(A) Family pedigree of kindred A with allele segregation of the mutation. The HSE patient is shaded in black. Roman numerals (left margin) indicate generations. An arrow indicates the proband. (B) Automated sequencing profiles for the TRIF C421>T mutation in gDNA isolated from leukocytes from a healthy unrelated control; the patient, P1; and the father, I.2. The arrow indicates the position of the mutation. (C) A schematic representation of the TRIF protein (1–712 amino acids) indicating the amino acid position, R141X, affected by the C421>T mutation. Proline-rich domains (pro rich) are shaded in gray; functional domains are shaded in black (TRAF6 binding, TIR, RIP homotypic interacting motif [RHIM]). (D) TRIF protein expression by immunoblot analysis of SV40 fibroblast cell lysates from a healthy control (C+) and P1. Samples were migrated on the same blot. TRIF expression levels were quantified by densitometry results normalized with respect to ACTIN levels and expressed as relative intensity of TRIF. This is a representative blot from 3 independent experiments (mean ± SEM). (E) RT-PCR of full-length TRIF cDNA is shown with ACTB cDNA as an internal control. TRIF cDNA levels were assessed by real-time PCR in control fibroblasts (C+) and P1. Data are represented as relative fold change (ΔΔCt units), where GUS was used for normalization. An average of 3 independent experiments is represented (mean ± SEM).

(A) Family pedigree of kindred B with allele segregation of the mutation. The HSE patient is shaded in black. Roman numerals (left margin) indicate generations. An arrow indicates the proband, and “E?” indicates an undetermined genotype. (B) Automated sequencing profiles for the TRIF C557>T mutation in gDNA isolated from leukocytes from a healthy unrelated control and in the patient, P2. The arrow indicates the position of the mutation. (C) A representation of the TRIF protein (1–712 amino acids) indicating the amino acid position, S186L, affected by the C557>T mutation. Proline-rich domains are shaded in gray; functional domains are shaded in black (TRAF6 binding, TIR, and RHIM). (D) Multiple alignment of TRIF amino acid sequence surrounding the mutation S186L. (E) TRIF protein expression by immunoblot analysis of SV40 fibroblast cell lysates from a healthy control (C+) and P2. TRIF expression levels were quantified by densitometry results normalized with respect to ACTIN levels and expressed as relative intensity of TRIF. This is a representative blot from 3 independent experiments (mean ± SEM). (F) TRIF cDNA levels were assessed by real-time PCR in control fibroblasts and P2. Data are represented as relative fold change (ΔΔCt units), where GUS was used for normalization. An average of 3 independent experiments is represented (mean ± SEM).

(A) SV40 fibroblasts were stimulated with increasing doses of poly(I:C) for 24 hours, and production of cytokines was assessed. C+ is a healthy control; UNC93B1^–/– served as a negative control. Values (mean ± SEM) were calculated from 3 independent experiments. (B) Native gel Western blot showing IRF3 dimers from total fibroblast cell lysates after stimulation with poly(I:C) for 1 or 2 hours. C+ and MYD88^–/– cells were used as positive controls to poly(I:C) stimulation, and UNC93B1^–/– was used as a negative control. (C) Nuclear protein extracts from SV40 fibroblasts stimulated for 30 minutes with IL-1β or TNF-α and 120 minutes with poly(I:C) were tested for the presence of the p65 subunit of NF-κB by ELISA. NEMO^–/– cells were used as a negative control for all stimuli; MyD88^–/– was used as a negative control for IL-1β; and UNC93B1^–/– was used for poly(I:C) stimulation. Values (mean ± SEM) were calculated from 3 independent experiments. NS, nonstimulated. (D) SV40 fibroblasts were stimulated with a TLR3-specific ligand, poly(A:U), transfected poly(I:C), RIG-I–specific ligand (7sk-as), or lipofectamine alone (L) for 24 hours and tested for cytokine production. Values (mean ± SEM) were calculated from 3 independent experiments. (E) PBMCs from healthy controls, various family members from kindred A (AII.1 and AII.3) and B (BII.3), and P1 and P2 were stimulated with LPS for 2 hours. Induction of IFNB1 and IL6 mRNA was assessed by real-time PCR. Values are expressed as relative fold change using the ΔΔCt method, where GUS was used for normalization; values (mean ± SEM) from 3 independent experiments were calculated.

