Fig. 6: ZIKV NS1 induces mitochondrial transfer between cells via TNTs.
A Representative confocal image of HTR-8 cells transfected with pNS1-ZIKV showing mitochondria (arrow) and NS1 transfer via F-actin-rich TNTs. B Cells transfected with pNS1ΔTNT show limited TNT formation and transfer of mitochondria cargo at 24 hours (hrs). Merged images show nuclei (blue), actin (gray), mitochondria (green), and ZIKV-NS1 (red). C Schematic of 24 hrs co-culture assays for determining mitochondria transfer to NS1-expressing cells via flow cytometry as in D–I. Percentage of HTR-8 cells (NS1 + , Celltrace-, Mitotracker + ) that acquired mitochondria from untransfected D HTR-8, all groups n = 5; F JEG-3 (PRVABC-59, n = 4; remaining groups n = 5); and H THP-1 MΦ, all groups n = 5. E, G, and I Quantification of mitochondria transfer to HTR-8 NS1-expressing cells (NS1 + , Celltrace-, Mitotracker +) in relation to untransfected cells (NS1-, Celltrace-, Mitotracker + ) as represented by the mitochondria mean fluorescence ratio (MFI). The confocal images shown are representative of experiments (n = 3) independently repeated with similar results. Images were acquired using a Nikon A1R confocal microscope at 40X (A) and 60X (B) oil objectives lens at 1.4 normal aperture (NA) and processed using the NIS Elements software (Nikon). Quantification of mitochondria transfer was performed using the BD LSRFortessa™ cell analyzer, total events collected= 30,000 cells. Flow cytometry results were analyzed using FlowJo™ v10.8 Software (BD Life Sciences). Data are presented as mean ± SEM. Statistical significance was determined by ANOVA followed by Tukey’s multiple comparison test. *= P ≤ 0.05, ***= P ≤ 0.001, ****= P ≤ 0.0001. Bar = 25 μm. Figure 6C was created in BioRender129. Source data are provided as a Source Data file.
