Tick-borne encephalitis virus (TBEV) a member of the family is a

Tick-borne encephalitis virus (TBEV) a member of the family is a leading cause of viral encephalitis in Europe and Asia. early suppression was still observed in DCs lacking the IFN-I receptor (mRNA expression but rather diminished IRF-1 protein levels and nuclear localization. The effect on IRF-1 was also observed in DCs infected with the highly virulent Sofjin strain of TBEV. Thus antagonism of IRF-1 is a novel mechanism that synergizes with the noted ability of flaviviruses to suppress IFNAR-dependent signaling resulting in the orchestrated evasion of host innate immunity. Introduction TBEV the causative agent of tick-borne encephalitis (TBE) is a single-stranded positive-sense RNA virus belonging to the family mRNA expression. These data identify IRF-1 as a novel target of flavivirus-mediated antagonism of innate immunity. Materials and Methods Mice C57Bl/6 (WT) mice and C57BL/6-Tg (TcraTcrb)425Cbn/J (called OTII) mice that express a T-cell receptor (TCR) specific for peptide 323-339 of OVA in the context of I-Ab were purchased from The Jackson Laboratory and housed in the animal facility at Rocky Mountain Laboratories (RML). Interferon α/β receptor 1-deficient mice on a C57Bl/6 background (and either IRF-1 or guanylate-binding protein 2 (Gbp-2) firefly luciferase reporter plasmids (31). At 24 h post-transfection cells were mock-infected or infected with LGTV (MOI 3) and either left untreated Rubusoside or treated with IFNγ (100 μg/ml) at 24 hpi for 6 h. Cell extracts were prepared for measurement of luciferase activity using a Dual-Luciferase Reporter Assay System per the manufacturer’s instructions (Promega Madison WI). The reporter activity of each sample was normalized to the constitutive luciferase activity of pTK-and expressed as fold change over the luciferase activity in the mock-infected unstimulated control. Immunofluorescence assay DCs (1-2 × 105 cells per well) were plated on 8-well Lab-Tek chamber slides (Thermo Scientific Waltham MA) in complete DC medium Rubusoside and infected with LGTV for 24 h. Cells were then stimulated with LPS Rubusoside (1 μg/ml) plus IFNγ (20 ng/ml) for 3.0 h fixed with Tmeff2 4% formaldehyde/PBS and stored in PBS at 4°C. For detection of IRF-1 and viral protein slides were incubated with 100% methanol for 8 min at ?20°C followed by incubation with blocking buffer (1% bovine serum albumin [Sigma] 2 normal goat serum [Life Technologies] and 0.01M glycine/Dulbecco’s PBS) for 1-2 h at RT. Slides were stained using rabbit anti-IRF-1 antibody (clone D5E4) and mouse anti-envelope protein (clone 11H12). Bound antibodies were detected with Alexa Fluor 594-conjugated anti-rabbit IgG and Rubusoside Alexa Fluor 488-conjugated anti-mouse IgG respectively (Invitrogen). Nuclei were stained using Prolong Gold + DAPI mounting media (Invitrogen). Immunofluorescent images were obtained using a Zeiss LSM710 confocal microscope. Statistical Analyses The Student’s test was used for statistical analysis using Prism software (GraphPad Prism5 San Diego CA). Results LGTV infection inhibits DC maturation in response to TLR ligand stimulation Upon recognition of a pathogen immature DCs in the periphery up-regulate surface expression of MHCII and co-stimulatory molecules necessary for optimal development of adaptive immune responses. To investigate the effect of tick-borne flaviviruses on DC maturation DCs were infected with LGTV and analyzed by flow cytometry for expression of the Rubusoside DC maturation markers CD40 CD86 and MHCII. Intracellular staining for viral envelope protein was used to Rubusoside distinguish viral antigen positive cells (infected) from viral antigen negative cells (bystander) in the same culture. The percentage of DCs expressing LGTV envelope protein at 24 hpi was 15-25% (Fig. 1A). A similar percentage of cells positive for virus were found by intracellular staining for the nonstructural protein NS3 that is only detectable during virus replication confirming that these cells contain replicating virus (Fig. 1B). As shown in Fig. 1C LGTV-positive DCs showed negligible up-regulation of CD40 CD86 and MHCII surface expression compared to the positive control of DCs treated with.