Phenotypic Mutation 'Rakshasa' (pdf version)
AlleleRakshasa
Mutation Type missense
Chromosome8
Coordinate45,850,734 bp (GRCm39)
Base Change A ⇒ G (forward strand)
Gene Tlr3
Gene Name toll-like receptor 3
Chromosomal Location 45,848,702-45,864,112 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The protein encoded by this gene is a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity. TLRs are highly conserved from Drosophila to humans and share structural and functional similarities. They recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, and mediate the production of cytokines necessary for the development of effective immunity. The various TLRs exhibit different patterns of expression. This receptor is most abundantly expressed in placenta and pancreas, and is restricted to the dendritic subpopulation of the leukocytes. It recognizes dsRNA associated with viral infection, and induces the activation of NF-kappaB and the production of type I interferons. It may thus play a role in host defense against viruses. Use of alternative polyadenylation sites to generate different length transcripts has been noted for this gene. [provided by RefSeq, Jul 2008]
PHENOTYPE: Homozygotes for a null allele show alterations in innate immunity against different viruses, viral pathogenesis, anxiety, hippocampal synaptic plasticity, memory retention and neurogenesis. Homozygotes for another null allele show altered ds-RNA responses in dendritic and aorta smooth muscle cells. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_126166; MGI:2156367

MappedYes 
Amino Acid Change Valine changed to Alanine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000034056] [ENSMUSP00000126556] [ENSMUSP00000147738] [ENSMUSP00000147783]
AlphaFold Q99MB1
PDB Structure Crystal structure of mouse TLR3 ectodomain [X-RAY DIFFRACTION]
Mouse Toll-like receptor 3 ectodomain complexed with double-stranded RNA [X-RAY DIFFRACTION]
SMART Domains Protein: ENSMUSP00000034056
Gene: ENSMUSG00000031639
AA Change: V721A

DomainStartEndE-ValueType
LRRNT 28 56 1.14e1 SMART
LRR 50 74 1.33e1 SMART
LRR_TYP 99 122 4.72e-2 SMART
LRR 123 146 2.47e2 SMART
LRR 171 194 3.36e1 SMART
LRR 198 220 7.57e0 SMART
low complexity region 224 238 N/A INTRINSIC
low complexity region 252 263 N/A INTRINSIC
LRR 274 297 1.06e1 SMART
LRR_TYP 298 321 1.28e-3 SMART
LRR 355 378 6.23e1 SMART
LRR 379 404 3.18e2 SMART
LRR 405 430 8.98e1 SMART
LRR 431 455 6.78e1 SMART
LRR_TYP 506 529 1.79e-2 SMART
LRR 530 553 2.63e0 SMART
LRR_TYP 562 585 1.56e-2 SMART
LRR 586 609 1.37e1 SMART
LRR 611 633 8.48e0 SMART
LRRCT 646 698 1.07e-10 SMART
transmembrane domain 705 724 N/A INTRINSIC
TIR 756 901 2.43e-26 SMART
Predicted Effect probably benign

PolyPhen 2 Score 0.084 (Sensitivity: 0.93; Specificity: 0.85)
(Using ENSMUST00000034056)
SMART Domains Protein: ENSMUSP00000126556
Gene: ENSMUSG00000031639
AA Change: V721A

DomainStartEndE-ValueType
LRRNT 28 56 1.14e1 SMART
LRR 50 74 1.33e1 SMART
LRR_TYP 99 122 4.72e-2 SMART
LRR 123 146 2.47e2 SMART
LRR 171 194 3.36e1 SMART
LRR 198 220 7.57e0 SMART
low complexity region 224 238 N/A INTRINSIC
low complexity region 252 263 N/A INTRINSIC
LRR 274 297 1.06e1 SMART
LRR_TYP 298 321 1.28e-3 SMART
LRR 355 378 6.23e1 SMART
LRR 379 404 3.18e2 SMART
LRR 405 430 8.98e1 SMART
LRR 431 455 6.78e1 SMART
LRR_TYP 506 529 1.79e-2 SMART
LRR 530 553 2.63e0 SMART
LRR_TYP 562 585 1.56e-2 SMART
LRR 586 609 1.37e1 SMART
LRR 611 633 8.48e0 SMART
LRRCT 646 698 1.07e-10 SMART
transmembrane domain 705 724 N/A INTRINSIC
TIR 756 901 2.43e-26 SMART
Predicted Effect probably benign

