IKK?/? MEFs and WT MEFs were maintained in DMEM supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin and 1% L-Glutamine at 37C, 5% CO2. in formation of lower molecular weight complexes. Well-documented inhibitors of IKK function, BAY-11-7082, BAY-11-7085 and IKK2 compound IV, were employed to determine whether IKK function was required for the production of infectious progeny virus. A decrease in infectious viral particles and viral RNA copies was observed with inhibitor treatment in the attenuated and virulent strains of VEEV infection. In order to further validate the requirement of IKK for VEEV replication, we over-expressed IKK in cells and observed an increase in viral titers. In contrast, studies carried out using IKK?/? cells demonstrated a decrease in VEEV replication. studies demonstrated that inhibitor treatment of TC-83 infected mice increased their survival. Finally, proteomics studies have revealed that IKK may interact with the Prasugrel Hydrochloride viral protein nsP3. In conclusion, our studies have revealed that the host IKK protein may be critically involved in VEEV replication. Introduction The New World alphavirus VEEV belongs to the family and and is a BSL-2 model for the fully virulent BSL-3 VEEV TrD. Experiments with TC-83 were performed under BSL2 settings and those with the wild type viruses Prasugrel Hydrochloride were conducted under BSL3 requirements. Wild type Eastern Equine Encephalitis Virus (EEEV) GA97 was obtained from Dr. Tnc Jonathan Jacobs (MRIGlobal) and wild type Western Equine Encephalitis Virus (WEEV) (California 1930 strain) was obtained from ATCC. All select agents used in the manuscript are registered with the Centers for Disease Control and Prevention and conducted at George Mason University’s Biomedical Research Laboratory, which is registered in accordance with Federal select agent regulations. As a control virus TC-83 strain was inactivated by exposure to ultraviolet radiation and termed UV-TC-83. 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 Joules X 100 per dose five times with a 2 minute interval between dosing. Human astrocytoma cells (U87MG cells) and African Green Monkey kidney epithelial cells (Vero cells) were maintained in DMEM supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin and 1% L-Glutamine at 37C, 5% CO2. Inhibitory B kinase knockout (IKK?/?) and wild type mouse embryonic fibroblast (WT MEFs) cells were a kind gift from Dr. Cynthia Masison from NIH/NCI [25], [26]. IKK?/? MEFs and WT MEFs were maintained in DMEM supplemented with 10% Fetal Bovine Serum (FBS), 1% Prasugrel Hydrochloride Penicillin/Streptomycin and 1% L-Glutamine at 37C, 5% CO2. Rat AP7 neuronal cells (a gift from Dr. Diann Griffin) were cycled at 33C with 7% CO2 in DMEM supplemented with 10% FBS, 1% Penicillin/Streptomycin and 1% L-Glutamine. For differentiating the AP7 neuronal cells, the cycling media was modified with the addition of 1 g/mL insulin, 20 M dopamine and 100 M ascorbic acid. The cells were then incubated at 39C in 5% CO2 for 5 to 7 days for complete differentiation. Viral Infections Cells were seeded in a 96-well plate such that confluency was attained the next day. The media was removed and saved and was referred to as conditioned media. The cells were infected for 1 hour to allow for viral adsorption at 37C. The viral inoculum was removed and replaced with the conditioned media. The cells were incubated at 37C, 5% CO2. The supernatant was collected 24 hours later and stored at ?80C until analyzed. Inhibitor Studies Cells were seeded in a 96-well plate at a density of 10,000 cells per well. The next day the cells were pretreated with inhibitors, BAY-11-7082 (Sigma, Catalogue No. B5556), BAY-11-7085 (Sigma, Catalogue No. B5681), IKK2 compound IV (Santa Cruz Biotechnology, Catalogue No. sc-203083), 5,7-dihydroxy-4-methylcoumarin (DMC) (Santa Cruz Biotechnology, Catalogue No. sc-254863), pathology associated with VEEV infection. Therefore we investigated if infection with the live-attenuated strain of VEEV, TC-83 would result in activation of the NF-B signaling cascade. Phosphorylation of IB on serine 32/36, p65 on serine 536 and p65 nuclear enrichment were used as markers of cascade activation. As a control, a UV-inactivated form of TC-83, termed UV-TC-83, was used. Inactivation of the UV-TC-83 virus was validated by plaque assays. As can be seen in Figure 1A, no plaques could be detected with the UV.