MV C does not interfere with activation of IRF

For effective induction of IFN-β, a crucial transcription factor, IRF3, has to be fully

activated. The classical activation steps include its phosphorylation at C-terminal serine residues; especially phosphorylation at serine 386 and 396 was shown to be obligatory. The phosphorylated transcription factor then dimerizes and is imported actively into the nucleus. Within the nucleus activated IRF3 binds to the DNA at its recognition sites and is responsible for the recruitment of cofactors required for efficient transcription initiation such as e.g. p300 and CBP (Panne et al., 2007a; Yoneyama et al., 1998).

An IRF3 binding site reporter plasmid (p55c1b-Luciferase) was co-transfected with plasmids coding for Cwt and Cs. Induction was done using overexpression of the transcription factor IRF3 and the luciferase activity was measured afterwards. Only the Cwt protein was able to inhibit reporter gene induction in a dose dependent manner, whereas Cs mostly failed to do so. This indicates an inhibition downstream of IRF3 and upstream of interferon beta translation (Fig. 13 D).

Figure 14: MV C inhibits IRF3 stimulated interferon induction (A) but does not interfere with IRF3 activation by phosphorylation (B), dimerization (C) or nuclear accumulation (D).

(A) Dual luciferase assays were used to determine activation of the IRF3 response element (p55c1b) promoter. Plasmids encoding the indicated protein constructs (250 to 750 ng of C, 200 ng fl-IRF3) were transfected into 293T cells and induction of p55c1b-luc-directed FL activity was determined 24 h p.t.. All assays were normalized with co-expressed RL, mock values set to 1 and relative induction is shown. Standard deviations of triplicates are displayed. (B) 293T cells expressing MV C, RV P as a positive control, fl-MV H as a negative control, and flag-tagged IRF3 (fl-IRF3) from transfected plasmids as indicated, were stimulated by SeV DI H4 to activate IRF3. Induction of p125-luc-directed FL activity was determined 12 h p.t.. All assays were normalized with co-expressed RL, mock values set to 1 and relative induction is shown. Standard

deviations of triplicates are displayed (top panel). Phosphorylation of IRF3 S386 and S396 was analyzed by Western blotting following SDS-PAGE of whole cell lysates harvested 12 h post transfection using specific antibodies. The expression level of the overexpressed proteins MV C, RV P, fl-IRF3 and MV fl-H was additionally monitored in the same western blot experiment, as well as the cellular control protein actin (lower panel). (B) Native PAGE of whole lysates collected from 293T cells 24 h post transfection. IRF3 dimerization was stimulated by overexpression of fl-TBK-1. IRF3 was stained with anti-IRF3 antibodies. The sizes of the IRF3 dimer and the monomer are indicated next to the blot. (C) HeLa cells were mock-infected or infected with SeV DI-H4. At 18 h p.i. endogenous IRF3 was visualized by staining with anti- IRF3 antibody and Alexa 488 (green) and fl-Cwt expressed from transfected plasmids by anti- flag and Alexa-555 (red). The nuclei were stained with ToPRO3 (white). The figure shows a representative example of multiple experiments.

To assay the phosphorylation status of overexpressed flag-IRF3, cells stimulated with SeV defective interfering particles H4 were lysed after 12h. A luciferase reporter gene assay of the same lysates showed, that even the initial low level induction of interferon beta by SeV DI H4 is blocked by MV Cwt, whereas RV P is less effective in blocking these initial steps (Fig. 14 A). RV P, however, is able to completely prevent phosphorylation of IRF3 at position 386 and 396 as indicated by phosphorylation-specific antibody western blots (Fig. 14 B). MV Cwt instead does not block visibly any phosphorylation of IRF3 compared to the mock stimulated cells or the negative control (MV fl-H). All inhibitory proteins were confirmed as expressed in this assay by Western blot analysis and subsequent detection of the proteins by specific antibodies.

To assess the dimerization of IRF3, native gel electrophoresis experiments were conducted. RV P co-expression induced a complete loss of IRF3 dimerization after stimulation with fl-TBK1 overexpression. Both MV Cwt and Cs could obviously not inhibit this dimerization step compared to the empty vector control (Fig. 14 C).

The nuclear import is thought to be a final step in activation of IRF3. In non- stimulated cells IRF3 was distributed in the cytoplasm and co-expression of MV Cwt does not alter this localisation. Upon stimulation - with SeV Dis - IRF3 accumulated visibly in the nucleus. Co-expression of MV Cwt again did not alter this behaviour, resulting in both Cwt and IRF3 residing inside the nucleus of activated cells (Fig. 14 D).

Taken together, these results indicate that the mechanism of Cwt does not involve inhibition of phosphorylation, dimerization or nuclear import of IRF3.

2.

Identification of cellular interaction partners of MV C

Most mechanisms employed by viral proteins to disturb cellular signalling pathways use protein-protein interaction as an interface to manipulate the host. In the case of MV for example, V binds strongly to the cellular helicase MDA-5, thereby inhibiting its ATPase activity and subsequent signalling (Andrejeva et al., 2004). It is therefore likely, that the C protein also interacts with cellular proteins to modify their functions.

Binding assays were mainly done using co-immunoprecipitation experiments in 293T cells, where both putative binding partners were overexpressed. One of the tagged interaction partners was then bound to a matrix either coated with HA or with flag antibodies. The lysates of these pulldowns were then subjected to Western blot analysis followed by immunodetection to show whether the precipitation worked. Possible co-purified protein interaction partners were detected by Western blotting as well. Whole cell lysates were also analysed in a similar fashion to show the presence of the proteins in the input of the pulldown. Alternatively these pulldowns were analysed by mass spectrometry instead of Western blotting to identify endogenous proteins bound to the tagged protein.

Another method employed to identify binding partners of C was overexpression of putative binding partners together with C in Hep2 cells. These cells were stained with fluorescent antibodies and then subjected to confocal microscopy analysis. A co-localisation of two proteins would then indicate an interaction.