PolyPhen 2 Score 0.084 (Sensitivity: 0.93; Specificity: 0.85)
(Using ENSMUST00000167106)
Predicted Effect probably benign

PolyPhen 2 Score 0.084 (Sensitivity: 0.93; Specificity: 0.85)
(Using ENSMUST00000209772)
Predicted Effect probably benign

PolyPhen 2 Score 0.032 (Sensitivity: 0.95; Specificity: 0.82)
(Using ENSMUST00000210013)
Meta Mutation Damage Score 0.0692 question?
Is this an essential gene? Probably nonessential (E-score: 0.153) question?
Phenotypic Category Autosomal Semidominant
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All mutations/alleles(6) : Targeted(6)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00162:Tlr3 APN 8 45853727 missense probably damaging 0.99
IGL01820:Tlr3 APN 8 45851376 missense probably benign
IGL02504:Tlr3 APN 8 45850944 missense probably damaging 1.00
IGL02523:Tlr3 APN 8 45851428 splice site probably null
IGL03166:Tlr3 APN 8 45855965 missense probably benign 0.05
IGL03287:Tlr3 APN 8 45855817 missense probably benign
Ultraman UTSW 8 45856018 missense probably damaging 1.00
E0354:Tlr3 UTSW 8 45853857 missense probably damaging 1.00
R0960:Tlr3 UTSW 8 45850452 missense probably damaging 1.00
R1175:Tlr3 UTSW 8 45850171 missense probably damaging 1.00
R1332:Tlr3 UTSW 8 45851774 missense probably damaging 0.99
R1477:Tlr3 UTSW 8 45851202 missense probably damaging 1.00
R1667:Tlr3 UTSW 8 45853874 missense probably benign 0.00
R1755:Tlr3 UTSW 8 45851010 missense probably benign
R1996:Tlr3 UTSW 8 45850734 missense probably benign 0.08
R2012:Tlr3 UTSW 8 45855823 missense possibly damaging 0.91
R2288:Tlr3 UTSW 8 45850705 missense probably damaging 0.98
R2895:Tlr3 UTSW 8 45850629 missense possibly damaging 0.89
R3837:Tlr3 UTSW 8 45849976 missense probably damaging 1.00
R4905:Tlr3 UTSW 8 45852260 critical splice acceptor site probably null
R4934:Tlr3 UTSW 8 45850072 missense probably benign 0.10
R5025:Tlr3 UTSW 8 45856075 missense probably benign 0.00
R5086:Tlr3 UTSW 8 45855862 missense probably damaging 0.96
R5129:Tlr3 UTSW 8 45856018 missense probably damaging 1.00
R5320:Tlr3 UTSW 8 45852137 missense possibly damaging 0.95
R5411:Tlr3 UTSW 8 45849992 missense probably benign 0.01
R5497:Tlr3 UTSW 8 45851851 missense possibly damaging 0.60
R5498:Tlr3 UTSW 8 45851851 missense possibly damaging 0.60
R5499:Tlr3 UTSW 8 45851851 missense possibly damaging 0.60
R5501:Tlr3 UTSW 8 45851851 missense possibly damaging 0.60
R5731:Tlr3 UTSW 8 45851157 missense probably benign 0.00
R5761:Tlr3 UTSW 8 45855808 missense probably benign 0.00
R5992:Tlr3 UTSW 8 45850851 missense probably benign
R6031:Tlr3 UTSW 8 45851565 missense probably damaging 1.00
R6031:Tlr3 UTSW 8 45851565 missense probably damaging 1.00
R6104:Tlr3 UTSW 8 45856130 missense probably benign 0.00
R6289:Tlr3 UTSW 8 45849966 missense probably benign 0.04
R6372:Tlr3 UTSW 8 45850048 missense probably damaging 1.00
R6470:Tlr3 UTSW 8 45850422 missense probably damaging 1.00
R6486:Tlr3 UTSW 8 45851650 splice site probably null
R6504:Tlr3 UTSW 8 45850486 missense possibly damaging 0.79
R6721:Tlr3 UTSW 8 45851917 missense probably benign 0.00
R7089:Tlr3 UTSW 8 45850810 missense probably benign 0.02
R7169:Tlr3 UTSW 8 45850056 missense probably damaging 1.00
R7679:Tlr3 UTSW 8 45852088 missense probably benign
R7771:Tlr3 UTSW 8 45856076 missense probably benign
R7863:Tlr3 UTSW 8 45850774 missense probably benign 0.00
R7896:Tlr3 UTSW 8 45850090 nonsense probably null
R8009:Tlr3 UTSW 8 45853819 missense not run
R8219:Tlr3 UTSW 8 45851016 missense possibly damaging 0.95
R8397:Tlr3 UTSW 8 45851896 missense possibly damaging 0.94
R8411:Tlr3 UTSW 8 45849978 missense probably damaging 1.00
R8539:Tlr3 UTSW 8 45851553 missense probably damaging 1.00
R8786:Tlr3 UTSW 8 45851286 missense possibly damaging 0.94
R8916:Tlr3 UTSW 8 45856076 missense probably benign
R9282:Tlr3 UTSW 8 45851643 missense probably benign 0.12
R9609:Tlr3 UTSW 8 45850117 missense probably benign 0.02
R9731:Tlr3 UTSW 8 45850944 missense probably damaging 1.00
Z1177:Tlr3 UTSW 8 45851020 missense probably damaging 1.00
Mode of Inheritance Autosomal Semidominant
Local Stock
MMRRC Submission 038219-MU
Last Updated 2019-09-04 9:45 PM by Katherine Timer
Record Created 2015-05-07 10:52 AM by Lei Sun
Record Posted 2015-09-22
Phenotypic Description

