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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

Analysis demonstrating BRCA1 is a member of the IFI16-inflammasome complex in cells latently infected with KSHV.(A-D) Cytoplasmic fractions of KSHV latently infected B lymphoma BCBL-1 (A) and endothelial TIVE-LTC (B) cells, and KSHV-negative B lymphoma BJAB (C) and endothelial TIVE (D) cells were IP-ed with anti-IFI16, BRCA1 or ASC antibodies and western blotted for IFI16, BRCA1 and caspase-1. IgG antibodies were used for specificity controls. Equal inputs for IPs were assessed by BRCA1, IFI16, ASC and Caspase-1 WBs, while tubulin and TBP WBs were done to confirm purity of cytoplasmic fractions. (E-I) PLA detecting IFI16 and BRCA1 complexes (E), IFI16 and ASC complexes (F), BRCA1 and ASC complexes (G), ASC and Caspase-1 complexes (H) and BRCA1 and Caspase-1 complexes (I) in KSHV-negative TIVE and KSHV positive TIVE-LTC cells. Boxed areas are enlarged. Red dots represent IFI16-BRCA1, IFI16-ASC, BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1 complexes. Yellow arrows indicate cytoplasmic localization of IFI16-BRCA1, IFI16-ASC, BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1 complexes. Red arrows indicate nuclear localization of IFI16-BRCA1, IFI16-ASC or BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1. Quantitative analysis of the average number of PLA spots/cell and that of nucleus vs cytoplasm of infected cells is presented in the right most columns. *: p<0.05; ***: p<0.001 and ns: not significant.
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ppat.1005030.g005: Analysis demonstrating BRCA1 is a member of the IFI16-inflammasome complex in cells latently infected with KSHV.(A-D) Cytoplasmic fractions of KSHV latently infected B lymphoma BCBL-1 (A) and endothelial TIVE-LTC (B) cells, and KSHV-negative B lymphoma BJAB (C) and endothelial TIVE (D) cells were IP-ed with anti-IFI16, BRCA1 or ASC antibodies and western blotted for IFI16, BRCA1 and caspase-1. IgG antibodies were used for specificity controls. Equal inputs for IPs were assessed by BRCA1, IFI16, ASC and Caspase-1 WBs, while tubulin and TBP WBs were done to confirm purity of cytoplasmic fractions. (E-I) PLA detecting IFI16 and BRCA1 complexes (E), IFI16 and ASC complexes (F), BRCA1 and ASC complexes (G), ASC and Caspase-1 complexes (H) and BRCA1 and Caspase-1 complexes (I) in KSHV-negative TIVE and KSHV positive TIVE-LTC cells. Boxed areas are enlarged. Red dots represent IFI16-BRCA1, IFI16-ASC, BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1 complexes. Yellow arrows indicate cytoplasmic localization of IFI16-BRCA1, IFI16-ASC, BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1 complexes. Red arrows indicate nuclear localization of IFI16-BRCA1, IFI16-ASC or BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1. Quantitative analysis of the average number of PLA spots/cell and that of nucleus vs cytoplasm of infected cells is presented in the right most columns. *: p<0.05; ***: p<0.001 and ns: not significant.

Mentions: Primary Effusion Lymphoma (PEL) derived BCBL-1 cells are KSHV latently infected B-cells with >80 copies of nuclear KSHV genomes and we have shown previously that IFI16 colocalizes with nuclear KSHV genome in latently infected cells resulting in the constitutive activation of IFI16 inflammasomes [3]. To examine the IFI16-BRCA1 interaction and redistribution in cells latently infected with KSHV, we used a combination of IP reactions with anti-IFI16,-BRCA1 or-ASC antibodies and Western blot reactions with cytoplasmic fractions from latent KSHV positive BCBL-1 and TIVE-LTC (endothelial) cells (Fig 5A and 5B) and also de novo KSHV infected (24 h) human foreskin fibroblast (HFF) cells (S4A Fig). We observed the association of BRCA1 with IFI16, ASC and Caspase-1 in the cytoplasm of infected cells (Fig 5A and 5B and S4A Fig) but not in the cytoplasmic fractions of control KSHV negative BJAB and TIVE (endothelial) (Fig 5C and 5D) or uninfected HFF cells.


