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.</p

VEEV capsid protein is ubiquitinated on K48.

<p>A) U87MG cells were seeded in an 8-well chambered slide at 20,000 cells per well. The cells were uninfected (Mock) or infected with TC-83 at an MOI of 10. At 1, 2, 5 and 6 hours post infection, cells were fixed and processed as described in the materials and methods section. The cells were probed with K48 ubiquitin and capsid antibodies followed by incubation with Alexa-Fluor 488 and Alexa-Fluor 568 respectively. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U with a 60X objective and are representative of 2 replicates in an experiment. Red boxes are parts of the image that have been zoomed in and displayed in Z-stacks. B) Number of infected U87MG cells showing co-localization tabulated. C) U87MG cells were seeded in an 8-well chambered slide at 20,000 cells per well. The cells were untreated (Mock), DMSO treated or Bortezomib treated (0.1μM) for 2 hours. The cells were uninfected (Mock) or infected with TC-83 at an MOI of 10. At 2 hours post infection, cells were fixed and processed as described in the materials and methods section. The cells were probed with K48 ubiquitin and capsid antibodies followed by incubation with Alexa-Fluor 488 and Alexa-Fluor 568 respectively. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U with a 60X objective and are representative of 2 independent experiments performed in duplicate. D) Number of infected U87MG cells showing co-localization tabulated. E) U87MG cells were treated with Bortezomib (0.1μM) or DMSO for 2 hours and then infected with TC-83 (MOI: 5) for 1 hour. At 2 hours post infection cell lysates were collected, lysed and quantified. Equal amounts of total protein were immunoprecipitated with capsid antibody and resolved by SDS-PAGE and subsequently immunoblotted for K48 ubiquitin (top panel) and capsid (bottom panel). The image is representative of 2 independent experiments. Protein bands were quantified using Image J software and normalized to capsid. Average percent differences of 2 independent experiments are depicted graphically.</p

Other proteasomal inhibitors decrease TC-83 multiplication in U87MG cells.

<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 MG132 at 0.01μM, 0.1μM, or 1μM concentrations for 2 hours. Treated cells were infected with TC-83 at MOI: 0.1 for 1 hour. Supernatants were collected 24 hours post infection and infectious viral titers (PFU/mL) were assessed by plaque assay. B) U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were treated with either DMSO or varying concentrations of MG132. Cell viability was determined 24 hours post-treatment using Cell-Titer-Glo Reagent as per manufacturer’s instructions. The graphs are representative of 3 independent experiments, each performed in triplicate. Standard deviations were calculated from 3 independent experiments and are represented thusly. C) U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were infected in triplicate with TC-83 at MOI: 0.1 for 1 hour. Infected cells were treated with 1μM of MG132 at 2 hour intervals from 0–8 hours post infection. As controls infected cells were treated with DMSO. Supernatants were collected 24 hours post infection and analyzed by plaque assay for infectious viral particles (PFU/mL). The arithmetic means are illustrated graphically. The graph is representative of 2 independent experiments performed in triplicate. Standard deviations were calculated accordingly. p<0.05(*).</p

Inhibition of wild type alphaviruses by Bortezomib.

<p>U87MG cells were pre-treated with Bortezomib at 0.1μM for 2 hours and then infected with VEEV TrD, EEEV strain GA97, and WEEV California 1930 strain at an MOI: 0.1 for 1 hour. Supernatants were collected 24 hours post infection and analyzed by plaque assay for infectious viral particles (PFU/mL). The arithmetic means are illustrated graphically. The graph is representative of 3 independent experiments where each experiment was performed in triplicate. Standard deviations were calculated accordingly. p<0.05(*).</p

Bortezomib inhibition is independent of viral load.

<p>U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were pre-treated with Bortezomib (0.1μM) or DMSO in triplicate for 2 hours. Treated cells were infected with TC-83 at MOI: 0.1 (A) MOI: 1 (B) or MOI: 5 (C). The viral inoculum was removed and replaced with conditioned media. At 6, 12, 18 and 24 hours post infection supernatants were collected and analyzed by plaque assay. The graph is representative of 2 independent experiments performed in triplicate. Standard deviations were calculated accordingly. p<0.05(*). p≤0.0001 (**). U87MG cells were seeded in a 12-well plate at a density of 100,000 cells per well. Cells were pre-treated with Bortezomib (0.1μM) or DMSO for 2 hours. Treated cells were infected with TC-83 at MOIs (0.1, 1 or 5) (right panels of A, B and C respectively). The viral inoculum was removed and replaced with conditioned media. At 6, 12, 18 and 24 hours post infection cell lysates were collected and analyzed by western blot. The images are representative of 2 independent experiments.</p

Ubiquitination of a viral protein.

<p>A) U87MG cells were transfected with HA-Ub for 24 hours and then infected with TC-83 (MOI: 5) for 1 hour. At 6 hours post infection, cell lysates were collected and quantified for protein concentration. Two milligrams of total protein was immunoprecipitated with HA probe antibody and mouse IgG as a control. LC/MS-MS was performed and the results tabulated. B) U87MG cells were seeded in an 8-well chambered slide at 20,000 cells per well. The cells were uninfected (Mock) or infected with TC-83 at an MOI of 10. At 2 hours post transfection, cells were fixed and processed as described in the materials and methods section. The cells were probed with ubiquitin and capsid antibodies followed by incubation with Alexa-Fluor 488 and Alexa-Fluor 568 respectively. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U with a 60X objective and are representative of 2 independent experiments performed in duplicate. C) U87MG cells were infected with TC-83 (MOI: 5) for 1 hour. At 6 hours post infection, cell lysates were collected and quantified for protein concentration. Two milligrams of total protein was immunoprecipitated with ubiquitin antibody and an isotype IgG as a control. Immunoprecipitated samples were resolved by SDS-PAGE and subsequently immunoblotted for capsid (top panel) and ubiquitin (bottom panel). The image is representative of 3 independent experiments.</p

Bortezomib inhibits TC-83 multiplication in U87MG cells.

<p>A) U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were either pre-treated with DMSO or 0.01μM, 0.1μM, or 1μM of Bortezomib in triplicate for 2 hours. The conditioned media were removed prior to infection with TC-83 (MOI: 0.1) for 1 hour. The viral inoculum was removed and replaced with conditioned media. Supernatants were collected 24 hours post infection and stored at -80°C until analyzed by plaque assays to determine infectious viral titers as PFU/mL. B) U87MG cells were seeded in a white walled 96-well plate at a density of 10,000 cells per well. Cells were either pre-treated with DMSO or 0.01μM, 0.1μM, or 1μM of Bortezomib in triplicate for 24 hours. Cell viability was measured using a Cell-Titer-Glo luminescent cell viability kit whereby the reagent was added to the cells in a 1:1 ratio. The plate was shaken for 2 minutes and incubated for 10 minutes at room temperature. Luminescence was detected using the DTX 880 multimode detector (Beckman Coulter). The arithmetic means are illustrated graphically. The graphs are representative of 2 independent experiments performed in triplicate. Standard deviations were calculated from 2 independent experiments. p<0.05(*).</p

Ubiquitinated capsid in virions.

<p>VEEV-containing supernatants obtained from infected VERO cells were subjected to sucrose density centrifugation as described in the materials and methods section. A) The collected fractions were analyzed by plaque assay and are represented graphically. Fraction 3 with the highest titer of virus was used for immunoprecipitation with capsid antibody and an isotype IgG as a control. Immunoprecipitated samples were resolved by SDS-PAGE and subsequently immunoblotted for ubiquitin (top panel) and capsid (bottom panel). The image is representative of duplicate immunoprecipitation runs.</p

IKKβ inhibitors decrease TC-83 replication in rat AP7 neuronal cells.

<p>A) Neurons were pre-treated with DMSO or with IKK inhibitors (1 µM) for 2 hours and 24 hours later cell viability was measured using the Cell-Titer-Glo Luminescent Cell Viability Assay. B) Neurons were pretreated with IKK inhibitors (1 µM), BAY-11-7082, BAY-11-7085 and IKK2-IV for 2 hours. Following the pretreatment, the conditioned media (media containing inhibitor) was removed and the cells infected at MOI: 1 for 1 hour. The viral inoculum was removed and the conditioned media replaced. Supernatants were collected 24 hours post-infection, and infectious viral titers were determined by plaque assay. C) Neurons were pretreated with IKK inhibitors (1 µM) for 2 hours and infected with TC-83 for 1 hour. Conditioned media was replaced after removal of the viral inoculum. Cell viability assay was performed 48 hours later using the Cell-Titer-Glo Luminescent Cell Viability Assay. The red line is representative of the base line for luminescence units, such that luminescence units above this are indicative of increased cell viability. The graphs are representative of 2 independent experiments. Error bars for the independent experiments were calculated and are represented thusly. *** p≤0.005, ** p≤0.01 and * p≤0.05.</p

VEEV nsP3 interaction with host IKKβ.

<p>A) IKKβ was immunoprecipitated from VEEV infected U87MGs and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) was performed. Control immunoprecipitations were performed with an IgG antibody. Mass spectrometry analysis revealed that the viral nonstructural protein nsP3 interacted with IKKβ. B) U87MGs were transfected in a 6-well plate with 5 µg of pcDNA3.1 and VEEV_nsP3_HA for 24 and 48 hours. Cell lysates were resolved using SDS-PAGE and subsequently immunoblotted with HA and β-actin served as a loading control. C) U87MGs were transfected in duplicate with (0.2 µg) VEEV_nsP3_HA and pcDNA3.1 (control), cells were fixed after 24 hours and probed with antibodies against the endogenous IKKβ and the HA tag. Cells were subsequently incubated with appropriate secondary Alexa Fluor antibodies and the nuclei stained with DAPI. Co-localization of IKKβ with nsP3 (yellow) was observed as shown by the arrows. The co-localization was confirmed by Z-stack analysis. Panels E–H and J–M serve as examples of transfected cells in a given field of view that show co-localization of IKKβ and VEEV_nsP3_HA at 24 hours post transfection. Panels I and N are magnified images of the outlined cells in red boxes in panels H and M respectively. Co-localization was found to be approximately in 71% of cells (72 cells were counted of which 55% demonstrated expression of nsP3. Of those cells that expressed nsP3, 71% showed co-localization of both IKKβ and VEEV_nsP3_HA proteins). D) U87MGs were transfected in duplicate with (0.2 µg) VEEV_nsP3_HA and pcDNA3.1 (control); cells were treated with BAY-11-7082 (1 µM and 0.1 µM). The cells were fixed 24 hours post transfection and probed with antibodies against the endogenous IKKβ and the HA tag. Cells were subsequently incubated with appropriate secondary Alexa Fluor antibodies and the nuclei stained with DAPI. Co-localization of IKKβ with nsP3 (yellow) was observed as shown by the arrows. The co-localization was confirmed by Z-stack analysis. Panels E–H and I–L serve as examples of transfected cells in a given field of view that show co-localization of IKKβ and VEEV_nsP3_HA at 24 hours post transfection. Panel M is a magnified image of the outlined cells in red boxes in panel L. Panels N–Q and S–V are examples of transfected and treated cells in a given field of view. Panels R and W are magnified images of the outlined cells in red boxes in panels Q and V respectively. Images were taken using Nikon Eclipse TE2000-U at 60× magnification and are representative of 3 independent experiments.</p