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. 2022 Apr 20;298(6):101956. doi: 10.1016/j.jbc.2022.101956

Figure 7.

Figure 7

CLINT1 binds DENV NS3 and mediates intracellular trafficking of DENV particles.A, CLINT1 binding to individual DENV proteins via PCAs in 293T cells. The dotted line represents the cutoff used for positive interactions. B and C, IPs with anti-FLAG antibody or IgG control from lysates of DENV-infected Huh7 cells ectopically expressing FLAG-tagged WT (B and C) or CLINT1-T294A mutant (C) or empty plasmid. Molecular weight markers are indicated on the right (kDa). Membranes were blotted with antibodies against NS3, CLINT1, and capsid. D, schematic of the TC-capsid construct. E and F, Quantitative data of the percentage of DENV capsid colocalizing with CLINT1 (E) and of the static and cotrafficking capsid/CLINT1 colocalized particles (F). The data were obtained from 3 fields of view (equivalent to 3 independent movies). Moreover, an average of 61 total DENV-capsid, 22 total colocalizing, 6 total cotrafficking, and 16 total static particles were counted across the three movies. G, representative live cell fluorescence microscopy montages of TC-capsid DENV (green) cotrafficking with CLINT1-mCherry (red) over 27 s. Distance traveled (μm) during video acquisition is indicated. Solid arrow heads indicate cotrafficking capsid/CLINT1 particles; open arrow heads indicate static capsid/CLINT1 colocalizing particles. CLINT1, clathrin-interacting protein 1; DENV, dengue virus; IP, immunoprecipitation; NLR, normalized luminescence ratio; PCA, protein-fragment complementation assay; WCL, whole-cell lysate.