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The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA.

Diner BA, Li T, Greco TM, Crow MS, Fuesler JA, Wang J, Cristea IM - Mol. Syst. Biol. (2015)

Bottom Line: We discover that IFIX detects viral DNA in both the nucleus and cytoplasm, binding foreign DNA via its HIN domain in a sequence-non-specific manner.Furthermore, IFIX contributes to the induction of interferon response.Our results highlight the value of integrative proteomics in deducing protein function and establish IFIX as an antiviral DNA sensor important for mounting immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ, USA.

No MeSH data available.


Related in: MedlinePlus

IFIX is a DNA-binding protein that functions as a viral DNA sensorA Biotinylated ISD was incubated with either recombinant GST-tagged IFIX-PY (aa 1–100) or IFIX-HIN200 (aa 200–492) and competed off with unlabeled ISD. DNA–protein complexes were resolved by non-denaturing PAGE, and biotinylated substrates were visualized.B Biotinylated ISD, pcDNA3.1, or BamHI-linearized pcDNA3.1 were incubated with recombinant GST-tagged IFIX-HIN200 and competed off with unlabeled ISD. DNA–protein complexes were resolved as above.C Recombinant GST-tagged IFIX-HIN200 was incubated with a dsDNA array consisting of all possible 10mer oligonucleotide sequences, and DNA–protein binding was visualized with a fluorescent GST-specific antibody. To illustrate IFIX-HIN200 sequence preferences, fluorescence intensity is plotted against oligomer sequence content.D IFIX-GFP HEK293 cells were transfected with Cy3-labeled VACV 70mer and imaged by fluorescence confocal microscopy (315× zoom factor). White arrows indicate co-localization between IFIX-GFP and VACV 70mer.E Control or IFIX-GFP HEK293 cells were transfected with VACV 70mer, and total RNA was collected after 6 h. Relative induction of ifn-β was quantified by RT–qPCR. mRNA levels are normalized to cellular gapdh levels. The basal expression levels in the IFIX-inducible HEK293 cells for ifn-β (right bar at +Tet/-VACV70) and ifix (left bar at -Tet/-VAVC70) relative to gapdh were 2E-6 and 3E-6, respectively. This corresponds to raw CT values of ˜28 and 27 for ifn-β and ifix, respectively. Mean values ± SEM (n = 3) are shown. **P ≤ 0.01 compared to transfected control cells and mock-transfected IFIX-GFP-expressing cells (Student's unpaired t-test; two-tailed).F GFP or GFP-IFIX HEK293 cells were infected with HSV-1 (MOI = 5) and cross-linked at 7 h post-infection. Cell lysates were subjected to α-GFP immunoaffinity isolation, and viral DNA binding was assessed by RT–PCR using HSV-1-specific genome sequences (top). Isolated GFP or GFP-IFIX (green arrows) was assessed by Western blotting (bottom).G Localization of IFIX-GFP in HSV-1-infected HEK293 cells (MOI = 1; 4 h post-infection) was visualized by fluorescence confocal microscopy. Viral protein ICP27: marker for infection. Scale bars, 5 μm.
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fig06: IFIX is a DNA-binding protein that functions as a viral DNA sensorA Biotinylated ISD was incubated with either recombinant GST-tagged IFIX-PY (aa 1–100) or IFIX-HIN200 (aa 200–492) and competed off with unlabeled ISD. DNA–protein complexes were resolved by non-denaturing PAGE, and biotinylated substrates were visualized.B Biotinylated ISD, pcDNA3.1, or BamHI-linearized pcDNA3.1 were incubated with recombinant GST-tagged IFIX-HIN200 and competed off with unlabeled ISD. DNA–protein complexes were resolved as above.C Recombinant GST-tagged IFIX-HIN200 was incubated with a dsDNA array consisting of all possible 10mer oligonucleotide sequences, and DNA–protein binding was visualized with a fluorescent GST-specific antibody. To illustrate IFIX-HIN200 sequence preferences, fluorescence intensity is plotted against oligomer sequence content.D IFIX-GFP HEK293 cells were transfected with Cy3-labeled VACV 70mer and imaged by fluorescence confocal microscopy (315× zoom factor). White arrows indicate co-localization between IFIX-GFP and VACV 70mer.E Control or IFIX-GFP HEK293 cells were transfected with VACV 70mer, and total RNA was collected after 6 h. Relative induction of ifn-β was quantified by RT–qPCR. mRNA levels are normalized to cellular gapdh levels. The basal expression levels in the IFIX-inducible HEK293 cells for ifn-β (right bar at +Tet/-VACV70) and ifix (left bar at -Tet/-VAVC70) relative to gapdh were 2E-6 and 3E-6, respectively. This corresponds to raw CT values of ˜28 and 27 for ifn-β and ifix, respectively. Mean values ± SEM (n = 3) are shown. **P ≤ 0.01 compared to transfected control cells and mock-transfected IFIX-GFP-expressing cells (Student's unpaired t-test; two-tailed).F GFP or GFP-IFIX HEK293 cells were infected with HSV-1 (MOI = 5) and cross-linked at 7 h post-infection. Cell lysates were subjected to α-GFP immunoaffinity isolation, and viral DNA binding was assessed by RT–PCR using HSV-1-specific genome sequences (top). Isolated GFP or GFP-IFIX (green arrows) was assessed by Western blotting (bottom).G Localization of IFIX-GFP in HSV-1-infected HEK293 cells (MOI = 1; 4 h post-infection) was visualized by fluorescence confocal microscopy. Viral protein ICP27: marker for infection. Scale bars, 5 μm.

