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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 interactions with PML bodies and DNA damage effectors reflect antiviral propertiesA SAINT-filtered IFIX interactions related to PML body function or DNA damage response were assessed by STRING and visualized in Cytoscape. Node colors correspond to the log2-transformed NSAF/PAX values. Several interactions failing to pass SAINT, but enriched ≥ twofold relative to GFP control immunoisolations were added manually (gray color).B Co-localization of IFIX-GFP with selected interactions in HEK293 cells was assessed by fluorescence confocal microscopy (white arrows). Scale bars, 5 μm.C IFIX-GFP-expressing HEK293 cells were infected at MOI = 0.1 with HSV-1. At 72 h post-infection, progeny virus was harvested and titered. Mean values ± SEM (n = 3) are shown. *P ≤ 0.05, compared to control GFP cells.D PML knockdown efficiency in HFFs relative to shScrambled (shSCR)-HFFs was assessed by Western blot (left). shPML-HFFs were infected at MOI = 0.1 with HSV-1. Progeny virus was harvested at 48 h post-infection and titered (right). Mean values ± SEM (n = 3) are shown. **P ≤ 0.01, compared to shSCR-HFFs.E ifixmRNA levels in shIFIX-HFFs were assessed by RT–qPCR to determine knockdown efficiency (left). mRNA levels were normalized to cellular β-actin levels. The basal level of ifix expression in shSCR HFF cells relative to actin was 1.8E-5, which corresponds to a raw Ct value of ˜31. shIFIX-HFFs were infected at MOI = 0.1 with HSV-1. Progeny virus was harvested at 48 h post-infection and titered (right). Mean values ± SEM (n = 3) are shown. *P ≤ 0.05, compared to shSCR-HFFs.Data information: In (C–E), Student's unpaired t-test; two-tailed.
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fig05: IFIX interactions with PML bodies and DNA damage effectors reflect antiviral propertiesA SAINT-filtered IFIX interactions related to PML body function or DNA damage response were assessed by STRING and visualized in Cytoscape. Node colors correspond to the log2-transformed NSAF/PAX values. Several interactions failing to pass SAINT, but enriched ≥ twofold relative to GFP control immunoisolations were added manually (gray color).B Co-localization of IFIX-GFP with selected interactions in HEK293 cells was assessed by fluorescence confocal microscopy (white arrows). Scale bars, 5 μm.C IFIX-GFP-expressing HEK293 cells were infected at MOI = 0.1 with HSV-1. At 72 h post-infection, progeny virus was harvested and titered. Mean values ± SEM (n = 3) are shown. *P ≤ 0.05, compared to control GFP cells.D PML knockdown efficiency in HFFs relative to shScrambled (shSCR)-HFFs was assessed by Western blot (left). shPML-HFFs were infected at MOI = 0.1 with HSV-1. Progeny virus was harvested at 48 h post-infection and titered (right). Mean values ± SEM (n = 3) are shown. **P ≤ 0.01, compared to shSCR-HFFs.E ifixmRNA levels in shIFIX-HFFs were assessed by RT–qPCR to determine knockdown efficiency (left). mRNA levels were normalized to cellular β-actin levels. The basal level of ifix expression in shSCR HFF cells relative to actin was 1.8E-5, which corresponds to a raw Ct value of ˜31. shIFIX-HFFs were infected at MOI = 0.1 with HSV-1. Progeny virus was harvested at 48 h post-infection and titered (right). Mean values ± SEM (n = 3) are shown. *P ≤ 0.05, compared to shSCR-HFFs.Data information: In (C–E), Student's unpaired t-test; two-tailed.

Mentions: Considering what little is known about the cellular functions of IFIX, we were intrigued by its interactions with several components of nuclear PML body structures and with DDR effectors. The prominence of these interactions is supported by their presence in both N- and C-terminally tagged IFIX IPs (Fig2A, Supplementary Fig S6, and Supplementary Table S4) and by their enrichment as assessed by NSAF/PAX analysis (Fig5A). Furthermore, although they failed to pass our SAINT filter criteria, several additional prey proteins integral to these cellular pathways had enriched spectral counts relative to the GFP control isolations. These included CBX5 interacting partners CBX1 (2.7-fold) and TRIM28 (3.8-fold), DNA damage response effector proteins PRKDC (3.4-fold) and histone variant H2AFX (2.8-fold), small ubiquitin-like modified SUMO2 (3.0-fold), and tumor suppressor TP53 (2.0-fold enriched). In agreement with its association with DNA repair pathway components, we observed a partial co-localization of IFIX with proteins known to be enriched at DSB foci—MRE11A and histone variant H2AFX phosphorylated at serine 139 (Fig5B). IFIX was also excluded from several DSB foci, suggesting its selective recruitment to sites of dsDNA repair.


