Fig. 3.

DGP blocks ZIKV MR766 during fusion or at a pre-fusion step. (A) Detection of ZIKV RNA during infection by in situ hybridization. VERO cells were infected with ZIKV at an MOI of 0.5 in the presence of DGP. At 48 h post-challenge, cells were fixed/permeabilized and stained for the detection of positive single-stranded RNA ZIKV molecules (green). Cell nuclei were counterstained using DAPI (blue). Images were obtained using a Leica TCP SP8 inverted confocal fluorescence microscope using the 63×/1.4 oil-immersion objective. (B) The percentage of cells containing ZIKV-positive single-strand RNA (green) was determined by counting 400 DAPI-positive (blue) cells. The fraction of infected cells per treatment is shown. (C) ZIKV RNA levels were measured using qRT-PCR. HT1080 and VERO cells were challenged by ZIKV MR766 at an MOI of 1 in the presence of DGP. At 48 h post-challenge, cells were lysed and total RNA was extracted using trizol. Total RNA was used to determine the levels of ZIKV RNA by qRT-PCR using specific primers against ZIKA. ZIKA viral RNA levels were normalized to actin (upper panels). In parallel, similar infections were used to determine infectivity via flow cytometry using anti-4G2 antibodies (lower panels). Experiments were performed at least three times, and results of a representative experiment are shown. (D) Kinetics of ZIKV entry. ZIKV-RVPs were pre-bound to HT1080 cells at 4 °C for 1 h. Infection was initiated by raising the temperature to 37 °C. At the indicated time points, cells were treated with 1 μM of DGP, 1 μM of Nanchangmycin, or 20 mM ammonium chloride(NH₄Cl). 48 h post-challenge, infection was determined by measuring the percentage of GFP-positive cells. Experiments were repeated at least three times and results of a representative experiment are shown. NT, not treated. (E) CHME3 cells were infected with ZIKV MR766 at an MOI of 1 in the presence of the indicated concentrations of DGP. At 48 h post-challenge, the levels of IFN-β were measured using qRT-PCR. The levels of IFN-β were normalized to actin (upper panel). In parallel, ZIKV infection was measured by determining the percentage of infected cells using anti-4G2 antibodies (lower panel). Experiments were repeated at least three times and results of a representative experiment are shown. (F) CHME3 cells were infected with Sendai virus (SeV) at an MOI of ~2.5, ~5 or ~10 in the presence of 1 μM DGP. At 48 h post-infection, the levels of IFN-β were quantified by qRT-PCR and normalized to actin (upper panel). In parallel, SeV infection was measured by qRT-PCR using specific primers against SeV (lower panel). Experiments were repeated at least three times and results of a representative experiment are shown. Bars represent the Mean ± SD. P < .05 (*), P < .01 (**), P < .001 (***), or not significant (ns), using two-tailed Student's t-test are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)