(A) IFN-β, IFN-λ1/3, and IL-6 production in SV40 fibroblasts 24 hours after infection with VSV. Values (mean ± SEM) were calculated from 3 independent experiments. (B) Cell viability of SV40 fibroblasts after 24 hours of infection with VSV in the absence (left) or presence (right) of recombinant IFN-α. The percentage of surviving cells was assessed using resazurin. C+ is a healthy control; UNC93B1^–/– and STAT1^–/– cells were used as negative controls. Values (mean ± SEM) were calculated from 3 independent experiments. (C) VSV replication in SV40 fibroblasts was estimated at various time points after infection using an MOI of 10. Cells were pretreated with IFN-α or media alone. VSV titer estimation was carried out on Vero cells. Values (mean ± SEM) were calculated from 3 independent experiments. (D) IFN-β, IFN-λ1/3, and IL-6 production after infection with 1 MOI of HSV-1 in SV40 fibroblasts after 24 hours. Values (mean ± SEM) were calculated from 3 independent experiments. (E) Cell viability of SV40 fibroblasts was assessed using resazurin after 24 hours of HSV-1 infection. C+ is a healthy control; UNC93B1^–/– and STAT1^–/– cells were used as negative controls. Cells were pretreated with media (left) or recombinant IFN-α (right). Values (mean ± SEM) were calculated from 3 independent experiments. (F) Replication of HSV-1 GFP was assessed in SV40 fibroblasts after 24 hours of infection. Cells were pretreated with media (left) or recombinant IFN-α (right). Values (mean ± SEM) were calculated from 3 independent experiments.

(A) 293 HEK-TLR3 cells were transfected with 10 ng empty vector, WT, R141X, or ΔNC TRIF along with IFN-β–Luc/NF-κB–Luc and RL-TK vectors to assess induction upon overexpression of TRIF. ΔNC TRIF serves as a dominant-negative construct of TRIF, containg only the TIR domain. Transfected cells were left unstimulated or stimulated with 50 μg/ml poly(I:C) for 4 hours. Firefly luciferase values were normalized using renilla values. Total transfected DNA was held constant by adding empty vector. Values (mean ± SEM) were calculated from 3 independent experiments. (B) Immunoblot analysis of protein lysates from 293HEK-TLR3–transfected cells, using an antibody recognizing the N-terminal of TRIF. CFP was cotransfected as a control for transfection efficiency. Samples were migrated on the same blot. (C) 293HEK-TLR4-MD2-CD14 cells were transfected with 10 ng TRIF vectors, including WT, R141X, and ΔNC TRIF, and the induction of IFN-β–Luc/NF-κB–Luc was assessed by luciferase. Values (mean ± SEM) were calculated from 3 independent experiments. (D) Control (C+) SV40 fibroblasts and P1’s fibroblasts retrovirally transduced with empty vector, WT TRIF, or R141X TRIF were stimulated with 25 μg/ml poly(I:C) for 24 hours, and production of cytokines was assessed by ELISA. Values (mean ± SEM) were calculated from 3 independent experiments. (E) HSV-1 GFP replication was assessed after 24 hours of infection in control SV40 fibroblasts (C+) and SV40 fibroblasts from P1 retrovirally transduced with empty vector, WT, or R141X TRIF. Cells were pretreated with media (left) or recombinant IFN-α (right).Values (mean ± SEM) were calculated from 3 independent experiments.

(A) 293HEK-TLR3 cells were transfected with empty vector, WT, or S186L TRIF plasmids along with IFN-β–Luc/NF-κB–Luc and RL-TK vectors to assess IFN-β and NF-κB promoter induction upon overexpression of TRIF. Values (mean ± SEM) were calculated from at least 3 independent experiments. (B) SV40 fibroblasts from control (C+) and P1’s fibroblasts retrovirally transduced with either empty vector, WT TRIF, or S186L TRIF were stimulated with 25 μg/ml poly(I:C) for 24 hours, and the production of cytokines was assessed by ELISA. Values (mean ± SEM) were calculated from at least 3 independent experiments. (C) SV40 fibroblasts from control (C+) and P2’s fibroblasts retrovirally transduced with empty vector, WT TRIF, or S186L TRIF were stimulated with 25 μg/ml poly(I:C) for 24 hours, and the production of cytokines was assessed by ELISA. Values (mean ± SEM) were calculated from at least 3 independent experiments. (D) Replication of HSV-1 GFP was assessed after 24 hours of infection in control SV40 fibroblasts (C+); SV40 fibroblasts from P1 retrovirally transduced with empty vector, WT, or S186L TRIF; and (E) SV40 fibroblasts from P2 retrovirally transduced with empty vector, WT, or S186L. Cells were pretreated with media (left) or recombinant IFN-α (right). Values (mean ± SEM) were calculated from at least 3 independent experiments.