Figure 1. Rakshasa mice exhibited decreased TNFα secretion in response to TLR3 ligand, poly(I:C). TNFα levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The Rakshasa phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R1996, some of which exhibited decreased TNFα secretion from macrophages in response to the Toll-like receptor 3 (TLR3) ligand, poly(I:C) (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of reduced TNFα secretion after poly(I:C) stimulation using an additive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 88 mutations (X-axis) identified in the G1 male of pedigree R1996.  Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity.  Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 88 mutations. The diminished poly(I:C)-induced TNFα secretion phenotype was linked by continuous variable mapping to a mutation in Tlr3:  a T to C transversion at base pair 45,397,697 (v38) on chromosome 8, or base pair 14,253 in the GenBank genomic region NC_000074.  Linkage was found with a dominant model of inheritance (P = 1.87 x 10-4), wherein 2 variant homozygotes and eight heterozygotes departed phenotypically from 14 homozygous reference mice (Figure 2).  


 

The mutation corresponds to residue 2,523 in the mRNA sequence NM_126166 within exon 6 of 7 total exons.

 
2507 GTTTTTATACTTGTGGTACTGCTCATTCACATC

216  -V--F--I--L--V--V--L--L--I--H--I-

The mutated nucleotide is indicated in red.  The mutation results in a valine (V) to alanine (A) substitution at position 721 (V271A) in the TLR3 protein, and is strongly predicted by PolyPhen-2 to be benign (score = 0.084) (1).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 3. Protein and domain structure of TLR3. (A) Schematic representation of TLR3 based on crystalized structures of mouse TLR3 LRR (PBD 3CIY) and human TLR2 TIR (1FYW) domains. The residue affected by the Rakshasa mutation is highlighted. 3D image was created using UCSF Chimera. (B) TLR3 is a 905 amino acid protein with an extracellur domain (pink) of leucine rich repeats (LRR), a short transmembrane (TM) domain (blue) and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain (green). The Rakashasa mutation (red asterisk) results in valine (V) to alanine (A) substitution at position 721 of the TLR3 protein in the transmembrane domain.

