected with 62.5 pmol of either IRF3 siRNA or siC for 48 hours. Cultures were harvested for SDS-PAGE analysis and membranes were reacted with rabbit polyclonal anti- IRF3 Abs. B. ISG56 mRNA in EV cells with or without stimulation.Cells were transfected with 62.5 pmol of either IRF3 siRNA or siC for 48 hours then either stimulated with poly I:C for 16 hours or left without treatment. Total ISG56 mRNA was quantified by RT-qPCR using 10 ng/well of complementary DNA (cDNA) as a template. Data were presented by the ∆∆Ct method, using β-actin as an internal control. All PCR products were subjected to dissociation curve analysis to ensure primer specificity. C. Ad5 hexon expression in PML-II Kd and EV cells. Cells were transfected with 62.5 pmol of either IRF3 siRNA or siC for 48 hours, then infected with Ad5 at moi of 5 for 20 hours. Cultures were harvested for SDS-PAGE analysis and membranes were probed with polyclonal anti-late rabbit Abs or monoclonal anti- GAPDH mouse Abs.

To reach to a final conclusion about the effect of IFN response on Ad5 infection and because of the clear relationship between PML-II, IFN and E4Orf3 (Chen et al., 2015, Ullman et al., 2007), infection was evaluted in PML-II Kd and EV cells in the presence and absence of E4Orf3 with and without IFN treatment. The idea was that E4Orf3 deletion, as it counteracts the interferon antiviral effect, would make the mutant virus more sensitive to interferon treatment than the wild type, especially in the EV cells where the level of interferon response would be normal. PML-II Kd and EV cells were both treated with 1000 U/ml of IFN-α for 24 hours prior to

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infection with Ad5wt300 or inOrf3 viruses at a moi of 5 for 20 hours. In essence, there was a clear increase in late viral gene expression in PML-II Kd cells for both viruses compared with the EV cells, this is consistent with all previous results as PML-II plays a negative role in the Ad5 life cycle (Fig.4.7A). The inOrf3 mutant displayed less gene expression in EV cells than the wild type and this is also expected as it lacks E4Orf3, the protein that compromises the interferon activity (Fig.4.7A). The wt300 was affected to a lesser extent with the interferon treatment than the mutant in both cell lines despite it was more striking in EV cells which is also the expected scenario as the control cells express more interferon than PML-II Kd cells (Fig4.7B). The IFN treatment had more effect on the mutant virus in both cell lines, especially in the control cells and it showed a more significant effect than the wild type as shown by the hexon band quantitation (Fig.4.7B). Hence, the mutant showed a greater level of late gene expression in PML-II Kd cells despite the interferon treatment because these cells are deficient in the interferon response due to the lack of PML-II protein. As an overall conclusion from this part of the work, the mutant was more vulnerable to the interferon treatment in the EV cells than in PML-II Kd cell, which suggests that the E4Orf3-PML-II interaction is necessary to overcome the antiviral functionof interferon since the mutant was affected to a lesser extent when the interferon responsewas low due to PML-II removal.

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Fig.4.7. Late gene expression in PML-II Kd and EV cells with and without IFN type I treatment. A. PML-II Kd and EV cells were plated for 24 hours, treated or not with 1000 U of IFN -α for another 24 hours (+I,-) and infected with either wt300 or InOrf3 mutant at moi of 5 for 20 hours. Protein samples were harvested for WB analysis; the membranes were probed with polyclonal anti-late rabbit Abs or monoclonal anti-GAPDH mouse Abs. B.

Hexon band density. The upper main band with a molecular weight of about 100 kDa was quantified using Quantity one software to compare its intensity in all 8 samples. The background of the negative film was subtracted and then the results were normalised to the related GAPDH bands, and then further normalised to the value for wt300 in EV cells with no treatment

4.3.2 Role of heat shock protein 70 (hsp70) in adenovirus gene expression