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)

Analysis demonstrating BRCA1 is a member of the IFI16-inflammasome complex in cells latently infected with KSHV.(A-D) Cytoplasmic fractions of KSHV latently infected B lymphoma BCBL-1 (A) and endothelial TIVE-LTC (B) cells, and KSHV-negative B lymphoma BJAB (C) and endothelial TIVE (D) cells were IP-ed with anti-IFI16, BRCA1 or ASC antibodies and western blotted for IFI16, BRCA1 and caspase-1. IgG antibodies were used for specificity controls. Equal inputs for IPs were assessed by BRCA1, IFI16, ASC and Caspase-1 WBs, while tubulin and TBP WBs were done to confirm purity of cytoplasmic fractions. (E-I) PLA detecting IFI16 and BRCA1 complexes (E), IFI16 and ASC complexes (F), BRCA1 and ASC complexes (G), ASC and Caspase-1 complexes (H) and BRCA1 and Caspase-1 complexes (I) in KSHV-negative TIVE and KSHV positive TIVE-LTC cells. Boxed areas are enlarged. Red dots represent IFI16-BRCA1, IFI16-ASC, BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1 complexes. Yellow arrows indicate cytoplasmic localization of IFI16-BRCA1, IFI16-ASC, BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1 complexes. Red arrows indicate nuclear localization of IFI16-BRCA1, IFI16-ASC or BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1. Quantitative analysis of the average number of PLA spots/cell and that of nucleus vs cytoplasm of infected cells is presented in the right most columns. *: p<0.05; ***: p<0.001 and ns: not significant.
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Related In: Results  -  Collection

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ppat.1005030.g005: Analysis demonstrating BRCA1 is a member of the IFI16-inflammasome complex in cells latently infected with KSHV.(A-D) Cytoplasmic fractions of KSHV latently infected B lymphoma BCBL-1 (A) and endothelial TIVE-LTC (B) cells, and KSHV-negative B lymphoma BJAB (C) and endothelial TIVE (D) cells were IP-ed with anti-IFI16, BRCA1 or ASC antibodies and western blotted for IFI16, BRCA1 and caspase-1. IgG antibodies were used for specificity controls. Equal inputs for IPs were assessed by BRCA1, IFI16, ASC and Caspase-1 WBs, while tubulin and TBP WBs were done to confirm purity of cytoplasmic fractions. (E-I) PLA detecting IFI16 and BRCA1 complexes (E), IFI16 and ASC complexes (F), BRCA1 and ASC complexes (G), ASC and Caspase-1 complexes (H) and BRCA1 and Caspase-1 complexes (I) in KSHV-negative TIVE and KSHV positive TIVE-LTC cells. Boxed areas are enlarged. Red dots represent IFI16-BRCA1, IFI16-ASC, BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1 complexes. Yellow arrows indicate cytoplasmic localization of IFI16-BRCA1, IFI16-ASC, BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1 complexes. Red arrows indicate nuclear localization of IFI16-BRCA1, IFI16-ASC or BRCA1-ASC, ASC-Caspase-1 or BRCA1-Caspase-1. Quantitative analysis of the average number of PLA spots/cell and that of nucleus vs cytoplasm of infected cells is presented in the right most columns. *: p<0.05; ***: p<0.001 and ns: not significant.
Mentions: Primary Effusion Lymphoma (PEL) derived BCBL-1 cells are KSHV latently infected B-cells with >80 copies of nuclear KSHV genomes and we have shown previously that IFI16 colocalizes with nuclear KSHV genome in latently infected cells resulting in the constitutive activation of IFI16 inflammasomes [3]. To examine the IFI16-BRCA1 interaction and redistribution in cells latently infected with KSHV, we used a combination of IP reactions with anti-IFI16,-BRCA1 or-ASC antibodies and Western blot reactions with cytoplasmic fractions from latent KSHV positive BCBL-1 and TIVE-LTC (endothelial) cells (Fig 5A and 5B) and also de novo KSHV infected (24 h) human foreskin fibroblast (HFF) cells (S4A Fig). We observed the association of BRCA1 with IFI16, ASC and Caspase-1 in the cytoplasm of infected cells (Fig 5A and 5B and S4A Fig) but not in the cytoplasmic fractions of control KSHV negative BJAB and TIVE (endothelial) (Fig 5C and 5D) or uninfected HFF cells.

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