3D, a Novel Mutation That Confers Defective Sensing by Toll-Like Receptors 3, 7 and 9.

The mammalian Toll-like receptors (TLRs) comprise a key interface between cells of the host and all classes of microbial pathogen. By sensing nucleic acids, the endosomal TLRs 3, 7, and 9 play a particularly important role in the detection of viral infection, and permit the host to mount an immediate and efficacious anti-viral response. In an effort to identify novel components of the TLR signaling apparatus, we have pursued a program of germline saturation mutagenesis with N-ethyl-N-nitrosourea (ENU) in mice. The germline mutants are produced on the C57BL/6 background, and peritoneal macrophages from individual animals are screened for their competence to respond to TLR-dependent microbial inducers. A strong phenodeviant called 3d was identified in the F3 generation as a non-responder to nucleoside-based molecules such as unmethylated CpG oligodeoxynucleotides, Resiquimod (a drug of the imidazoquinoline class) and Poly I:C. TNFa production induced by TLRs 3, 7, and 9 was completely prevented in 3d homozygotes. However, heterozygotes were unaffected, and the ability to produce TNFa in response to other bacterial compounds, sensed by TLRs 1, 2, 4, and 6, was intact. 3d homozygotes show extreme susceptibility to infection by mouse cytomegalovirus (MCMV) infection in vivo. After IP inoculation with MCMV (10⁴ PFU), 3d mutants showed impaired production of type I and type II interferons, TNF, and IL-12 in serum (p<0.001). With larger inocula (5 x 10⁵ PFU) a splenic viral titer approximately ten-thousand fold higher than that in controls (p<0.05) and rapid mortality (p<0.0001) were observed. This novel recessive phenotype is fully penetrant on the C57BL/6 and C3H/HeN x C57BL/6 hybrid backgrounds. The 3d mutation was mapped to mouse chromosome 19 using a panel of microsatellite markers and at present, has been confined to 0.4 a Mbp critical region on 3000 meioses. 3d appears to encode a protein essential for TLR-mediated detection of nucleic acids, which is indispensable for effective innate defense against viral infection.