Journal: iScience
Article Title: Dengue virus and Zika virus alter endoplasmic reticulum-mitochondria contact sites to regulate respiration and apoptosis
doi: 10.1016/j.isci.2024.111599
Figure Lengend Snippet: ERMC proteins negatively regulate mitochondrial respiration and viral replication (A–C) Huh7.5 cells were transduced with lentiviruses expressing shRNAs which target the indicated proteins (MOI = 4). Four days later, cells were trypsinized, counted, and processed for measurements of various parameters of the oxygen consumption rate using the Seahorse technology as described in the section. Data were normalized to the mean basal OCR of the non-target shRNA (shNT) control conditions as in B–4E. ∗: p ≤ 0.05; ∗∗: p ≤ 0.01; Kruskal-Wallis test; N = 4–5. (D) Cells were transduced as in (a-c). Two days post-transduction, cells were infected with DENV-R2A reporter viruses which express Renilla reniformis luciferase (Rluc) at an MOI of 0.01. Two days post-infection, the luciferase activity was measured as a readout of viral replication and normalized to the shNT control condition. ∗∗∗: p ≤ 0.001; ∗∗: p ≤ 0.01; Kruskal-Wallis test; N = 6–10. (E–F) Two days post-transduction, cells were infected with DENV 16681s (E) or ZIKV H/PF/2013 (F) at a MOI of 0.1). 48 h later, the infectious titers of extracellular viral particles were determined by plaque assays. All values were normalized to the shNT condition. ∗∗: p ≤ 0.01; ∗: p ≤ 0.05; Kruskal-Wallis test; N = 3–6. (G and H) Primary human monocytes were transduced with lentiviruses encoding shNT, shSYNJ2BP or shRRBP1. 2 days post-transduction cells were infected with either DENV 16681s or ZIKV H/PF/2013 at an MOI of 1 in the presence of the panflaviviral anti-E antibody to enhance the infection. One day post-infection cells were collected and analyzed for their content in (G) RRBP1 and SYNJ2BP mRNAs and (H) viral RNAs using RT-qPCR. As control conditions, monocytes were treated with 10 μM of the NS5 RNA polymerase inhibitor NITD008 to demonstrate a productive replication in these cells. Mean values with standard deviations are shown.
Article Snippet: As replication control, NS5 inhibitor NITD008 (Tocris Small Molecules) was added at a final concentration of 10μM.
Techniques: Transduction, Expressing, shRNA, Control, Infection, Luciferase, Activity Assay, Quantitative RT-PCR
