Phenotypic Mutation 'macro-1' (pdf version)
Allelemacro-1
Mutation Type missense
Chromosome16
Coordinate91,499,885 bp (GRCm38)
Base Change A ⇒ C (forward strand)
Gene Ifnar1
Gene Name interferon (alpha and beta) receptor 1
Synonym(s) Ifar, Ifrc, IFN-alpha/betaR
Chromosomal Location 91,485,238-91,507,441 bp (+)
MGI Phenotype Homozygotes for targeted null mutations exhibit increased susceptibility to viral infection, elevated levels of myeloid lineage cells in the peripheral blood and bone marrow, and reduced immune response to immunostimulatory DNA.
Accession Number

NCBI RefSeq:  NM_010508;  MGI: 107658

Mapped Yes 
Amino Acid Change Threonine changed to Proline
Institutional SourceBeutler Lab
Ref Sequences
T341P in Ensembl: ENSMUSP00000023689 (fasta)
Gene Model not available
PDB Structure
Murine Ifnar1 in complex with interferon-beta [X-RAY DIFFRACTION]
SMART Domains

DomainStartEndE-ValueType
low complexity region 2 16 N/A INTRINSIC
FN3 29 110 6.97e0 SMART
FN3 128 213 7.02e1 SMART
low complexity region 267 275 N/A INTRINSIC
FN3 332 409 3.23e0 SMART
transmembrane domain 427 449 N/A INTRINSIC
low complexity region 550 562 N/A INTRINSIC
Predicted Effect possibly damaging

PolyPhen 2 Score 0.556 (Sensitivity: 0.88; Specificity: 0.91)
(Using Ensembl: ENSMUSP00000023689)
Phenotypic Category adenovirus infection of macrophages- increased, immune system, influenza proliferation in macrophages- increased, MCMV proliferation in macrophages- increased, MCMV susceptibility, RVFV proliferation in macrophages- increased
Penetrance 100% 
Alleles Listed at MGI

All alleles(8) : Targeted, knock-out(2) Targeted, other(1) Gene trapped(4) Chemically induced(1)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00229:Ifnar1 APN 16 91489782 missense probably benign 0.11
IGL02183:Ifnar1 APN 16 91505146 missense possibly damaging 0.94
IGL02828:Ifnar1 APN 16 91505416 unclassified probably null
shook UTSW 16 91499537 nonsense probably null
R0124:Ifnar1 UTSW 16 91499537 nonsense probably null
R0502:Ifnar1 UTSW 16 91501751 missense probably damaging 1.00
R0617:Ifnar1 UTSW 16 91501682 missense probably damaging 1.00
R1509:Ifnar1 UTSW 16 91503496 missense probably damaging 1.00
R4111:Ifnar1 UTSW 16 91496158 missense probably damaging 1.00
R4473:Ifnar1 UTSW 16 91495170 missense probably damaging 0.98
R4964:Ifnar1 UTSW 16 91505086 missense probably benign 0.08
R5497:Ifnar1 UTSW 16 91505364 missense probably benign 0.01
R6135:Ifnar1 UTSW 16 91501620 critical splice acceptor site probably null
X0057:Ifnar1 UTSW 16 91495424 missense probably damaging 0.98
X0057:Ifnar1 UTSW 16 91505283 missense possibly damaging 0.92
Mode of Inheritance Autosomal Recessive
Local Stock Live Mice, Sperm, gDNA
MMRRC Submission 030878-UCD
Last Updated 04/11/2017 2:16 PM by Katherine Timer
Record Created unknown
Record Posted 12/18/2008
Phenotypic Description
Macro-1 was discovered in an ex vivo macrophage screen for control of viral infection.  Macrophages from ENU-mutagenized G3 mice were infected with mouse cytomegalovirus (MCMV), influenza virus, Rift Valley Fever virus (RVFV), and an adenoviral vector and examined for effective viral clearance.  Peritoneal macrophages from macro-1 mice are highly permissive to MCMV, influenza, RVFV (Figure 1) and adenoviral infection.  These macrophages produce normal amounts of tumor necrosis factor (TNF)-α after Toll-like receptor (TLR) stimulation (TLR Signaling Screen). 
 
Preliminary results suggest that mutant macrophages produce lower levels of type I interferons (IFNs) than wild type macrophages upon adenovirus infection.  This result needs confirmation.