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 interactions with PML bodies and DNA damage effectors reflect antiviral propertiesA SAINT-filtered IFIX interactions related to PML body function or DNA damage response were assessed by STRING and visualized in Cytoscape. Node colors correspond to the log2-transformed NSAF/PAX values. Several interactions failing to pass SAINT, but enriched ≥ twofold relative to GFP control immunoisolations were added manually (gray color).B Co-localization of IFIX-GFP with selected interactions in HEK293 cells was assessed by fluorescence confocal microscopy (white arrows). Scale bars, 5 μm.C IFIX-GFP-expressing HEK293 cells were infected at MOI = 0.1 with HSV-1. At 72 h post-infection, progeny virus was harvested and titered. Mean values ± SEM (n = 3) are shown. *P ≤ 0.05, compared to control GFP cells.D PML knockdown efficiency in HFFs relative to shScrambled (shSCR)-HFFs was assessed by Western blot (left). shPML-HFFs were infected at MOI = 0.1 with HSV-1. Progeny virus was harvested at 48 h post-infection and titered (right). Mean values ± SEM (n = 3) are shown. **P ≤ 0.01, compared to shSCR-HFFs.E ifixmRNA levels in shIFIX-HFFs were assessed by RT–qPCR to determine knockdown efficiency (left). mRNA levels were normalized to cellular β-actin levels. The basal level of ifix expression in shSCR HFF cells relative to actin was 1.8E-5, which corresponds to a raw Ct value of ˜31. shIFIX-HFFs were infected at MOI = 0.1 with HSV-1. Progeny virus was harvested at 48 h post-infection and titered (right). Mean values ± SEM (n = 3) are shown. *P ≤ 0.05, compared to shSCR-HFFs.Data information: In (C–E), Student's unpaired t-test; two-tailed.
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fig05: IFIX interactions with PML bodies and DNA damage effectors reflect antiviral propertiesA SAINT-filtered IFIX interactions related to PML body function or DNA damage response were assessed by STRING and visualized in Cytoscape. Node colors correspond to the log2-transformed NSAF/PAX values. Several interactions failing to pass SAINT, but enriched ≥ twofold relative to GFP control immunoisolations were added manually (gray color).B Co-localization of IFIX-GFP with selected interactions in HEK293 cells was assessed by fluorescence confocal microscopy (white arrows). Scale bars, 5 μm.C IFIX-GFP-expressing HEK293 cells were infected at MOI = 0.1 with HSV-1. At 72 h post-infection, progeny virus was harvested and titered. Mean values ± SEM (n = 3) are shown. *P ≤ 0.05, compared to control GFP cells.D PML knockdown efficiency in HFFs relative to shScrambled (shSCR)-HFFs was assessed by Western blot (left). shPML-HFFs were infected at MOI = 0.1 with HSV-1. Progeny virus was harvested at 48 h post-infection and titered (right). Mean values ± SEM (n = 3) are shown. **P ≤ 0.01, compared to shSCR-HFFs.E ifixmRNA levels in shIFIX-HFFs were assessed by RT–qPCR to determine knockdown efficiency (left). mRNA levels were normalized to cellular β-actin levels. The basal level of ifix expression in shSCR HFF cells relative to actin was 1.8E-5, which corresponds to a raw Ct value of ˜31. shIFIX-HFFs were infected at MOI = 0.1 with HSV-1. Progeny virus was harvested at 48 h post-infection and titered (right). Mean values ± SEM (n = 3) are shown. *P ≤ 0.05, compared to shSCR-HFFs.Data information: In (C–E), Student's unpaired t-test; two-tailed.
Mentions: Considering what little is known about the cellular functions of IFIX, we were intrigued by its interactions with several components of nuclear PML body structures and with DDR effectors. The prominence of these interactions is supported by their presence in both N- and C-terminally tagged IFIX IPs (Fig2A, Supplementary Fig S6, and Supplementary Table S4) and by their enrichment as assessed by NSAF/PAX analysis (Fig5A). Furthermore, although they failed to pass our SAINT filter criteria, several additional prey proteins integral to these cellular pathways had enriched spectral counts relative to the GFP control isolations. These included CBX5 interacting partners CBX1 (2.7-fold) and TRIM28 (3.8-fold), DNA damage response effector proteins PRKDC (3.4-fold) and histone variant H2AFX (2.8-fold), small ubiquitin-like modified SUMO2 (3.0-fold), and tumor suppressor TP53 (2.0-fold enriched). In agreement with its association with DNA repair pathway components, we observed a partial co-localization of IFIX with proteins known to be enriched at DSB foci—MRE11A and histone variant H2AFX phosphorylated at serine 139 (Fig5B). IFIX was also excluded from several DSB foci, suggesting its selective recruitment to sites of dsDNA repair.

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