PMC

(A) IFNB, IFNL1, and IL6 mRNA levels in SV40 fibroblasts from a control (C+), patients 1–6, and a TLR3−/− patient, not stimulated (NS) or stimulated for 2, 4, and 6 hours with 25 μg/mL polyinosine:polycytidylic acid [poly(I:C)]. GUS was included for normalization. (B) IFNB, IFNL1, and IL6 mRNA induction without stimulation (NS) or after 16 hours of stimulation with vesicular stomatitis virus (VSV) M51R at a multiplicity of infection (MOI) of 1. (C) IFNL1 mRNA induction without stimulation (NS), after 4 hours of stimulation with 25 μg/mL poly(I:C), or after 16 hours of stimulation with VSV M51R. Fibroblasts from patients 1, 5, and 6 were left untransfected, mock-transfected (mock), or transfected with a vector encoding hemagglutinin–tagged wild-type (WT) TLR3. (D) TLR3 mRNA levels were assessed by quantitative reverse transcription PCR. Mean values ± SD were calculated from 3 (A, B) or 2 (C, D) independent experiments.

(A) Family pedigrees with allele segregation in the 6 families. The patients are indicated in black. Healthy TLR3 wild-type relatives of patients 1 and 5 and heterozygous parents of patient 6 are shown in white. Heterozygous carriers of the patient 1 and 5 mutation and a homozygous sibling of patient 6 are indicated by bold vertical lines in each pedigree, respectively. The other family members of patients 2, 3, and 4 were not tested. (B) Schematic diagram of the human TLR3 gene, with the previously reported (blue) and new (red) mutations indicated at the corresponding location of each mutation. The coding exons are numbered with Roman numerals and delimited by a vertical bar. The regions corresponding to the leader sequence (L), leucine-rich repeats (LRR), transmembrane domain (TM), linker region (LR), and Toll/interleukin-1 receptor (TIR) domain are shaded in light gray and are delimited by dark gray lines. The 2 LRRs with an insertion are indicated by asterisks.

(A) TLR3 mRNA levels were determined by quantitative reverse transcription PCR (RT-qPCR) in P2.1 TLR3-deficient fibrosarcoma cells with or without transfection with various TLR3 alleles (WT TLR3, G743D, R811I, G743D+R811I, D592N, M374T, L360P, R867Q, E746X mutant TLR3), or a mock vector. GUS was included for normalization. (B) TLR3 expression, as assessed by immunoblotting (IB) after immunoprecipitation (IP), in P2.1 TLR3-deficient fibrosarcoma cells not transfected (P2.1) or stably transfected with wild-type (WT) or mutant TLR3, or mock vector, with an anti-TLR3 N-terminal (N) antibody and an antihemagglutinin C-terminal tag antibody. Ins12 served as an uncleavable form lacking the entire LRR12 insertion and 346Cterm served as a C-terminal cleaved fragment, as previously established and characterized.^, The experiment shown is representative of 6 experiments performed. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal expression control for immunoblotting. (C) IFNL1 mRNA induction without stimulation (NS) or after 2 and 4 hours of stimulation with 25 μg/mL polyinosine:polycytidylic acid [poly(I:C)], as assessed by RT-qPCR, in P2.1 TLR3-deficient fibrosarcoma cells not transfected (P2.1) or transfected with WT TLR3, G743D, R811I, G743D+R811I, D592N, M374T, L360P, R867Q or E746X mutant TLR3, or mock vector. The E746X mutant served as a loss-of-function control. All transfections generated stable cell lines. GAPDH was included for normalization. (D) IFNL1 mRNA induction without stimulation (NS) or after 16 hours of stimulation with vesicular stomatitis virus (VSV) M51R at a multiplicity of infection (MOI) of 1. Mean values ± SD were calculated from 2 (A, D) or 3 (C) independent experiments.

(A) The induction of mRNA for IFNB and IFNL1 was assessed by quantitative reverse transcription PCR (RT-qPCR) in the absence of stimulation (NS) or after 4 hours of stimulation with 25 μg/mL polyinosine:polycytidylic acid [poly(I:C)] in SV40 fibroblasts from a healthy control transfected with an empty vector (C-mock) or with various TLR3 alleles, and in cells from a TLR3−/− patient. (B) Production of interferon (IFN)-β and IFN-λ in the absence of stimulation (NS), after 24 hours of stimulation with 25 μg/mL poly(I:C) in the presence of lipofectamine [poly(I:C)+L] or without lipofectamine [poly(I:C)], or lipofectamine alone (L), as assessed by ELISA. (C) The production of TLR3 mRNA was assessed by RT-qPCR in SV40 fibroblasts from healthy controls stably transfected with various TLR3 alleles (wild-type [WT] TLR3, G743D+R811I, L360P mutant TLR3), or a mock vector. GUS was included for normalization. (D) Induction of mRNA for IFNB, IFNL1, and IFIT2 in the absence of stimulation (NS) or after 24 hours of stimulation with herpes simplex virus 1 (HSV-1) at an multiplicity of infection (MOI) of 1, in SV40 fibroblasts from 3 healthy controls, the patients (1, 5, and 6), a TLR3−/− patient, and a NEMO−/− patient. (E) HSV-1 replication, quantified by green fluorescent protein (GFP) measurement, in SV-40 fibroblasts from 3 healthy controls, the patients, a TLR3−/− patient, and a STAT1−/− patient, 24 hours after HSV-1 GFP infection at MOI of 0.1, 1, and 10, with (lower panel) or without (upper panel) 16 hours of pretreatment with IFN-α2b. The data shown are representative of 3 (A, D, E) or 2 (B, C) independent experiments.