 

Nature of Mutation
The Ifnar1 gene on chromosome 16 of macro-1 mice was sequenced, and an A to C transversion was identified in exon 8 (of 11 total exons) at position 1115 of the Ifnar1 transcript.
 
2834 CCGGTCATTACTGTCACCGCCATGAGTGACACC
852  -P--V--I--T--V--T--A--M--S--D--T-
 
The mutated nucleotide is indicated in red lettering, and results in a conversion of threonine to proline at residue 341 of the IFNAR1 protein. 
Protein Prediction
Figure 2. Domain structure of IFNAR1. IFNAR1 belongs to the class II helical cytokine receptor (hCR) family. The extracellular ligand-binding region of IFNAR1 contains four fibronectin type III subdomains known as SD1-4. The micro-1 mutation results in a conversion of threonine to proline at residue 341 in SD4 of the IFNAR1 protein.
Ifnar1 encodes a 590 amino acid protein that, along with IFNAR2 (see record for macro-2), forms the heterodimeric type I interferon (IFN) receptor (IFNAR) (Figure 2).  The IFNAR complex binds to all type I interferons including 13 IFN-α subtypes, as well as IFN-β, w, ε, ω, κ and others in some species.  Type I IFNs consist of five α-helices (A-E), which are linked by one overhand loop (AB loop) and three shorter segments (BC, CD, and DE loops).  Helices A, B, C, and E are arranged in an antiparallel fashion (1).  Type I IFNs are hypothesized to bind to IFNAR1 and IFNAR2 on opposing surfaces (2-4). 
 
Figure 3. A, Schematic drawing depicting the organization of the IFNAR1 and IFNAR2 extracellular domains bound to type I IFN. IFNAR1 domains are green; IFNAR2 domains are blue; type I IFN is in pink. The location of the residue altered by the macro-1 mutation is indicated by a red asterisk. Model is based on Li et al J. Mol. Biol. 377, 715-724 (2008). B, NMR structure of the human IFNAR2 extracellular domain complexed with IFNa2. The two extracellular domains of IFNAR2 are labeled, and the three residues that are most critical for binding IFNa2 are shown in yellow (see text). UCSF Chimera model is based on PDB 2HYM, Chill et al. Protein Science 15, 2656-2668 (2006). This image is interactive. Click on the 3D structure to view it rotate.
Both IFNAR1 and IFNAR2 belong to the class II helical cytokine receptor (hCR) family, which includes receptors for type II IFN, tissue factor (TF), and interleukin (IL)-10.  The extracellular ligand-binding regions of these receptors all share fibronectin type III (FBN-III) domains similar to the constant region of immunoglobin (Ig).  Most of these receptors contain two of these domains, each containing ~100 amino acids with seven β-strands and connecting loops.  The 403 amino acid extracellular domain of mouse IFNAR1 is atypical in that it contains four FBN-III subdomains known as SD1-4 (2;5).  Based on the human sequence, SD1 in mouse IFNAR1 comprises amino acids 29-129, SD2 amino acids 130-229, SD3 residues 230-334, and SD4 residues 335-427 (6).  Relative to IFNAR2, IFNAR1 binds to type I IFNs with low affinity (7), but binding of ligand to IFNAR1 is necessary to form the proper ligand-receptor complex (Figure 3).  It is likely that the differential binding of the many type I IFNs to IFNAR1 provides the mechanism for their varying biological activities, as type I IFNs with higher affinity to IFNAR1 have higher antiproliferative activities (7;8).  Several studies, including structural and mutational analyses, suggest that IFNAR1 binding to ligand occurs through critical amino acids found in SD1-3 (6;8-11), while SD4 is necessary for ternary complex formation and signal activation (8;9;12).  SD1 also contains conserved residues implicated in binding membrane glycosphingolipids (13). 
 
The IFNAR receptor is typical of class II hCRs and lacks intrinsic kinase activity.  Instead, the intracellular domains of the IFNAR receptor subunits are associated with Janus activating kinases (JAKs) that phosphorylate receptors and signal transducing molecules [reviewed in (14;15)] (see Background).  In particular, IFNAR1 binds to tyrosine kinase 2 (TYK2) at amino acids 474-498 (16-18).  This binding domain contains a proline-rich sequence (box 2 motif) that has been shown in other receptors to recruit and activate tyrosine kinases (16).  IFNAR1 has a similar motif at amino acids 461-466 (box 1), but this has only a minor role in TYK2 recruitment (17).  IFNAR1 contains several tyrosine residues that become phosphorylated by activated TYK2, and then are able to recruit signal transduction molecules such as signal transducer and activator of transcription (STAT)1 (see the records for domino and poison) and STAT2 (19;20).  However, mutational analysis of these tyrosine residues in mouse IFNAR1 suggests that these residues are not necessary for type I IFN activity (18), although it remains possible that phosphorylation of other tyrosine residues may provide STAT docking sites on mouse IFNAR1.  IFNAR1 also associates with negative regulators of type I IFN signaling such as suppressor of cytokine signaling 1 (SOCS1) (21).    
 
