Manifestaciones y configuraciones subjetivas en contexto de guerra: acalladas en un tiempo y en un espacio, pero no silenciadas para siempre.

To study the significance of the nuclear localisation signal of C several mutants were generated (Fig. 12).

Figure 12: A functional NLS is a prerequisite for nuclear accumulation of MV C.

The intracellular localization of the MV Cwt and Cs proteins and of the indicated mutants was analyzed by confocal laser scan microscopy in HeLa cells 24 h p.t. transfected with the indicated plasmids. C proteins were stained with the C-specific peptide/rabbit antiserum C1242 and visualized with anti-rabbit-Alexa488 secondary antibody (green). Nuclei were stained with ToPro3 iodine (white). The sequences of the NLS are displayed next to the scheme of the respective mutants.

The functional MV Cwt NLS was destroyed by site-specific mutation of the basic cluster motif (44RK to 44AA) exchanging arginine and lysine to alanine. Additionally, the defective NLS of MV Cs was repaired to the wildtype NLS situation by exchanging the G44 residue to an R. Finally a heterologous NLS (SV40 large T antigen NLS) was added to the N-terminus of a MV Cwt mutation with destroyed NLS (44AA). The subcellular localisation of these mutants was assayed by immunofluorescence and detection of the overexpressed C protein

was done by using specific antibodies. As expected from the predictions, MV Cwt was located exclusively in the nucleus (Fig. 12 B). MV Cs, although possessing a NES signal, was distributed over cytoplasm and nucleus. It still can shuttle to the nucleus, being small enough to fit through the nuclear pores (Fig. 12 C). Mutations in the nuclear localisation signal of Cwt (MV CwtmNLS) abrogate its dominant nuclear accumulation, yielding a localisation phenotype similar to MV Cs (Fig. 12 D). Repairing the basic cluster of the NLS sequence of MV Cs back to the wildtype situation promotes the accumulation of MV CswtNLS in the nucleus (Fig. 12 E). This mutant exhibits the same localisation as MV Cwt. Upon fusing the SV40 large T antigen NLS to the N terminus of CwtmNLS, the localisation of this protein to the nucleus is restored (Fig. 12 F). For all these localisations it has to be considered, that the C protein possesses both an NLS (mutated or not mutated) as well as an unchanged NES. This means that it still shuttles to some degree, whereas the confocal images portrait the localisation of C at an exact timepoint picturing C in its preferential location. Therefore in the case of Cwt the balance between nuclear and cytoplasmic protein is definitely on the side of the nucleus, however it does not mean that the same C molecule is not present at some timepoint in the cytoplasm, as well.

The functional consequences of these mutations and different intracellular localisations were assayed using luciferase reporter gene assays (Fig. 13). The P protein of rabies virus was used again as the positive control, whereas the MV fl-H protein served as the

negative control. For normalization purposes the CMV promoter controlled Renilla luciferase

encoding plasmid was always co-transfected. The expression levels were equal as checked by Western blot analysis (below the luciferase assays).

The p125-luciferase plasmid (Fig. 13 A-B) was co-transfected with plasmids encoding for the different C mutants as well as the stimulus fl-TBK1. MV Cwt was able to inhibit induction of the interferon beta promoter more efficiently than MV Cs, as seen in previous experiments. The wildtype C NLS mutant (CwtmNLS), however, clearly showed a loss of function compared to Cwt, whereas the repaired NLS Cs (CswtNLS) gained function. The inhibitory effect of CwtmNLS was comparable to Cs and the effect of CswtNLS is comparable to Cwt. This indicates that the decreased efficiency of Cs is only due to its cytoplasmic localisation. The protein still can decrease induction of interferon beta, upon its relocation to the nucleus by repairing the NLS.

Figure 13: Nuclear localization of MV C is required for efficient inhibition of IFN-β

promoter activity.

(A, B) Dual luciferase assays were used to determine activation of the IFN-β promoter. Plasmids

encoding the indicated protein constructs (300 and 600 ng of C, 200 ng fl-TBK-1, 600 ng RV P or fl-MV H) were transfected into 293T cells and induction of p125-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. (A) The inhibitory

activity of Cwtis completely lost by after disruption of the NLS and Cs gains full activity after

repair of the NLS R44G mutation. (B) An ectopic heterologous SV40 large T antigen NLS can substitute for the authentic internal C protein NLS in SV40NLS-CwtmNLS. (C) Quantitative RT-

PCR from 293T cells stimulated by expression of ΔRIG-I (100ng) in the presence of the indicted

viral protein constructs (400ng). IFN-β mRNA normalized with GAPDH mRNA. Columns show

average values from two experiments, error bars represent standard deviation. Lysates were harvested 24h post transfection for luciferase assays and the protein content analysed by western blotting and immunodetection with specific antisera (lower panels).

Adding an exogenous NLS (SV40 large T antigen NLS) to the N-terminus of CwtmNLS also restored the function of CwtmNLS (Fig. 13 B), reflecting the Cwt situation. This indicates that the exact sequence of the NLS is not important but only the spatial distribution of C inside the cell.

Quantitative realtime PCR of interferon beta messenger RNA showed the same results as the luciferase assays (Fig. 13 C). Cwt and CswtNLS were more efficiently inhibiting the transcription of interferon beta messenger RNA than Cs and CwtmNLS. This time a plasmid coding for a constitutive active mutant of RIG-I, comprising only the CARD domains was used as a transfectable stimulus. The normalisation was done using GAPDH mRNA levels, again indicating that MV C does not induce a general shutdown of transcription as the GAPDH mRNA values of mock and the C transfected cells were similar.

In summary the NLS sequence drives the preferential accumulation of C in the nucleus and the mutation in the vaccine C abrogates the function of the NLS, resulting in a different intracellular distribution of the protein. The inhibition of IFN-β induction clearly correlates

with the accumulation of C in the nucleus and occurs at a transcriptional level downstream of TBK1.