Journal: Journal of Virology
Article Title: Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication
Figure Lengend Snippet: Induced S-phase arrest of SNB-19 cells enhances ZIKV, but not DENV or WNV replication. (A) Schematic of two treatment conditions used to synchronize SNB-19 cells in S phase. Cells were initially treated with 2 mM thymidine for 16 h and then were released back into the cell cycle for 12 h. Following release, the cells were treated with either 2 mM thymidine or 12 μM aphidicolin for an additional 16 h to induce S-phase arrest prior to infection. (B) Representative flow cytometry analysis of SNB-19 cells treated as indicated in panel A. Samples were stained with propidium iodide (PI) and analyzed by flow cytometry. (C, D, and F) SNB-19 cells were infected for (C) 24 h, (D) 4 to 32 h, or (F) 12 to 24 h with ZIKV PR (MOI of 0.5), ZIKV M (MOI of 0.5), or DENV (MOI of 0.5). (C) Western blot images and quantification of intracellular viral protein expression. Viral protein was detected by anti-ZIKV NS1 or anti-DENV NS3 for each respective sample. Viral protein band intensities were normalized to the DMSO control, representing the mean from three biological replicates ± SD. (D and F) Time course of relative intracellular viral RNA copies in (D) ZIKV M - or (F) DENV-infected SNB-19 cells as measured by qPCR. Error bars are the mean ± SD from three biological replicates. (E and G) Infectivity titers of culture supernatants at (E) 48 h postinfection as measured by focus-forming units (ZIKV PR and ZIKV M [MOI of 0.5]) and at 36 h postinfection as measured by PFU (DENV [MOI of 0.5]) or (G) at 12 and 24 h postinfection as measured by PFU (WNV [MOI of 0.5]) on (E) Vero cells or (F) BHK cells. Error bars are the mean ± SD from three biological replicates. In panels C to G, * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, and **** indicates P ≤ 0.0001. (C, E, and G) One-way ANOVA. (D and F) Two-way ANOVA.
Article Snippet: Total cellular RNA was purified from mock-, ZIKVM -, or DENV-infected SNB-19 cells using an RNeasy Plus kit (Qiagen) per the manufacturer’s instructions. cDNA was synthesized from purified RNA using random hexamers and a SuperScript III First-Strand kit (Invitrogen).
Techniques: Infection, Flow Cytometry, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction