Activation of NF-κB complex in TC-83 infected cells.

<p>A) UV inactivation of the virus was carried out using a Stratalinker UV crosslinker (model 1800). The inactivation was achieved by delivering an energy dose equivalent to 1200 µJoules X 100 per dose five times with a 2 minute interval between dosing. UV-TC-83 and TC-83 were serially diluted and used to infect Vero cells. UV-TC-83 inactivation was confirmed by plaque assay. Plaques were photographed and counted 48 hours post-infection. Plaque counts are represented graphically. B) U87MG cells were either mock infected, treated with LPS (1 µg/mL) or infected with TC-83 or UV-TC-83 (MOI: 1). At 30 minutes, 1 and 2 hours post-infection cells were lysed and protein extracts were resolved by SDS-PAGE and subsequently immunoblotted with antibodies against phosphorylated p65 and phosphorylated IκBα. Total p65, total IκBα and β-actin served as controls. The western blot is representative of 2 independent experiments. C) U87MGs were either mock infected, treated with LPS (1 µg/mL) or UV-TC-83 or TC-83 infected (MOI: 3). One hour post-infection cells were fixed, probed with p65 antibody followed by incubation with Alexa-Fluor 568. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U at 60× magnification and are representative of 2 independent experiments. ND = not detectable.</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

IKKβ inhibitors are effective in decreasing viral load of wild type VEEV.

<p>A) U87MG cells (A) or neuronal rat AP7 cells (B) were pretreated with 1 µM IKK inhibitors, BAY-11-7082, BAY-11-7085 and IKK2-IV for 2 hours. The cells were infected with the wild type strain of VEEV (TrD) at a MOI: 0.1 (A) or MOI: 1 (B) for 1 hour. The conditioned media (media containing inhibitor) was removed prior to the viral infections and replaced after the viral inoculum was removed. The cells were incubated for an additional 24 hours. The supernatants were collected from all samples and viral titers were determined by plaque assay. The graphs are representative of 2 independent experiments. Error bars (Standard deviations) for 3 replicates within the 2 independent experiments were calculated and are represented thusly. ** p≤0.01.</p

Macromolecular reorganization of the IKKβ complex in TC-83 infected cells.

<p>U87MG cells were infected with TC-83 or UV-TC-83 (MOI: 5) or the cells were treated with Poly I∶C (10 µg/mL) or Imiquimod (2 µg/mL). Cells were lysed 24 hours post-infection and post treatment and protein extracts were quantified. Two milligrams of total protein was subjected to chromatography using a Superose 6 HR 10/30 size-exclusion chromatography column and an AKTA purifier system. Every 5th fraction was acetone precipitated using 4 volumes of ice-cold 100% acetone and incubated for 15 minutes on ice. Lysates were centrifuged at 4°C for 10 minutes at 12,000 rpm, supernatants were removed, and the pellets were allowed to dry for a few minutes at room temperature. The pellets were resuspended in Laemmli buffer and analyzed by immunoblotting using IKKα (A), IKKβ (B), IKKγ (C) and β-actin (D) antibodies. Every fifth fraction ranging from 2.2 mDa to 100 kDa is represented on the western blot. Smaller IKKβ complexes (highlighted in the red box) in the higher fractions suggests a rearrangement of the IKKβ complex in TC-83 infected cells.</p

BAY-11-7082 decreases replication of wild type alphaviruses.

<p>U87MGs were pre-treated with DMSO or BAY-11-7082 (1 µM) for 2 hours. The conditioned media (media containing inhibitors) was removed and the cells infected with VEEV TrD, EEEV GA97 and WEEV (California 1930 strain) for 1 hour at an MOI of 0.1. The conditioned media was replaced and the supernatants collected 24 hours later. Plaque assays were performed to determine viral titers. The graph is the average of n = 3 from a single experiment. Error bars for 3 replicates were calculated and are represented thusly.</p

IKKβ inhibitors decrease viral load in TC-83 infected cells pre and post exposure.

<p>A) U87MGs were untreated, DMSO treated or pretreated 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) U87MG cells were pretreated with IKK inhibitors (1 µM), BAY-11-7082, BAY-11-7085 and IKK2-IV and non-IKK specific inhibitors (1 µM), O-Phe and DMC for 2 hours. The conditioned media (media containing inhibitor) was removed and viral infections proceeded (MOI: 0.1) for 1 hour. The viral inoculum was removed and replaced with the conditioned media. The cells were incubated for an additional 24 hours. The supernatants were collected from infected and inhibitor treated cells. Infectious viral titers were determined by plaque assay. O-phe and DMC served as positive control inhibitors. C) Supernatants from B) were subjected to q-RT-PCR analysis to determine viral RNA copies using VEEV specific primers. D) U87MGs were infected at MOI: 0.1 for 1 hour and then treated with BAY-11-7082 (1 µM), BAY-11-7085 (1 µM) and IKK2-IV (1 µM) 4 hours post-infection. Supernatants were collected 24 hours post-infection and viral titers were determined by plaque assay. E) U87MGs were pretreated with IKK inhibitors (1 µM) and non-IKK inhibitors (1 µM) for 2 hours and followed by a 1 hour infection. Conditioned media was replaced and cell viability assay was performed 72 hours later using the Cell-Titer-Glo Luminescent Cell Viability Assay. The red line is representative of the base line for luminescence units, where luminescence units above this value are indicative of increased cell viability. F) U87MGs seeded in an 8-well chambered glass slide were either pre-treated with DMSO or BAY-11-7082 (1 µM) for 2 hours and then infected with UV-TC-83 or TC-83 (MOI: 0.1) for 1 hour. The conditioned media was replaced after the infection. One hour post-infection the cells were fixed and probed for p65 with subsequent incubation with Alexa Fluor 568. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U at 60× magnification and are representative of 2 replicate samples within the same experiment. The graphs represent an average of 3 independent experiments. Error bars for the 3 independent experiments were calculated and are represented thusly. **** p≤0.0001, *** p≤0.005, ** p≤0.01 and * p≤0.05.</p