Journal: Journal of Virology

Article Title: Decapod iridescent virus 1 (DIV1) enters hematopoietic Cherax quadricarinatus cells via caveola-mediated endocytosis in a pH-dependent manner

doi: 10.1128/jvi.01681-25

Figure Lengend Snippet: Confocal microscopy analysis of DIV1 internalization into HPT cells. ( A ) Time-course analysis of DIV1 internalization into HPT cells and its co-localization with caveolin-1. HPT cells were pre-chilled and incubated with Cy5-labeled DIV1 at 4°C for 1 h to allow binding. After removing the unbound virus, cells were shifted to 27°C to initiate internalization, then fixed with methanol at 0, 0.5, and 3 h post-internalization. IFA was performed using an anti-caveolin-1 antibody, followed by a FITC-conjugated secondary antibody. Uninfected cells served as a negative control. Confocal imaging was conducted, and the correlation between red fluorescence (DIV1) and green fluorescence (caveolin-1) was analyzed using ImageJ software. Pearson correlation coefficient (PCC) values are shown in the upper-left corner of the merged images. ( B ) Pull-down assays to verify the interaction between DIV1 and caveolin-1. Pull-down assays were performed with two groups: one using caveolin-1 as the bait protein, and the other without caveolin-1, with purified DIV1 included in both reactions. Eluted proteins from the caveolin-1 pull-down fraction and input samples were analyzed by WB. Blots were probed with antibodies against DIV1-168L or caveolin-1. ( C ) Localization of DIV1 in the Golgi apparatus. HPT cells were pre-chilled and co-incubated with DIV1 and NBD-labeled C6-ceramide (Golgi marker) at 4°C for 1 h to allow binding. Unbound substances were removed, and cells were shifted to 27°C for 4 h (to allow viral trafficking to the Golgi apparatus). After fixation with methanol and nuclear staining with DAPI, confocal imaging was performed. The correlation between red fluorescence (DIV1)-green fluorescence (Golgi) was analyzed using ImageJ software. PCC values are shown in the upper left corner of merged images. Scale bar: 10 μm. ( D ) Localization analysis of DIV1 and transferrin-AF647 during internalization. HPT cells were pre-chilled and co-incubated with DIV1 and transferrin-AF647 at 4°C for 1 h to allow binding. Unbound substances were removed, and cells were shifted to 27°C for 30 min to induce endocytosis. After fixation with methanol, IFA was performed to label DIV1 with an anti-DIV1-168L antibody, followed by a FITC-conjugated goat anti-rabbit antibody. Nuclei were counterstained with DAPI. Confocal images were acquired, and the correlation between red fluorescence (transferrin-AF647) and green fluorescence (DIV1) was analyzed via ImageJ software. PCC values are shown in the upper right corner of merged images. Scale bar: 10 μm.

Article Snippet: Subsequently, the cells were incubated at room temperature for 1 h with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (Bioss, China), which was diluted 1:500 in 1% normal goat serum.

Techniques: Confocal Microscopy, Incubation, Labeling, Binding Assay, Virus, Negative Control, Imaging, Fluorescence, Software, Purification, Marker, Staining