Fig. 6: MERS-CoV blocks autophagic flux.

a MERS-CoV decreases BECN1 and increases pSKP2. VeroB4 cells were infected with MERS-CoV (MOI = 0.001) or mock-infected and harvested 48 h later. Representative western blots are shown, quantification in Supplementary Fig. 3a, b. b MERS-CoV increases K48-linked poly-ubiquitination of BECN1. BECN1 was immune-precipitated from cell extracts 48 h p.i. and its ubiquitination determined by a ubiquitin K48 linkage specific antibody. c, d MERS-CoV blocks fusion of AP with lysosomes. VeroB4 cells were transfected with tandem fluorescent-tagged LC3B (mRFP and EGFP) and infected with MERS-CoV (MOI = 0.001). Twenty-four hours later, cells were fixed and analyzed for fluorescence. Vesicles with both green and red fluorescence (autophagosomes, AP) and with red fluorescence only (autolysosomes, AL) were counted. d, representative images, scale bar 25 µm. e, f MERS-CoV decreases ATG14 oligomerization. VeroB4 cells were infected with MERS-CoV (MOI = 0.001), cross-linked with disuccinimidyl suberate (DSS, 75 µM) 48 h p.i. for 30 min and harvested. ATG14 homo-oligomerization was examined after western blotting (ProteinSimple). g MERS-CoV decreases autophagic flux. VeroB4 cells were infected with MERS-CoV (MOI = 0.001) and incubated with bafilomycin A1 (BafA1, 0.1 µM) for 2 h before samples were taken at 48 h p.i.. The ratios of LC3B-II/I were determined by western blotting. h MERS-CoV affects SNARE protein interactions. VeroB4 cells were infected with MERS-CoV (MOI = 0.001), cross-linked with disuccinimidyl suberate (DSS, 75 µM) 48 h p.i. for 30 min and harvested. The SNARE complex protein STX17 was immunoprecipitated and the eluate was probed for interacting VAMP8 and SNAP29 by western blotting (the quantification represents the bands detected at the combined molecular weights of the cross-linked proteins, i.e. STX17 + VAMP8 and STX17 + SNAP29). In all panels, error bars denote the standard error of the mean. derived from n = 3 biologically independent experiments for b, f, g, h, and n = 13 (control) or n = 12 (CoV) different cells for c. WCE = whole cell extract of vehicle exposed cells, i.e. no cross-linking. *p < 0.05, **p < 0.01, ***p < 0.001 (t-test in b, c, f, h, two-way ANOVA in g, details in Supplementary Tables 1, 2). Source data and blot collections are provided as a Source Data file.