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BRCA1 Regulates IFI16 Mediated Nuclear Innate Sensing of Herpes Viral DNA and Subsequent Induction of the Innate Inflammasome and Interferon-β Responses.

Dutta D, Dutta S, Veettil MV, Roy A, Ansari MA, Iqbal J, Chikoti L, Kumar B, Johnson KE, Chandran B - PLoS Pathog. (2015)

Bottom Line: The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β).The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production.These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

View Article: PubMed Central - PubMed

Affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America.

ABSTRACT
The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

No MeSH data available.


Related in: MedlinePlus

Enhanced interaction of IFI16 with BRCA1 but not with other DNA damage response proteins during de novo KSHV infection.(A-C) Analysis of de novo infection with BrdU or EdU genome labeled KSHV. (A) HMVEC-d cells were infected with BrdU genome labeled KSHV (30 DNA copies/cell) for 0.25 h and processed for IFA. Cells were fixed, permeabilized, treated with 4 N HCl and reacted with anti-BrdU antibodies followed by Alexa Fluor-488 secondary antibodies. Green spots represent BrdU KSHV genomes of representative virus infected cells. Yellow arrows: nuclear KSHV genome; white arrows: cytoplasmic KSHV genomes. (B) HFF cells uninfected or infected with EdU genome labeled virus for the indicated time points were processed for detection of EdU viral genome by Click-reaction with Alexa 594 labeled azide. Red spots and arrows indicate cytoplasmic or nuclear viral genome. Nuclei were stained with DAPI (blue). (C) Nuclear and cytoplasmic distribution of IFI16 during EdU KSHV infection (4 h p.i.) as shown in (B). HFF cells infected with EdU genome labeled KSHV (30 DNA copies/cell) were processed for IFA to detect IFI16 with anti-IFI16 antibodies followed by Alexa Fluor-488 secondary antibodies and then EdU labeled viral genomes were detected with Alexa 594 labeled azide by Click-reaction. Green staining represents IFI16; white arrows indicate cytoplasmic IFI16 in EdU (red) virus infected cells but not in uninfected cells. Nuclei were stained with DAPI (blue). (D and E) HMVEC-d cells infected with KSHV (30 DNA copies/cell) for 4 and 24 h. Lysates from uninfected (UI) and infected cells immunoprecipitated (IP-ed) with (D) anti-IFI16 and (E) BRCA1 antibodies and western blotted (WB) for BRCA1, IFI16, CHK2 and H2AX proteins. (F) Input controls for IP reactions in D and E. Whole cell-lysates (WCL) were blotted with anti-BRCA1, IFI16, CHK2, H2AX or β-actin antibodies. (G) Lysates from the above experiments (D and E) were used to IP with species specific IgG antibodies and WB for BRCA1, IFI16, CHK2 and H2AX for specificity controls (Ga). IP inputs were assessed by BRCA1, IFI16, CHK2, H2AX and β-actin WBs (Gb).
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ppat.1005030.g001: Enhanced interaction of IFI16 with BRCA1 but not with other DNA damage response proteins during de novo KSHV infection.(A-C) Analysis of de novo infection with BrdU or EdU genome labeled KSHV. (A) HMVEC-d cells were infected with BrdU genome labeled KSHV (30 DNA copies/cell) for 0.25 h and processed for IFA. Cells were fixed, permeabilized, treated with 4 N HCl and reacted with anti-BrdU antibodies followed by Alexa Fluor-488 secondary antibodies. Green spots represent BrdU KSHV genomes of representative virus infected cells. Yellow arrows: nuclear KSHV genome; white arrows: cytoplasmic KSHV genomes. (B) HFF cells uninfected or infected with EdU genome labeled virus for the indicated time points were processed for detection of EdU viral genome by Click-reaction with Alexa 594 labeled azide. Red spots and arrows indicate cytoplasmic or nuclear viral genome. Nuclei were stained with DAPI (blue). (C) Nuclear and cytoplasmic distribution of IFI16 during EdU KSHV infection (4 h p.i.) as shown in (B). HFF cells infected with EdU genome labeled KSHV (30 DNA copies/cell) were processed for IFA to detect IFI16 with anti-IFI16 antibodies followed by Alexa Fluor-488 secondary antibodies and then EdU labeled viral genomes were detected with Alexa 594 labeled azide by Click-reaction. Green staining represents IFI16; white arrows indicate cytoplasmic IFI16 in EdU (red) virus infected cells but not in uninfected cells. Nuclei were stained with DAPI (blue). (D and E) HMVEC-d cells infected with KSHV (30 DNA copies/cell) for 4 and 24 h. Lysates from uninfected (UI) and infected cells immunoprecipitated (IP-ed) with (D) anti-IFI16 and (E) BRCA1 antibodies and western blotted (WB) for BRCA1, IFI16, CHK2 and H2AX proteins. (F) Input controls for IP reactions in D and E. Whole cell-lysates (WCL) were blotted with anti-BRCA1, IFI16, CHK2, H2AX or β-actin antibodies. (G) Lysates from the above experiments (D and E) were used to IP with species specific IgG antibodies and WB for BRCA1, IFI16, CHK2 and H2AX for specificity controls (Ga). IP inputs were assessed by BRCA1, IFI16, CHK2, H2AX and β-actin WBs (Gb).