Several splice variants of human IFNAR1 have been identified in cell lines (22;23), but other data suggests that normal cells only express full-length IFNAR1 (2).  Two of these alternative splice variants may produce proteins that lack the transmembrane domain (22), while one is predicted to produce a protein that lacks the amino-terminal region as well as a portion of SD2 (23).  It is possible these aberrant IFNAR1 transcripts are expressed only in particular tumor cell lines.  
 
The residue mutated in macro-1 mice is in the extracellular SD4 domain of the IFNAR1 protein.  It is unknown whether this protein is expressed and localized normally.
Expression/Localization
The Ifnar1 mRNA transcript is expressed in all tissues and organs and most cell lines (2;24).  In mice, the thymus, bone marrow, testes, embryonic day (E) 10 placenta, L929 cells and spleen had higher levels of Ifnar1 mRNA than other tissues (24).  The IFNAR receptor is localized to caveolae on the plasma membrane where there is a concentration of cytoplasmically oriented signaling molecules (25), but is subject to ligand-induced internalization and lysosomal degradation (26).
Background
Type I IFNs are a critical class of cytokines that have potent antiviral, growth-inhibitory and immunomodulatory functions [reviewed in (1;27)].  Classically, the production of type I IFNs (notably IFN-α/β) in response to viral infections is considered to be essential for antiviral defense, a role highlighted by the phenotype of Ifnar1 knockout mice that lack type I IFN signaling (28;29).  These mice display high susceptibility to viral infection by vesicular stomatitis virus (VSV), Semliki Forest virus (SFV), Vaccinia virus, lymphocytic choriomeningitis virus (LCMV) and mouse cytomegalovirus (MCMV).  The production of type I IFNs in response to viral infection leads to inhibition of viral propagation and increases the vulnerability of infected cells to virus-induced apoptosis (30), a mechanism that is suggested to limit viral spread and may also increase the delivery of antigen to professional antigen-presenting cells (APCs) and be important to the onset of adaptive immunity.  Type I IFNs activate natural killer (NK) cells, macrophages and dendritic cells (DCs), all of which play an essential role in the innate immune system (31;32).  In addition to their antiviral effects, type I IFNs are also produced in response to infection with bacterial pathogens and have an important role in the host response to bacterial infection [reviewed by (33)].  IFNAR1-deficient mice display susceptibility to some bacterial infections, although they have been shown to be resistant to others including Listeria monocytogenes (33-35).  The antiproliferative and apoptotic effects of type I IFNs on macrophages may be the reason why type I IFNs promote susceptibility to certain bacterial infections (36). 
 
All cell types that are susceptible to viral infection are able to release type I IFNs.  However, in the immune system, plasmacytoid dendritic cells (pDCs) produce the largest amounts of IFN-α/β in response to nucleic acids derived from pathogenic organisms [reviewed by (37)].  In general, the induction of type I IFN genes is transcriptionally controlled and depends on two members of the IFN regulatory factor (IRF) family, IRF-3 and IRF-7. In response to microbial infections, these factors become phosphorylated, undergo nuclear translocation and induce transcription of genes encoding members of the IFN-α/β family (38).  IRF-3 is relatively specific in its induction of the solitary Ifnβ gene; IRF-7 is relatively specific for induction of the Ifnα cluster.  Upstream of these events, the recognition of various pathogens leading to type I IFN induction occurs through the toll-like receptors (TLRs) as well as the nucleic-acid cytosolic sensors retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA5) (39;40).  The TLRs engage various adaptors including myeloid differentiation (MyD) 88 (see pococurante and lackadaisical), TIRAP for toll-interleukin 1 receptor domain containing adaptor protein (also called MAL for MyD88 adaptor-like) (see torpid), TRIF for Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-β (also known as TICAM-1 for TIR domain-containing adaptor molecule-1) (see Lps2), and TRAM for TRIF related adaptor molecule (also called TICAM-2) (see Tram KO).  With the exception of TLR3, which signals solely by activating TRIF, all TLRs recruit MyD88, which triggers signaling pathways leading to nuclear translocation of NF-κB and phosphorylation of IRF-7 (38).  Both the MyD88-dependent and TRIF mediated pathways are used by TLR4 (39).  The TRIF pathway mediates the phosphorylation of IRF-3 by activating the TANK-binding kinase 1 (TBK1) and IκB kinase (IKK)-i/ε (41).  Interestingly, pDCs produce both IFN-α/β, while conventional or myeloid DCs produce only IFN-β.  Differences in expression of the receptors that sense viral components play a major role in these differences (37).  TLR7 and TLR9 are the critical sensing components in pDCs and result in the activation of the MyD88/IRF-7 pathway in the endosomal compartment, while type I IFN production in cDCs is generally MyD88-independent/IRF-3 dependent and occurs through TLR3, TLR4 and the nucleic-acid cytosolic sensors (15;37).
 