Figure 4. Crystal structure of a TLR3 homodimer complexed with its ligand dsRNA (dark blue). TLR3 ectodomains are shown in pink and the two long LRR insertions in each ectodomain are in yellow. The dsRNA ligand binds to the concave surface of the homodimer complex at both N- and C-terminal locations on each ectodomain. UCSF Chimera structure is based on PDB:3CIY, Liu et al. Science 320, 379-381 (2008). Click on the 3D structure to view it rotate.

TLR3 is a type I integral membrane glycoprotein containing 905 amino acids (Figure 3). Like the other TLRs, its cytoplasmic domain (at its C terminus) shares similarity with the interleukin-1 and IL-18 receptors (IL-1R and IL-18R) in a conserved region of approximately 200 amino acids known as the Toll/IL-1R (TIR) domain (2-4), which mediates homo- and heterotypic protein interactions during signal transduction. TIR domains in TLRs and in IL receptors contain 3 conserved boxes (boxes 1, 2 and 3), which are required for signaling (5). In addition, TIR domains contain six α-helices (αA, αB, αC, αC’, αD and αE) and five β-strands (βA, βB, βC, βD and βE) that are connected by seven loops (named for the α-helix and β-strand they connect; e.g. AA connects βA with αA). The crystal structures of the TLR1 and TLR2 TIR domains reveal that they fold into a structure with a central five-stranded parallel β-sheet surrounded by five helices (6) (see the record for languid for a picture of the TLR2 TIR domain). Many of the α-helices and connecting loops are predicted to participate in binding partner recognition, and their mutation is expected to abrogate specific binding interactions. This is true of an alanine to proline mutation in the BB loop of TLR3 (Ala795Pro), which has been reported to abolish TRIF-dependent IRF3 responses and promote MyD88-dependent responses (7).

The extracellular domains of TLRs, unlike those of the interleukin receptors, contain multiple leucine-rich repeats (LRRs), which mediate ligand recognition by TLRs. LRRs consist of 24-29 amino acids with two conserved leucine-rich sequences: XLXXLXLXXN (residues 1-10, present in all LRR subtypes) followed by XØXXØX4FXXLX (residues ~11-24, but variable in length, sequence and structure), where X is any amino acid and Øis a hydrophobic amino acid [discussed in (8)]. TLR3 has 23 predicted LRRs in its ectodomain along with the LRR-NT (LRR N-terminal) and LRR-CT (LRR-C-terminal) regions encoded by the N-terminal half of the protein. Crystal structures of several TLRs reveal that each LRR forms a loop such that the juxtaposition of several LRR loops forms a horseshoe structure, with the hydrophobic residues of the LRR consensus sequence pointed inward (9-12). In addition, the XLXXLXLXX sequence folds into a β-strand, with the remaining LRR residues oriented on the convex side of the structure. Some LRRs contain insertions of up to 16 amino acids following positions 10 or 15 in the LRR consensus sequence, a common occurrence among LRRs of many TLRs. In TLR3, insertions in two of the LRRs extend outward from the convex face of the protein [Figure 4; PDB ID 3CIY; (10;12;13)]. For TLR7, 8 and 9, LRRs 2, 5 and 8 contain long insertions following the tenth residue. These insertions, along with an insertion in LRR11, are positioned proximal to the β-strands formed by the first ten residues. These four insertions may contribute to the ligand-binding site (8).  The two TLR3 monomers interact between the two LRR-CT domains. Each extracellular domain of TLR3 binds dsRNA at two sites at opposite ends of the structure. LRR19 and LRR20 comprise the first dsRNA binding site in TLR3. The second dsRNA binding site is comprised of LRR-NT, LRR1, LRR2, and LRR3. Deletion of the insertion within LRR12 did not have an effect on TLR3 stimulation, but deletion of the LRR20 insertion resulted in a significant loss of TLR3 activity (14). The loss of the LRR20 insertion may disrupt the structure of TLR3 near residues essential for ligand binding. The consensus leucine side chains of the LRRs point towards the interior and form a hydrophobic core. The first eight residues of each LRR contribute a β-strand to an extended parallel β-sheet that forms the concave, inner surface of the molecule. The LRR-NT motif has a hairpin loop that is stabilized by a disulfide bond. The LRR-CT motif is globular in structure, and contains an internal α-helix and two disulfide bonds, both of which are essential for TLR3 function (15).