Mentions: To test this hypothesis, we utilized de novo KSHV infection that is well-characterized in our earlier studies [2, 3, 14] in which infected cells are identified by a variety of methods such as i) entry of KSHV into the cytoplasm measured by immunofluorescence assay (IFA) for viral envelope and capsid proteins or 5-bromo-2’-deoxyuridine (BrdU) or 5-ethynyl-2’-deoxyuridine (EdU) labeled KSHV genome, and ii) entry of viral DNA into the nucleus measured by IFA for BrdU or EdU and nuclear expression of viral latency associated LANA-1 protein by IFA [2, 3, 14]. In addition, relocalization of nuclear IFI16 into the cytoplasm is also considered as an indicator of infection [2, 3]. When HMVEC-d cells were infected with KSHV containing BrdU genome (30 DNA copies/cell) and immunostained with anti-BrdU antibodies (Table 1), several BrdU labeled viral particles (Fig 1A, green spots) were detected both in the cytoplasm as well as in the infected cell nucleus at 15 min (0.25h) p.i. (Fig 1A, white and yellow arrows, respectively). Similarly, when HFF cells were infected with KSHV containing EdU labeled viral genome (30 DNA copies/cell), we observed a gradual increase in nuclear entry of viral DNA from the cytoplasm during the observed 0.25 h, 0.5 h and 4 h p.i. (Fig 1B, arrows). When these cells were stained for IFI16, we observed the colocalization of IFI16 with the viral genomes in the nucleus (Fig 1C, panels 3 and 4). In addition, IFI16 was observed only in the cytoplasm of cells with nuclear-EdU KSHV genome (Fig 1C, white arrows) and not in the uninfected HFF cells. These observations also support our assertion that relocalization of nuclear IFI16 into the cytoplasm is an indication of KSHV infection. We utilized similar concentrations of labeled and unlabeled virus in all our experiments.


BRCA1 Regulates IFI16 Mediated Nuclear Innate Sensing of Herpes Viral DNA and Subsequent Induction of the Innate Inflammasome and Interferon-β Responses.

Dutta D, Dutta S, Veettil MV, Roy A, Ansari MA, Iqbal J, Chikoti L, Kumar B, Johnson KE, Chandran B - PLoS Pathog. (2015)