 Figure 4. Diagram of the type I interferon receptor complex composed of IFNAR1 and IFNAR2 and type I IFN signaling pathways. The extracellular regions of the IFNAR receptor subunits consist of Fibronectin III domains, which are labeled as subdomain (SD) 1-4 in IFNAR1. The intracelluar regions of the IFNAR receptor subunits can interact with several factors that are important from type I IFN signaling. IFNAR1 and 2 contain proline-rich regions that bind to JAK tyrosine kinases (TYK2 binds to IFNAR1 and JAK1 binds to IFNAR2.) Phosphorylation of the IFNAR intracellular domains by these kinases leads to the activation of STATs, which are then able to form homo- and heterodimeric complexes. The interaction of SOCS1 with IFNAR1 inhibits STAT activation, while UBP43 binds to the JAK1 binding site. The STAT1/STAT2 heterodimer forms the ISGF3 complex with IRF9 that translocates into the nucleus where it activates genes containing the IFN-stimulated response element (ISRE). STAT1/STAT1 homodimers can activate genes containing the IFN-γ-activated sequence (GAS). The JAK kinases also activate other substrates, which initiate many pathways. For example, phosyphorylation of insulin receptor substrate (IRS) proteins leads to PI3K activation, wh ich has multiple downstream effectors. Phosphorylation of guanine nucleotide exchange factor (VAV) leads to activation of the small G protein, RAC1, which in turn activates MAP kinase pathways leading to p38 activation. Other factors that are activated in response to type I IFNs include the transcription factor NF-κB, which is downstream of PI3k or TRAF2, and AP1, which is activated by the ERK pathway. Please see text for further details. This image is interactive. Click on the image to view mutations within the pathway (red) and the genes affected by these mutations (black). Click on the mutations for more specific information.
Type I IFN signaling modulates the expression of hundreds of IFN-stimulated genes (ISGs), accounting for the diverse biological properties of these cytokines and their highly pleiotropic and diverse effects.  Some of these antimicrobial gene products include proteins that can bind directly to viral RNA and inactivate it.  Other type I IFN-induced proteins affect the intracellular transport of viral particles, regulate transcription and translation and induce apoptosis.  ISGs include MHC class I, which contribute to T cell responses (33).  Type I IFNs induce ISGs by activating several signal transduction pathways including the classical JAK/STAT signaling pathway (Figure 4).  As discussed above, the IFNAR receptor associates with protein tyrosine kinases including JAK1 in the case of IFNAR2 and TYK2 in the case of IFNAR1.  Indeed, the presence of TYK2 is important for the proper localization and stabilization of IFNAR1 expression at the cell surface (42).  Binding of ligand to the IFNAR complex results in conformational changes that juxtapose these protein tyrosine kinases, resulting in auto and cross-phosphorylation and activation [reviewed by (15;43)].  Phosphorylation of critical tyrosine residues on the IFNAR receptor results in recruitment of signal transducing molecules such as STAT1 and STAT2 to the complex (18-20).  Phosphorylation of these signal transducing molecules by the IFNAR-associated protein kinases results in formation of STAT1/STAT2 heterodimers and STAT1 homodimers that dissociate from the receptor and translocate into the nucleus to form transcriptional complexes with other factors.  STAT1/STAT2 heterodimers, along with IRF-9, forms the IFN-stimulated gene factor 3 (ISGF3) complex that binds to upstream regulatory consensus sequences (IFN-stimulated response elements or ISRE) of type I IFN-inducible genes to initiate transcription.  ISGF3 regulates the transcription of IRF-7 (44;45), providing the type I IFN system a positive feedback loop that allows massive amplification of the type I IFN response.  STAT1 homodimers stimulate the transcription of genes containing the IFN-γ-activated sequence (GAS) (46). 
 