The TLR3 ectodomain has 15 putative N-glycosylation sites. The glycans are distributed on the concave, convex, and on the N-terminal side of the β-sheet of the ectodomain. The ribose-phosphate backbone of dsRNA interacts with the glycan-free surfaces of a TLR3 ectodomain homodimer. His539 is essential for ligand-dependent activation of TLR3 (14). Asn541 is essential for dsRNA recognition (14). Mutation of His539 to glutamic acid (H539E) and Asn541 to alanine (N541A) results in loss of dsRNA binding. Similar to other endosomal TLRs, TLR3 undergoes proteolytic processing to confer stability and endosomal localization (16). The ectodomain of TLR3 is cleaved between residues 342 and 343 (16). Phosphorylation of TLR3 is required for the recruitment of the adaptor protein TICAM-1 (TRIF), which mediates downstream signaling. Phosphorylation of Tyr759 and Tyr858 are sufficient to promote downstream signaling (17). The tyrosines c-Src (18) and epidermal growth factor receptor (EGFR; see the record for Velvet) (19) mediate TLR3 phosphorylation.

The Rakshasha mutation results in a valine (V) to alanine (A) substitution at position 721 (V271A) in the TLR3 protein, which lies within the transmembrane domain.

Expression/Localization

TLR3 is expressed in myeloid dendritic cells (20), macrophages (21), mast cells, CD8+ T cells (22), γδ T cells, and natural killer cells (23) as well as fibroblasts, epithelial cells (24;25), the retina (26), hepatocytes (27;28), keratinocytes (29), and the nervous system including neurons, oligodendrocytes, astrocytes, and microglia (30-32). TLR3 mRNA is expressed in human placenta, pancreas, lung, liver, heart, lymph nodes, and brain (20;33).

In resting cells, TLR3 is localized in the endoplasmic reticulum and at endosomal/lysosomal membranes. Upon TLR3 stimulation, TLR3 interacts with UNC-93B (see the record for 3d), which assists in trafficking of TLR3 to the endosome. TRL3 localizes to the early endosome in myeloid DCs (34), but on both the cell surface and early endosome in macrophages, splenic CD8+ dendritic cells, marginal zone B cells, lung fibroblasts, and some epithelial cell lines (16;35;36).

TLR3 expression in DCs is upregulated by viral infection and exogenous addition of poly(I:C) or type I IFN (37).

Background

Figure 5. Overview of Toll-like receptor (TLR) signaling pathways. Shown are the signaling events downstream of TLR activation that ultimately lead to the induction of thousands of genes including TNF and type I IFN, which are critical in activating innate and adaptive immune responses. TLR1,2,4,5 and 6 are located at the cell surface, while TLR3,7, and 9 are localized in the endosome. Once TLR complexes recognize their ligands, they recruit combinations of adaptor proteins (MyD88, TICAM, TRAM, TIRAP) via homophilic TIR domain interactions.