Enhanced interaction of IFI16 with BRCA1 but not with other DNA damage response proteins during de novo KSHV infection.(A-C) Analysis of de novo infection with BrdU or EdU genome labeled KSHV. (A) HMVEC-d cells were infected with BrdU genome labeled KSHV (30 DNA copies/cell) for 0.25 h and processed for IFA. Cells were fixed, permeabilized, treated with 4 N HCl and reacted with anti-BrdU antibodies followed by Alexa Fluor-488 secondary antibodies. Green spots represent BrdU KSHV genomes of representative virus infected cells. Yellow arrows: nuclear KSHV genome; white arrows: cytoplasmic KSHV genomes. (B) HFF cells uninfected or infected with EdU genome labeled virus for the indicated time points were processed for detection of EdU viral genome by Click-reaction with Alexa 594 labeled azide. Red spots and arrows indicate cytoplasmic or nuclear viral genome. Nuclei were stained with DAPI (blue). (C) Nuclear and cytoplasmic distribution of IFI16 during EdU KSHV infection (4 h p.i.) as shown in (B). HFF cells infected with EdU genome labeled KSHV (30 DNA copies/cell) were processed for IFA to detect IFI16 with anti-IFI16 antibodies followed by Alexa Fluor-488 secondary antibodies and then EdU labeled viral genomes were detected with Alexa 594 labeled azide by Click-reaction. Green staining represents IFI16; white arrows indicate cytoplasmic IFI16 in EdU (red) virus infected cells but not in uninfected cells. Nuclei were stained with DAPI (blue). (D and E) HMVEC-d cells infected with KSHV (30 DNA copies/cell) for 4 and 24 h. Lysates from uninfected (UI) and infected cells immunoprecipitated (IP-ed) with (D) anti-IFI16 and (E) BRCA1 antibodies and western blotted (WB) for BRCA1, IFI16, CHK2 and H2AX proteins. (F) Input controls for IP reactions in D and E. Whole cell-lysates (WCL) were blotted with anti-BRCA1, IFI16, CHK2, H2AX or β-actin antibodies. (G) Lysates from the above experiments (D and E) were used to IP with species specific IgG antibodies and WB for BRCA1, IFI16, CHK2 and H2AX for specificity controls (Ga). IP inputs were assessed by BRCA1, IFI16, CHK2, H2AX and β-actin WBs (Gb).
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Show All Figures
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ppat.1005030.g001: Enhanced interaction of IFI16 with BRCA1 but not with other DNA damage response proteins during de novo KSHV infection.(A-C) Analysis of de novo infection with BrdU or EdU genome labeled KSHV. (A) HMVEC-d cells were infected with BrdU genome labeled KSHV (30 DNA copies/cell) for 0.25 h and processed for IFA. Cells were fixed, permeabilized, treated with 4 N HCl and reacted with anti-BrdU antibodies followed by Alexa Fluor-488 secondary antibodies. Green spots represent BrdU KSHV genomes of representative virus infected cells. Yellow arrows: nuclear KSHV genome; white arrows: cytoplasmic KSHV genomes. (B) HFF cells uninfected or infected with EdU genome labeled virus for the indicated time points were processed for detection of EdU viral genome by Click-reaction with Alexa 594 labeled azide. Red spots and arrows indicate cytoplasmic or nuclear viral genome. Nuclei were stained with DAPI (blue). (C) Nuclear and cytoplasmic distribution of IFI16 during EdU KSHV infection (4 h p.i.) as shown in (B). HFF cells infected with EdU genome labeled KSHV (30 DNA copies/cell) were processed for IFA to detect IFI16 with anti-IFI16 antibodies followed by Alexa Fluor-488 secondary antibodies and then EdU labeled viral genomes were detected with Alexa 594 labeled azide by Click-reaction. Green staining represents IFI16; white arrows indicate cytoplasmic IFI16 in EdU (red) virus infected cells but not in uninfected cells. Nuclei were stained with DAPI (blue). (D and E) HMVEC-d cells infected with KSHV (30 DNA copies/cell) for 4 and 24 h. Lysates from uninfected (UI) and infected cells immunoprecipitated (IP-ed) with (D) anti-IFI16 and (E) BRCA1 antibodies and western blotted (WB) for BRCA1, IFI16, CHK2 and H2AX proteins. (F) Input controls for IP reactions in D and E. Whole cell-lysates (WCL) were blotted with anti-BRCA1, IFI16, CHK2, H2AX or β-actin antibodies. (G) Lysates from the above experiments (D and E) were used to IP with species specific IgG antibodies and WB for BRCA1, IFI16, CHK2 and H2AX for specificity controls (Ga). IP inputs were assessed by BRCA1, IFI16, CHK2, H2AX and β-actin WBs (Gb).
Mentions: To test this hypothesis, we utilized de novo KSHV infection that is well-characterized in our earlier studies [2, 3, 14] in which infected cells are identified by a variety of methods such as i) entry of KSHV into the cytoplasm measured by immunofluorescence assay (IFA) for viral envelope and capsid proteins or 5-bromo-2’-deoxyuridine (BrdU) or 5-ethynyl-2’-deoxyuridine (EdU) labeled KSHV genome, and ii) entry of viral DNA into the nucleus measured by IFA for BrdU or EdU and nuclear expression of viral latency associated LANA-1 protein by IFA [2, 3, 14]. In addition, relocalization of nuclear IFI16 into the cytoplasm is also considered as an indicator of infection [2, 3]. When HMVEC-d cells were infected with KSHV containing BrdU genome (30 DNA copies/cell) and immunostained with anti-BrdU antibodies (Table 1), several BrdU labeled viral particles (Fig 1A, green spots) were detected both in the cytoplasm as well as in the infected cell nucleus at 15 min (0.25h) p.i. (Fig 1A, white and yellow arrows, respectively). Similarly, when HFF cells were infected with KSHV containing EdU labeled viral genome (30 DNA copies/cell), we observed a gradual increase in nuclear entry of viral DNA from the cytoplasm during the observed 0.25 h, 0.5 h and 4 h p.i. (Fig 1B, arrows). When these cells were stained for IFI16, we observed the colocalization of IFI16 with the viral genomes in the nucleus (Fig 1C, panels 3 and 4). In addition, IFI16 was observed only in the cytoplasm of cells with nuclear-EdU KSHV genome (Fig 1C, white arrows) and not in the uninfected HFF cells. These observations also support our assertion that relocalization of nuclear IFI16 into the cytoplasm is an indication of KSHV infection. We utilized similar concentrations of labeled and unlabeled virus in all our experiments.

Bottom Line: The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β).The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production.These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

View Article: PubMed Central - PubMed

Affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America.

ABSTRACT
The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

No MeSH data available.


Related in: MedlinePlus