Chemical intervention of influenza virus mRNA nuclear export
Fig 2
Schematic representation of a high-throughput screen to identify chemical inhibitors of viral M mRNA processing and nuclear export.
Screen was performed using a chemical library of 232,500 compounds in A549 cells. Cells were incubated with compounds for 30 min and, for robust imaging analysis, ~ 100% of the cells were infected with influenza virus (WSN), at MOI 2 for 7.5h. Viral M mRNA was detected by smRNA-FISH and images were systematically taken in a high throughput microscope (IN Cell Analyzer 6000). Samples on 384-well black clear-bottom plates were imaged at 20X magnification using the Hoechst and dsRed filters. 4 fields of view per well were collected for each channel. The distribution of fluorescent signal between the nucleus (N) and the cytoplasm (N/C ratio) as well as total cell signal intensity were quantified using GE IN Cell Analyzer Workstation (version 3.7.3) and Pipeline Pilot (version 9.5; Biovia). Data was imported into the GeneData’s Screener™ software analysis suite for quality control to ensure that data quality is high for all plates in each experimental run (Z’ > 0.4).