Fig. 8: SKP2 inhibition reduces replication of MERS-CoV and its effects on autophagy. | Nature Communications

Fig. 8: SKP2 inhibition reduces replication of MERS-CoV and its effects on autophagy.

From: SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection

Fig. 8

a The SKP2 inhibitor (SKP2i) efficiently inhibits MERS-CoV replication. VeroB4 cells were infected with MERS-CoV (MOI = 0.001) and treated with SKP2i (10 µM) or DMSO (Co = control). MERS-CoV GE were determined by real-time RT-PCR at 24 and 48 h p.i., data are presented as fold difference (black bars, SKP2i condition) in comparison to control (gray bars, Co = DMSO condition). Raw data presenting GE ml^-1 is shown in Fig. S5A. b, c The SKP2-inhibitor (SKP2i) restores autophagic flux in MERS-CoV-infected cells. VeroB4 cells were transfected with mRFP-EGFP-tagged LC3B, infected with MERS-CoV (MOI = 0.001) and treated with SKP2i (10 µM) or vehicle for 24 h. b Representative images (scale bar 25 µm). c The numbers of vesicles with both green and red fluorescence (autophagosomes, AP) and with red fluorescence only (autolysosomes, AL) were counted 24 h p.i.. d–f SKP2i enhances protein interactions indicative of autolysosome formation in MERS-CoV-infected cells. VeroB4 cells were infected with MERS-CoV (MOI = 0.001), treated with SKP2i for 48 h, cross-linked with disuccinimidyl suberate (DSS, 75 µM) 48 h p.i. for 30 min and harvested. ATG14 homo-oligomerization was assessed after western blotting (d) and quantified (e). f The SNARE complex protein STX17 was immunoprecipitated and the eluate was probed for interacting VAMP8 and SNAP29 by western blotting and quantified as in Fig. 6h. In all panels, error bars denote the standard error of the mean, derived from n = 3 (a, e) or n = 4 (f) biologically independent experiments, and n = 13 (c, vehicle, non-infected) or n = 12 (all other conditions in c) different cells. *p < 0.05, **p < 0.01, ***,###p < 0.001 (c, *,***refer to the statistical difference between the numbers autolysosomes, ###to the difference between the total numbers of fluorescing vesicles). Two-way ANOVA was performed in c, t-tests in a, e, f, details in Supplementary Tables 1, 2. Source data and blot collections are provided as a Source Data file.

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