In the MyD88-dependent pathway utilized by all TLRs except TLR3, MyD88 (lime green) recruits IRAK kinases through their death domains (DD). TRAF6 and IRF5 are also recruited to this complex. Phosphorylation of IRAK1 by IRAK4 allows dissociation of IRAK1 and TRAF6. K63 ubiquitination (small light blue circles) of TRAF6 recruits TAK1 and the TAK1 binding proteins, TAB1 and TAB2. Activation of TAK1 leads to activation of MAP kinase cascades and the IKK complex. NEMO polyubiquitination by TRAF6 is necessary for IKK complex function. The IKK complex phosphorylates IκB, p105, and TPL2 (or MAP3K8), resulting in IκB and p105 ubiquitination and degradation (small pink circles), releasing NF-κB into the nucleus and permitting TPL2 to become activated, respectively. Activation of the p38, JNK and ERK1/2 kinases leads to the activation of both CREB and AP1, which in turn induce many target genes. In pDCs, activation of TLR7 and 9 in endosomes recruits MyD88 and IRAK4, which then interact with TRAF6, TRAF3, IRAK1, IKKα, osteopontin (OPN), and IRF7. IRAK-1 and IKKα phosphorylate and activate IRF7, leading to transcription of interferon-inducible genes and production of large amounts of type I IFN.
 
In the TICAM-dependent pathway stimulated by TLR3 or 4 activation, TICAM (bright yellow) recruits polyubiquitinated RIP1, which interacts with the TRAF6/TAK1 complex and leads to NF-κB activation and proinflammatory cytokine induction. TICAM signaling also leads to type I IFN production through phosphorylation and activation of IRF3 by a complex containing TRAF3, TBK1 and IKKe; RIP1 is not required for TICAM-dependent activation of IRF3.

Note that TLR4 signals through the MyD88-dependent pathway from the cell membrane and is subsequently internalized into late endosomes to signal through the TICAM-dependent pathway. When bound to vesicular stomatitis virus glycoprotein G (VSV-G) (far left), TLR4 can signal through TRAM to induce IRF7 activation, a process that is partially dependent on TICAM. Upon viral stimulation, TLR2 may also be internalized into endosomes to activate both IRF3 and IRF7 by an unknown mechanism. LTA = lipoteichoic acid; LP2 = lipopeptide 2. PAM3CSK4 is a triacyl lipopeptide. Phosphorylation events are represented by small yellow circles labeled with a “P”. This image is interactive. Click on the image to view mutations found within the pathway (red) and the genes affected by these mutations (black). Click on the mutations for more specific information.

Figure 6. Toll-like receptor 3 (TLR3) signaling pathway. TLR3 is localized in the endosome.  TLR3 forms homodimers and recruits TICAM1 for the production of proinflammatory cytokines. TICAM recruits polyubiquitinated RIP1, which interacts with the TRAF6/TAK1 complex and leads to NF-κB activation and proinflammatory cytokine induction. TICAM signaling also leads to type I IFN production through phosphorylation and activation of IRF3 by a complex containing TRAF3, TBK1 and IKKe.

Toll-like receptors (TLRs) play an essential role in the innate immune response as key sensors of invading microorganisms by recognizing conserved molecular motifs found in many different pathogens, including bacteria, fungi, protozoa and viruses. The twelve mouse TLRs and ten human TLRs recognize a wide range of structurally distinct molecules, and all signal through only four adaptor proteins known to date: MyD88, Tirap (Mal), TICAM-1 (TRIF) and TRAM (Figure 5). TLR signaling through these adaptors initiates a cascade of signaling events involving various kinases, adaptors and ubiquitin ligases, ultimately leading to transcriptional activation of cytokine and other genes through the transcription factors NF-κB, AP-1, interferon responsive factor (IRF)-3, and IRF-7.

TLR3 is an endosomal TLR along with TLR7 (see the record for rsq1), TLR8, and TLR9 (see the record for Cpg1). The endosomal TLRs recognize exogenous nucleic acids:  double-stranded DNA unmethylated at CpG motifs [TLR9; (2)], single-stranded (ss) RNA viruses (TLR7 and TLR8; (38)] and double-stranded RNA (dsRNA; TLR3]. TLR3 recognizes both virus-derived dsRNA and the synthetic dsRNA, poly(I:C) [Figure 6; (35;39)]. TLR3 recognizes dsRNA in a sequence-independent manner. Recently, an RNA structure was identified that mediates TLR3 recognition of RNA. The RNA contains an incomplete stem with bulge and internal loops, but is able to induce type I interferons and pro-inflammatory cytokines (40). 5’-Triphosphorylation of dsRNA is essential for TLR3 recognition. In addition, 2’-hydroxy groups are essential for TLR3 activation by poly(I:C).

