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. Author manuscript; available in PMC: 2020 Aug 31.
Published in final edited form as: Nat Microbiol. 2017 Mar 27;2:17037. doi: 10.1038/nmicrobiol.2017.37

Figure 3 |. NS2B protease cofactor degrades cGAS in an autophagy–lysosome-dependent mechanism.

Figure 3 |

a, Evaluation of cGAS degradation by NS2B3 protease or its components. Expression analysis of human cGAS and empty vector, DENV NS2B, NS3 or NS2B3, performed in 293T cells (48 h) by SDS–PAGE/western blot. b, Evaluation of the mechanism of cGAS degradation by DENV NS2B. Co-expression of cGAS-FLAG and DENV NS2B-HA in 293T cells in the presence of DMSO, Z-VAD-FMK (5uM), MG-132 (5 μM), clasto-lactacystin β-lactone (lactacystin) (1 μM), DBeQ (5 μM), chloroquine (5 μM) or NH4Cl (2 mM). cGAS, DENV NS2B and α-tubulin expression levels were analysed by western blot. Bar graph: densitometry analysis of cGAS protein by ImageJ software. c,d, Analysis of cGAS degradation by NS2B (c) or DENV-2 infection (MOI = 10) (d) in the presence of 3-MA (autophagosome formation inhibitor). Densitometry analysis of cGAS protein by ImageJ software. e, Analysis of the interaction between DENV HA-tagged NS2B, NS3, NS2B3 protease complex WT or S135A mutant with human full-length cGAS-FLAG or cGAS domains by co-immunoprecipitation in 293T cells. f, Analysis of cGAS and viral proteins interaction during infection. Immunoprecipitation of cGAS-FLAG in 293T cells MOCK or DENV-2 infected for 12 h. Immunodetection of viral proteins NS3 and NS2B by western blot, using specific antibodies. g, Analysis of cGAS in DENV-infected cells by immunofluorescence: A549 cells expressing cGAS-V5 were infected MOCK or with DENV-2 or DENV-4. At 24 or 48 h.p.i., cells were fixed and processed for indirect immunofluorescence (see Methods). Primary antibodies against V5 (cGAS), autophagy marker (Atg12) or DENV NS3 protein were used. Alexa Fluor-conjugated secondary antibodies −488 (green), −568 (red) and −647 (magenta) were used to detect the primary antibodies, respectively. Nuclei were stained with DAPI (blue). Images on the right correspond to the squared zoomed images in each panel. Scale bars, 10 μm. A detailed three-dimensional reconstruction of the Z-stacks is shown top right. Scale bars, 0.5 μm. Arrows mark co-localization of cGAS with autophagosomes and viral protein. ND, not detected.