In addition to the positive feedback loop mediated by ISGF3-induction of IRF-7, constitutively low levels of IFN-α/β have been found to be expressed in several cell types and appear to be a prerequisite for enhanced production of type I IFNs in response to stimuli.  These low levels of IFN-α/β do not significantly activate downstream signaling events through IFNAR, but they do maintain tyrosine phosphorylation on IFNAR1, allowing for more efficient recruitment of STAT1 (25).  Basal levels of IFN-α/β are also correlated with IRF-7 expression, providing another mechanism for more efficient induction of type I IFN upon infection (15).  Constitutive IFN-α/β signaling appears to be required for more efficient type II IFN (IFN-γ) signaling as IFNAR1-deficient cells have a defective IFN-γ response associated with impaired dimerization of STAT1.  IFNAR1 has been shown to physically interact with a type II IFN receptor subunit (IFNGR2) in the plasma membrane, thus maintenance of the STAT docking sites on IFNAR1 are probably important for IFN-γ signaling as well (25).
 
Unique among all the cytokines, type I IFNs are able to promote the activation of all seven STAT family members resulting in the formation of a number of homo- and heterodimer combinations and differing gene induction responses.  The particular STAT(s) activated, STAT complexes formed, and the biological activities evoked are dependent on a variety of factors including cell type, cytokine milieu and stimulus [reviewed in (27)].  Indeed, some of the particular STATs have opposing activities and may compete with each other for binding to IFNAR and other STATs.  For instance, activated STAT1 is known to promote antiproliferative and apoptotic effects (47), while STAT4 promotes survival and proliferation in CD8+ T cells, particularly those with low levels of STAT1 (48-50).  In CD4+ T cells, activation of STAT3 and 5 in the presence of low STAT1 levels is necessary for the antiapoptotic and mitogenic effects of type I IFN (47), although STAT3 has also been shown to mediate apoptotic effects in still other cell types (51).  Unlike T cells, the activation of STAT1 in NK cells does not promote apoptosis, but is necessary for NK cytotoxicity (52).  Through these opposing effects, type I IFNs can mediate both the destruction of virally infected cells, while promoting the function of cells that are critical for mounting an appropriate immune response.   
 
In addition to the classical JAK-STAT pathway, type I IFN signaling through IFNAR is able to induce several other pathways that can act either in conjunction with STATs or independently [reviewed in (14;27)] (Figure 2).  For instance, some IRF family members can induce gene transcription independently of STATs in response to type I IFN signaling (ie IRF-1; mutated in Endeka) (53).  Additionally, type I IFN signaling activates p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol (PI) 3-kinase (PI3K), and extracellular signal regulated kinase (ERK), kinases that are known to be involved in the activation of a variety of critical factors including the transcription factors NF-κB and activator protein (AP)-1, as well as the serine/threonine kinases protein kinase B (PKB or Akt) and the mammalian target of rapamycin (MTOR).  Indeed, NF-κB is activated by type I IFNs both through the classical NF-κB pathway involving the IκB kinase (IKK) complex as well as an alternative pathway involving NF-κB inducing kinase (NIK) and TNF receptor associated factor(TRAF) 2 (see the record for panr2).  TRAF2 is able to directly interact with IFNAR1, and is important for the antiviral effects of type I IFN (54).  All of these pathways have been shown to be critical for many of the biological effects of type I IFNs including growth inhibition and antiviral responses. 
 
