Cogulantes-floculantes naturales

The second protein of interest was MDA5. Together with RIG-I it senses viral RNA in the cytoplasm and initiates the signaling cascade to produce IFNs and induce ISG expression. The CETSA experiments revealed Tm shifts of -1.00°C and -1.06 °C in the two experiments, respectively (Table 5). The calculated melting curves shown in Figure 26B revealed a clear destabilizing effect of CsA on MDA5. The generation of CRISPR/Cas9 knockouts in THP-1 based CypA -/- cells was successful, as was judged in Western blot analysis shown in Figure 28A. As for RIG-I knockout cells, A549 cell lines served as a reference for MDA5 knockout in immunoblotting (Figure 27A and Figure 28A). These cells were previously characterized for MDA5 knockout (unpublished work by the group of Marco Binder3_{) and indicate that the lower}

band observed in the anti-MDA5 blot is the MDA5 corresponding band. As can be seen for THP-1 and CypA -/- control cells, MDA5 expression was observed in THP-1 cells, however to a lesser extent than observed in A549 cells. Based on CypA -/- THP-1 cells a MDA5 double- knockout was created. As can be seen in Figure 28A not all single cell clones had the knockout phenotype, but some CypA-MDA5 -/- cell lines could be created (Figure 28A).

Infection assays were performed with several MDA5 knockout clones to control for clonal effects (data not shown). For the following infection assays, one representative double knockout cell line is shown (Figure 28 B-G). In HIV-1 LV infection a reduced infection of CypA-MDA5 double knockout cells compared to the two reference cell lines could be observed (15.1 % infection), as shown in Figure 28B. Infection of CypA -/- resulted in 36 % infection and THP- 1 parental cells showed 23.6 % infection. Additional knockout of MDA5 reverses this effect and infectivity was lower than in THP-1 wt cells. Infection of IFN2 and CsA stimulated cells was also lower compared to CypA single knockouts or THP-1 parental cells (Figure 28C and D, respectively). Infection after IFN2 pre-stimulation was almost completely lost in double knockout cells resulting in a 12.9-fold block to HIV-1 LV infection, indicating strong effects of MDA5 in the type I IFN response to HIV-1 infection. This magnitude of infection block by IFN2 was only observed in HIV-1 NL4.3 infection in this study. The effects of CsA were also enhanced in the double knockout cell line. CypA-MDA5 -/- cells showed a rescue of 4.1-fold (see Figure 28D) whereas CypA single knockouts only showed a rescue of HIV-1 LV infection of 1.4-fold. This suggests an inhibiting effect of CsA on MDA5, which might be direct or indirect.

Figure 28: CypA-MDA5 -/- show reduced HIV-1 infectivity, a strong response to IFN and hypersensitivity to the rescue of this block by CsA.

A: THP-1 wt, CypA -/- and CypA and MDA5 double knockout (CypA-MDA5 -/-) cell lines were analyzed by

Western blot for CypA, MDA5 and -actin expression. A569 parental cells and MDA5 -/- in these cells served as controls. B: THP-1 wt, CypA -/- or CypA-MDA5 -/- cells were treated or not with 500 U/ml IFN2 for 24 h. Prior to infection with VSV-G pseudotyped HIV-1 8.91 LV for 48 h, cells were treated with 2.5 µM CsA. Percentage of GFP positive cells was determined by flow cytometry. Bars indicate the average infectivity determined from three independent experiments and error bars indicate standard deviation. Unpaired two -tailed t test was performed (*, p< 0.05; ***, p< 0.001). C: Calculated fold changes of IFN2 induced block to HIV-1 LV infection. Bars indicate the average fold change and error bars indicate standard deviation. Unpaired two- tailed t test was performed (***, p< 0.001). D: Calculated fold changes of HIV-1 LV infection levels upon CsA treatment compared to IFN stimulated cells. Bars indicate the average fold change and error bars indicate standard deviation. Unpaired two-tailed t test was performed (***, p< 0.001). E: THP-1 wt cells or CypA- MDA5 -/- cells were treated or not with 500 U/ml IFN2. 24 h post IFN stimulation cells were treated with 2.5 µM CsA. At the time of CsA addition, cells were infected with VSV -G pseudotyped NL4.3 for 48 h. Percentage of GFP positive cells was determined by flow cytom etry. Bars indicate the average infectivity determined from independent experiments and error bars indicate standard deviation . Unpaired two-tailed t test was performed (**, p< 0.01; ***, p< 0.001; ns, not significant). F: Calculated fold changes of IFN2 induced block to NL4.3 infection. Bars indicate the average fold change and error bars indicate standard deviation. Unpaired two -tailed t test was performed (***, p< 0.001). G: Calculated fold changes of NL4.3 infection levels upon CsA treatment compared to IFN stimulated cells. Bars indicate the average fold change and error bars indicate standard deviation. Unpaired two-tailed t test was performed (**, p< 0.01; ***, p< 0.001).

Surprisingly, these effects were reversed in HIV-1 NL4.3 infection, pointing to a possible involvement of HIV-1 accessory proteins. A reduction of infection was observed in CypA- MDA5 -/- cells compared to CypA -/- cells (14.3 % compared to 22.3 %) but no difference in infection to THP-1 wt cells was found (12.6 %; Figure 28E). Interestingly, the type I IFN- induced block to infection was only 4.7-fold for CypA-MDA5 double knockout cells, which was significantly lower than the block observed in parental THP-1 cells or CypA -/- cells (6.6- fold and 14.7-fold, respectively; Figure 29F). Additionally, no rescue of this type I IFN-induced block to infection was observed upon CsA treatment in CypA-MDA5 -/- cells whereas CsA treatment of THP-1 parental cells reduced HIV-1 NL4.3 infection further. As CsA has no effect on IFN2 treated CypA-MDA5 double knockout cells, MDA5 is a possible candidate for a type I IFN-induced CsA affected protein involved in HIV-1 infection. However, CypA-MDA5 -/- cells were less sensitive to the type I IFN-mediated HIV-1 infection block and effects of CsA could be masked due to the smaller IFN-induced block. Nonetheless, MDA5 is an interesting candidate for further analysis. MDA5 effects could be investigated more thoroughly with MDA5 single knockout cells, which were already generated but remain to be screened.

8.2.4 CypA-MAVS -/- and CypA-IRF3 -/- cells show hypersensitivity to type I IFN-