Mentions: Considering that immune sensors are activated via direct interaction with their cognate ligand, we first assessed the ability of IFIX to bind pathogenic dsDNA in vitro. To determine the domain of IFIX mediating such binding, we generated PY-only (amino acids 1–100; herein IFIX-PY) and HIN200-only (amino acids 200–492; herein IFIX-HIN200) truncations. For the binding assays, we used the interferon-stimulating dsDNA (ISD) sequence, previously used in studies of IFI16 (Unterholzner et al, 2010). As shown by electrophoretic mobility shift assay (EMSA) (Fig6A), the purified IFIX-HIN200 effectively binds biotinylated ISD, and this association could be titrated away using unlabeled ISD probe. In contrast, no shift was observed using purified IFIX-PY. Thus, the HIN200 domain of IFIX is both necessary and sufficient for binding dsDNA.


The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA.

Diner BA, Li T, Greco TM, Crow MS, Fuesler JA, Wang J, Cristea IM - Mol. Syst. Biol. (2015)

IFIX is a DNA-binding protein that functions as a viral DNA sensorA Biotinylated ISD was incubated with either recombinant GST-tagged IFIX-PY (aa 1–100) or IFIX-HIN200 (aa 200–492) and competed off with unlabeled ISD. DNA–protein complexes were resolved by non-denaturing PAGE, and biotinylated substrates were visualized.B Biotinylated ISD, pcDNA3.1, or BamHI-linearized pcDNA3.1 were incubated with recombinant GST-tagged IFIX-HIN200 and competed off with unlabeled ISD. DNA–protein complexes were resolved as above.C Recombinant GST-tagged IFIX-HIN200 was incubated with a dsDNA array consisting of all possible 10mer oligonucleotide sequences, and DNA–protein binding was visualized with a fluorescent GST-specific antibody. To illustrate IFIX-HIN200 sequence preferences, fluorescence intensity is plotted against oligomer sequence content.D IFIX-GFP HEK293 cells were transfected with Cy3-labeled VACV 70mer and imaged by fluorescence confocal microscopy (315× zoom factor). White arrows indicate co-localization between IFIX-GFP and VACV 70mer.E Control or IFIX-GFP HEK293 cells were transfected with VACV 70mer, and total RNA was collected after 6 h. Relative induction of ifn-β was quantified by RT–qPCR. mRNA levels are normalized to cellular gapdh levels. The basal expression levels in the IFIX-inducible HEK293 cells for ifn-β (right bar at +Tet/-VACV70) and ifix (left bar at -Tet/-VAVC70) relative to gapdh were 2E-6 and 3E-6, respectively. This corresponds to raw CT values of ˜28 and 27 for ifn-β and ifix, respectively. Mean values ± SEM (n = 3) are shown. **P ≤ 0.01 compared to transfected control cells and mock-transfected IFIX-GFP-expressing cells (Student's unpaired t-test; two-tailed).F GFP or GFP-IFIX HEK293 cells were infected with HSV-1 (MOI = 5) and cross-linked at 7 h post-infection. Cell lysates were subjected to α-GFP immunoaffinity isolation, and viral DNA binding was assessed by RT–PCR using HSV-1-specific genome sequences (top). Isolated GFP or GFP-IFIX (green arrows) was assessed by Western blotting (bottom).G Localization of IFIX-GFP in HSV-1-infected HEK293 cells (MOI = 1; 4 h post-infection) was visualized by fluorescence confocal microscopy. Viral protein ICP27: marker for infection. Scale bars, 5 μm.