The responses to type I IFNs are negatively regulated by several mechanisms. These include dephosphorylation of critical tyrosine residues on the receptor subunits as well as receptor internalization and degradation.  Other mechanisms include dephosphorylation of JAKs and STATs by several phosphatases.  In addition, the association of IFNAR1 with SOCS1 prevents the tyrosine phosphorylation of STATs (21).  Further downstream, the inhibition of STAT functions in the nucleus by protein inhibitors of activated STATs (PIAS) proteins occurs (55).  Many viruses have similarly found a way to prevent host antiviral responses by producing factors that inhibit the production of type I IFNs as well as type I IFN signaling [reviewed in (15)].  For example, poxvirus encodes a soluble protein that binds with high affinity to type I IFNs, thus neutralizing type I IFN signaling (56), while the V proteins of paramyxoviruses prevent the activation of STAT proteins through a variety of mechanisms including proteasomal degradation, inhibition of phosphorylation, sequestration, and blockage of nuclear translocation (15).
 
Due to their potent antiviral and anti-proliferative effects, type I IFNs are widely used to treat certain viral infections and cancers.  In addition, type I IFNs have been found to play important roles in the etiology of some autoimmune diseases [reviewed in (43)].  Type I IFNs are widely used for the treatment of multiple sclerosis (MS), a chronic autoimmune demyelinating disease characterized by the infiltration of inflammatory cells such as macrophages and T cells into the CNS, resulting in the destruction of axonal myelin sheaths (57).  In an animal model of MS, animals deficient in IFN-β or IFNAR1 show an increased level of disease (58-60).  The mechanisms underlying this involvement are not well understood, but appear to involve TRIF-dependent induction of type I IFN and subsequent inhibition of the development of a certain T cell subtype (Th17 cells) through IFNAR-dependent signaling in macrophages (59).  Although another study studying IFNAR1-deficient mice in the same animal model of MS did not find a connection with Th17 cells, the absence of IFNAR1 on myeloid cells specifically led to severe disease and increased lethality (60).  Mice with a B or T cell-specific IFNAR1 deficiency did not show increased disease levels.  These results suggest that type I IFN signaling through IFNAR in myeloid cells plays a protective role against the development of multiple sclerosis.  By contrast, excess type I IFNs and IFN-stimulated gene expression have been linked to the pathogenesis of other autoimmune diseases such as systemic lupus erythematosus (SLE) and insulin-dependent diabetes mellitus (IDDM) [reviewed by (43)].  Furthermore, IFN-α treatment for viral infections and tumors can sometimes induce these diseases as well as other autoimmune symptoms.  The mechanisms behind these effects are not well understood, but likely include enhanced DC maturation and function, promotion of T and B cell differentiation, proliferation and survival, and changes in cytokine expression (43).    
 
Polymorphisms in the human IFNAR1 gene have been implicated in a number of diseases [reviewed in (2)], including protection or susceptibility against cerebral malaria (61), susceptibility to multiple sclerosis (62), as well as susceptibility to various viruses such as human immunodeficiency virus (HIV) (63), and hepatitis C (64).  Cells from Down syndrome patients are more sensitive to type I IFN treatment due to trisomy of Chromosome 21, which carries the IFNAR1 and IFNAR2 genes.  Accordingly, patients with Down syndrome have an aberrant immune response (65).  Human deficiencies in proteins that are involved in the type I IFN response, including various STATs (see records for domino and poison) and TYK2, result in immunodeficiency (66-68).  TYK2 deficiency in humans confirms the essential role TYK2 plays in type I IFN signaling, in contrast to results from mice that suggests that TYK2 plays a minor role in the type I IFN response (69).   

 

Putative Mechanism
As the macro-1 mutant was discovered in an ex vivo macrophage screen, it is not known whether macro-1 mice have a similar phenotype to those of Ifnar1 knockout animals.  The high permissiveness of macro-1 macrophages to viral infection suggests that IFNAR function in these cells is severely affected and that the macro-1 mutation may be equivalent to a severely hypomorphic or null allele.  However, it is not known whether other cell types such as NK cells and DCs will show a similar phenotype.  Preliminary results suggest that macro-1 macrophages also produce lower levels of type I IFN relative to wild type macrophages upon viral infection, perhaps due to the lack of the positive feedback loop through IRF-7 .  Again, this data suggests that the macro-1 mutation abrogates all type I IFN signaling through the IFNAR receptor. 
 