dsRNA exists both as a viral genome and can be generated in the cytosol during replication of positive-strand RNA viruses (e.g., poliovirus, coxsackievirus group B serotype 3, and encephalomyocarditis) and DNA viruses [e.g., herpes simplex virus 1 [HSV1; (41)] and murine cytomegalovirus (MCMV)] (42). In the case of negative-strand RNA viruses, TLR3-mediated signaling can intensify the immune response to infection (43;44). Tlr3-deficient mice are susceptible to encephalomyocardititis virus (ECMV) (45), coxsackievirus group B3 (CVB3) (46), CVB4 (47), poliovirus (48;49), MCMV (50), HSV-1 (51), Theiler’s murine encephalomyelitis virus (TMEV) (52), vesicular stomatitis virus (VSV) (53), LCMV (53), Vaccinia virus (54), influenza virus (43), T3 reovirus (53), Punta toro virus (PTV) (44), and West Nile Virus (WNV) (55). A polymorphism in TLR3 (rs3775291, Leu412Phe) confers resistance to HIV-1 infection (56). The 412Phe allele occurs in approximately 30% of the population with European and Asian ancestry. The 412Phe allele is more common among a population of Spanish HIV-1-exposed seronegative individuals when compared to controls. The TLR3412Phe protein exhibits reduced poly(I:C)-stimulated activation.

TLR3 mediates downstream signaling through TICAM-1 [Toll-interleukin 1 receptor (TIR) domain-containing adaptor molecule-1; hereafter TRIF (TIR domain-containing adaptor inducing IFN-β); see the record for Lps2] (57). In the TRIF-dependent pathway, TRIF recruits polyubiquitinated RIP1 (58), which interacts with TRAF6, an E3 ubiquitin ligase that also coordinates the activation of several kinases including TAK-1 and in turn MAP kinases and the IKK complex leading to NF-κB activation. TRIF signaling also leads to type I IFN production through phosphorylation and activation of IRF3 by a complex containing TRAF3, TBK1 and IKKe; RIP1 is not required for TICAM-dependent activation of IRF3. TRIF-mediated MyD88-independent pathway induces a late-phase activation of NF-κB and MAP kinases, while MyD88-dependent pathway induces an early-phase activation of NF-κB and MAP kinases.

Putative Mechanism

The defective poly(I:C)-induced TLR signaling defect observed in Rakshasa indicates that TLR3Rakshasa exhibits loss-of-function. The mutation affects amino acid 721 within the transmembrane domain and is not predicted to be a residue involved in dsRNA binding or recognition of downstream signaling proteins.

Primers PCR Primer
Rakshasa_pcr_F: CCATTGGGGAGAAATGTTCCC
Rakshasa_pcr_R: AATCCGTTCGACTGCACGTG

Sequencing Primer
Rakshasa_seq_F: TTGGGGAGAAATGTTCCCAGACC
Rakshasa_seq_R: CGACTGCACGTGTGAAAGTATTTCC
Genotyping

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold


The following sequence of 400 nucleotides is amplified (chromosome 8, - strand):


1   aatccgttcg actgcacgtg tgaaagtatt tcctggtttg ttaactggat caaccagacc
61  cacactaata tctctgagct gtccactcac tacctctgta acactccaca tcattattat
121 ggcttccccc tgaagctttt cgatacatca tcctgtaaag acagcgcccc ctttgaactc
181 ctcttcataa tcagcaccag tatgctcctg gtttttatac ttgtggtact gctcattcac
241 atcgagggct ggaggatctc tttttactgg aatgtttcag tgcatcggat tcttggtttc
301 aaggaaatag acacacaggc tgagcagttt gaatatacag cctacataat tcatgcccat
361 aaagacagag actgggtctg ggaacatttc tccccaatgg 


Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References
Science Writers Anne Murray
Illustrators Diantha La Vine, Peter Jurek, Katherine Timer
AuthorsLei Sun and Bruce Beutler