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fig06: IFIX is a DNA-binding protein that functions as a viral DNA sensorA Biotinylated ISD was incubated with either recombinant GST-tagged IFIX-PY (aa 1–100) or IFIX-HIN200 (aa 200–492) and competed off with unlabeled ISD. DNA–protein complexes were resolved by non-denaturing PAGE, and biotinylated substrates were visualized.B Biotinylated ISD, pcDNA3.1, or BamHI-linearized pcDNA3.1 were incubated with recombinant GST-tagged IFIX-HIN200 and competed off with unlabeled ISD. DNA–protein complexes were resolved as above.C Recombinant GST-tagged IFIX-HIN200 was incubated with a dsDNA array consisting of all possible 10mer oligonucleotide sequences, and DNA–protein binding was visualized with a fluorescent GST-specific antibody. To illustrate IFIX-HIN200 sequence preferences, fluorescence intensity is plotted against oligomer sequence content.D IFIX-GFP HEK293 cells were transfected with Cy3-labeled VACV 70mer and imaged by fluorescence confocal microscopy (315× zoom factor). White arrows indicate co-localization between IFIX-GFP and VACV 70mer.E Control or IFIX-GFP HEK293 cells were transfected with VACV 70mer, and total RNA was collected after 6 h. Relative induction of ifn-β was quantified by RT–qPCR. mRNA levels are normalized to cellular gapdh levels. The basal expression levels in the IFIX-inducible HEK293 cells for ifn-β (right bar at +Tet/-VACV70) and ifix (left bar at -Tet/-VAVC70) relative to gapdh were 2E-6 and 3E-6, respectively. This corresponds to raw CT values of ˜28 and 27 for ifn-β and ifix, respectively. Mean values ± SEM (n = 3) are shown. **P ≤ 0.01 compared to transfected control cells and mock-transfected IFIX-GFP-expressing cells (Student's unpaired t-test; two-tailed).F GFP or GFP-IFIX HEK293 cells were infected with HSV-1 (MOI = 5) and cross-linked at 7 h post-infection. Cell lysates were subjected to α-GFP immunoaffinity isolation, and viral DNA binding was assessed by RT–PCR using HSV-1-specific genome sequences (top). Isolated GFP or GFP-IFIX (green arrows) was assessed by Western blotting (bottom).G Localization of IFIX-GFP in HSV-1-infected HEK293 cells (MOI = 1; 4 h post-infection) was visualized by fluorescence confocal microscopy. Viral protein ICP27: marker for infection. Scale bars, 5 μm.
Mentions: Considering that immune sensors are activated via direct interaction with their cognate ligand, we first assessed the ability of IFIX to bind pathogenic dsDNA in vitro. To determine the domain of IFIX mediating such binding, we generated PY-only (amino acids 1–100; herein IFIX-PY) and HIN200-only (amino acids 200–492; herein IFIX-HIN200) truncations. For the binding assays, we used the interferon-stimulating dsDNA (ISD) sequence, previously used in studies of IFI16 (Unterholzner et al, 2010). As shown by electrophoretic mobility shift assay (EMSA) (Fig6A), the purified IFIX-HIN200 effectively binds biotinylated ISD, and this association could be titrated away using unlabeled ISD probe. In contrast, no shift was observed using purified IFIX-PY. Thus, the HIN200 domain of IFIX is both necessary and sufficient for binding dsDNA.

Bottom Line: We discover that IFIX detects viral DNA in both the nucleus and cytoplasm, binding foreign DNA via its HIN domain in a sequence-non-specific manner.Furthermore, IFIX contributes to the induction of interferon response.Our results highlight the value of integrative proteomics in deducing protein function and establish IFIX as an antiviral DNA sensor important for mounting immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ, USA.

No MeSH data available.


Related in: MedlinePlus