Bortezomib decreases intracellular viral RNA.

<p>A) U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were pre-treated with 0.1μM Bortezomib or DMSO for 2 hours and then infected with TC-83 (MOI: 0.1) for 1 hour. At 0, 2, 4, 6, and 8 hours post infection cells were lysed with MagMAX-96 Total RNA Isolation Kit. As controls, cells were either pre-treated with 0.1μM Bortezomib or DMSO for 2 hours, then infected with TC-83 (MOI: 0.1) and the cells lysed at 24 hours post infection. Levels of viral RNA were quantified by q-RT-PCR using VEEV specific primers. The cycling conditions and probe primer pair sequences are described in the methods section. The graphs represent an average of 2 independent experiments where each experiment was performed in triplicate. Standard deviations were calculated accordingly. p≤0.0001 (**). U87MG cells were seeded onto #1.0 coverslips in 24-well plates at a density of 54,000 cells per well. Cells were pretreated with Bortezomib for 2 hours and then infected with TC-83 (MOI: 5) (B and C). B) After 8 hours of infection, the cells were fixed and processed for RNA FISH as described in the materials and methods section. Following FISH staining, cells were imaged using a Zeiss 700 confocal microscope with a 20X objective and at least 10 high-powered fields (HPFs) were obtained for each sample. The percentage of infected to uninfected cells was calculated per HPF for each condition. A representative 20X objective image is shown with viral RNA in red and nuclei in blue. C) After 0, 4 and 8 hours of infection, the cells were fixed and processed for RNA FISH as described in the materials and methods section. Following FISH staining, cells were imaged using a Zeiss 700 confocal microscope. A representative 63X objective image is shown with viral RNA in red and nuclei in blue. D) VERO cells were infected with TC-83 or Rift Valley Fever Virus (RVFV) (strain MP-12) for 24 hours. Cells were then processed for RNA FISH and stained with VEEV vRNA specific FISH probes as described in the materials and methods section. Following the RNA FISH staining, the VEEV infected cells were immunostained with an anti- VEEV capsid antibody and the RVFV infected cells were immunostained with an anti-RVFV nucleocapsid antibody. Nuclei were detected using DAPI. Images are shown as condensed Z-stacks. The images are representative of 2 independent experiments performed in duplicate.