A Point Mutation in the Amino Terminus of TLR7 Abolishes Signaling without Affecting Ligand Binding

Abstract

TLR7 is the mammalian receptor for ssRNA and some nucleotide-like small molecules. We have generated a mouse by N-nitrose-N′-ethyl urea mutagenesis in which threonine 68 of TLR7 was substituted with isoleucine. Cells bearing this mutant TLR7 lost the sensitivity to the small-molecule TLR7 agonist resiquimod, hence the name TLR7rsq1. In this work, we report the characterization of this mutant protein. Similar to the wild-type counterpart, TLR7rsq1 localizes to the endoplasmic reticulum and is expressed at normal levels in both primary cells and reconstituted 293T cells. In addition to small-molecule TLR7 agonists, TLR7rsq1 fails to be activated by ssRNA. Whole-transcriptome analysis demonstrates that TLR7 is the exclusive and indispensable receptor for both classes of ligands, consistent with the fact that both ligands induce highly similar transcriptional signatures in TLR7wt/wt splenocytes. Thus, TLR7rsq1 is a bona fide phenocopy of the TLR7 null mouse. Because TLR7rsq1 binds to ssRNA, our studies imply that the N-terminal portion of TLR7 triggers a yet to be identified event on TLR7. TLR7rsq1 mice might represent a valuable tool to help elucidate novel aspects of TLR7 biology.

RNA and Imidazoquinolines Are Sensed by Distinct TLR7/8 Ectodomain Sites Resulting in Functionally Disparate Signaling Events

“…Of note, the sites identified here do not appear critical for ligand binding. Earlier studies showed a general promiscuity of TLR7 for both RNA and DNA (37) and in an ORN pull-down assay we found TLR7 binding was stronger for activating vs. non-activating ORN, but not altogether abrogated (19), indicative of a basal level of (non-specific) binding for nucleic acids. Similarly to TLR9 (10, 38) RNA ORN binding by TLR7 and TLR8 thus appears to be relatively non-specific with ligand recognition being a separate event dependent on the presence of activation determinants (e.g.…”

“…Indeed rsq2 peritoneal macrophages failed to respond to R848 (Fig. 7A,B) like the ‘functionally null’ Tlr7 strain rsq1 (mutation T68I; Ref. (19)).…”

Induction of an IFN-Mediated Antiviral Response by a Self-Amplifying RNA Vaccine: Implications for Vaccine Design

“…3), the role of this receptor in RNA vaccine activity was further evaluated. We compared the cytokine pattern of human PBMCs (hPBMCs) stimulated with a TLR7 agonist, the single-stranded ribonucleotide R0006 (9), with the pattern induced with SAM (RNA) or an amplification-deficient version of SAM (DRNA). Both of the molecules were delivered with DOTAP, because this transfection reagent delivers RNA to the endosomes (26), where TLR7 is localized.…”

“…In addition, we used a mouse genetic mutant (TLR7 rsq1 ) and KO models (MyD88 and TLR3) to elucidate the importance of the TLR7 pathway. TLR7 rsq1 mice contain a point mutation within TLR7 that blocks the ability of the receptor to respond to ssRNA and smallmolecule agonists (9). MyD88 is a key signaling adapter downstream of some TLRs including TLR7.…”

“…BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). TLR7 rsq1 mice (C57BL/6 genetic background) were described previously (8,9). Null type I IFN-a/b receptor (IFNAR) knockout (KO) mice (129/SvEv genetic background) were purchased from B&K Universal.…”