Fig. 4

CapZ deficiency compromises the maturation of early endosomes to inhibit ZIKV, DENV, or MHV infection. A The expression of CapZβ in Control, CapZβ-knockout, or CapZβ-reconstituted HeLa cells. The CapZβ-reconstituted HeLa cells were constructed by stably transfecting pCDNA3.1-CapZβ-His₆ into CapZβ-knockout cells. B Control, CapZβ-knockout, or CapZβ-reconstituted HeLa cells were infected with ~ 1 MOI ZIKV for 48 h. The cells were then immunolabeled with dsRNA antibodies and subjected to confocal imaging and analysis. The expression levels of CapZβ in these cells were also determined by immunoblot analysis. C Control or CapZβ-knockout cells were incubated with ~ 50 MOI ZIKV on ice for 1 h, and the cells were then incubated at 37 °C for indicated times, followed by in situ RNA hybridization of plus-strand RNA of ZIKV RNA genome and RAB5 immunostaining. The images were acquired by a confocal microscope and proceeded for colocalization analysis. The scale bar is 2 μm. D Control or CapZβ-knockout cells were infected with ~ 1 MOI DENV-3 for 48 h, and the cells were then immunolabeled with dsRNA antibodies and subjected to confocal imaging and analysis. E Lysates from Control or CapZβ-knockdown 4T1 cells were collected and subjected to SDS-PAGE followed by immunoblotting analysis against the indicated antibodies. F, G Control or CapZβ-knockdown 4T1 cells were infected with ~ 1 MOI of MHV for 24 h. The cells were either lysed and subjected to NSP9 and GAPDH immunoblot analysis (F) or were immunostained with dsRNA antibody followed by confocal imaging (G). The scale bar is 20 μm. The blots, images, and graphs represent data from at least three independent experiments. Data quantifications were analyzed using unpaired Student’s t-test (two-group comparison in D, E, F, and G) or ANOVA (multiple comparisons in B) and expressed as mean ± S.D., *P < 0.05, **P < 0.01, ***P < 0.001