The macro-1 mutation affects amino acid 341 of the IFNAR1 extracellular domain.  This residue occurs in SD4, which is critical for proper formation of the IFNAR complex but does not play a role in ligand-binding (8;9;12).  Although amino acid 341 is not conserved amongst species, it is present in the first β-strand (β1) of the SD4 domain.  As prolines are known to disrupt secondary structure (70), the substitution of a proline for a threonine at this position may disrupt the conformation of the β1 strand of SD4 and affect the ability of the entire receptor to form a proper ternary complex.
Primers Primers cannot be located by automatic search.
Genotyping
Macro-1 genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
 
Primers for PCR amplification
Macro-1(F): 5’- AGAACAGCTTGCCACTTCACTGG -3’
Macro-1(R): 5’- GCAGAGAAGCCTTAGCCTTAGAAGAAC -3’
 
PCR program (use SIGMA JumpStart REDTaq)
1) 94°C             2:00
2) 94°C             0:30
3) 56°C             0:30
4) 72°C             1:00
5) repeat steps (2-4) 29X
6) 72°C             7:00
7) 4°C               ∞
 
Primers for sequencing
Macro_seq(F): 5’- CCCAGGGTAGCTTCAAACTTATG -3’
Macro_seq(R): 5’- TCACAAAGTTCCTGGGTAGC -3’
 
The following sequence of 1717 nucleotides (from Genbank genomic region NC_000082 for linear DNA sequence of Ifnar1) is amplified:
 
13961                                             agaacagctt gccacttcac
13981 tgggaattcc ccacacactc tagagcaccc gtggccttcc tttgttgggg atctgtgtct
14041 cctccattgt accaggagtt ccctaagtgg ggaaactgcc ctcaggtcca cagtgcttac
14101 tacagtgtct taggtattat agagcctcag tacatagtga attaattcat gattggattg
14161 gattttgttt ttgctttctg ttttcaaagt ggctattcaa aaagcagttc tggaagccgt
14221 tcagataaat ggaaaccaat accaacctgt gcaaatgtcc agactacgca ctgtgtcttt
14281 tctcaagata ctgtctacac aggaacgttc tttctccatg tacaagcctc agagggaaat
14341 cacacatcct tttggtctga agagaagttt attgattctc aaaaacacag taagccgagt
14401 tttctttgag acagtctgac actgtagccc aggctggcct ggaactcacg gtgtagccca
14461 gggtagcttc aaacttatgg cagtcctcct gcctgagctc ctgagagctg aggttgtggg
14521 tgtggtccat gcctgctgta tagcaagcgc tttctggagt gtaattcctc atgtagggcg
14581 agtcccggaa ggttgtttga aggtgtctta gtgtgcaatt tctgtttgca ttcttcccca
14641 gttctccctc ctcctccggt cattactgtc accgccatga gtgacacctt gcttgtttat
14701 gtcaactgtc aggacagcac atgtgatgga ctcaattacg aaatcatctt ttgggaaaac
14761 acttccaata ctaaggtaaa aagctaccca ggaactttgt gacttagcct cataccggtg
14821 atgatgggaa agaaagttag tgggggaggg agggcaagag caagcagtca cagctctaag
14881 gtttgggagg ccttttaatc ttgatggtgg cctgtctgca gtagagcgaa tgtcagcctt
14941 ttccatggtc tgcatgacag tacaggtcgc ctgcatctgg gcctgacttt tctgtgtgtg
15001 aatatacagg agtgtgtgca agcatgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg
15061 tgtgtgtgtg tgtgactgtg catgagtatg tgaatgtatg tatgtgtgaa tatatgtgtg
15121 tgtatgtgtg aatgtacgtg tgtgtgtatg agtgtatgtg tgtgtgtgag tgtatgtgtg
15181 aatgtgtggg aggatgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgagtg actctccgtg
15241 agtatgtgag tatatgtatg tgaatctgtg tgaatgtgtg tgaggatgtg agtgtaagtg
15301 tgtgtgtgtg tgtgtgaatg actttgcatg agtatgtgtg tgtatatatg tgtgaatgta
15361 cgtgtttgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgag gatgtgaatg tgcgtgtgcg
15421 cgtgtgtgtt gacaattgct tatgtactat atgttcatca caggacactc atggcggcca
15481 gacgataagc tgtggaagtg agctctctct cctgccttat aactcccggg gattgaatta
15541 agtcatcagg ttttgcagca agcatcctta tgtactaggc catcttgatt ttcaactata
15601 tcttgagcct cctactgctg aattagaacc attattacac tttcccaagt gttcttctaa
15661 ggctaaggct tctctgc
 
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated A is shown in red text.
References
Science Writers Nora G. Smart
Illustrators Diantha La Vine
AuthorsSungyong Won, Celine Eidenschenk, Bruce Beutler
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