Limits...
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

PLA analysis demonstrating abrogation of KSHV genome recognition by IFI16 in the absence of BRCA1 during de novo infection.(A and B) Z-stack analysis of control Si-RNA (Si-control) and BRCA1 (Si-BRCA1) knockdown HMVEC-d cells infected with EdU labeled KSHV for 24 h. Images of ten 1μm thick Z planes were acquired and analyzed. EdU labeled KSHV genome was detected by reaction with Alexa 594 labeled picolylazide (red) and IFI16 was detected using PLA as described above. (A) The presence of a significant association of IFI16 with EdU labeled KSHV genome in Si-Control HMVEC-d cells (white arrows) is indicated by a yellow color in the XZ and YZ planes. (B) The absence of IFI16 association with EdU labeled KSHV genome in Si-BRCA1 HMVEC-d cells is shown by the absence of yellow spots (white arrows) in the XZ and YZ planes. (C-F) PLA analysis of the time course of KSHV de novo infection showing the effect of BRCA1 absence on KSHV genome recognition by IFI16. HMVEC-d cells with Si-Control or Si-BRCA1 (BRCA1 knockdown) were infected with EdU labeled KSHV for the indicated time periods. IFI16 was detected by PLA using mouse and goat anti-IFI16 antibodies (green). Nuclei were stained with DAPI. EdU labeled viral genome was detected using Alexa 594 labeled azide (red). Numbered boxed areas are enlarged and shown in the rightmost two panels. Green PLA reaction dots indicate IFI16. Arrows (yellow spots) indicate EdU labeled KSHV genome (red spots) and its association with IFI16 (PLA green dots).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4487893&req=5

ppat.1005030.g011: PLA analysis demonstrating abrogation of KSHV genome recognition by IFI16 in the absence of BRCA1 during de novo infection.(A and B) Z-stack analysis of control Si-RNA (Si-control) and BRCA1 (Si-BRCA1) knockdown HMVEC-d cells infected with EdU labeled KSHV for 24 h. Images of ten 1μm thick Z planes were acquired and analyzed. EdU labeled KSHV genome was detected by reaction with Alexa 594 labeled picolylazide (red) and IFI16 was detected using PLA as described above. (A) The presence of a significant association of IFI16 with EdU labeled KSHV genome in Si-Control HMVEC-d cells (white arrows) is indicated by a yellow color in the XZ and YZ planes. (B) The absence of IFI16 association with EdU labeled KSHV genome in Si-BRCA1 HMVEC-d cells is shown by the absence of yellow spots (white arrows) in the XZ and YZ planes. (C-F) PLA analysis of the time course of KSHV de novo infection showing the effect of BRCA1 absence on KSHV genome recognition by IFI16. HMVEC-d cells with Si-Control or Si-BRCA1 (BRCA1 knockdown) were infected with EdU labeled KSHV for the indicated time periods. IFI16 was detected by PLA using mouse and goat anti-IFI16 antibodies (green). Nuclei were stained with DAPI. EdU labeled viral genome was detected using Alexa 594 labeled azide (red). Numbered boxed areas are enlarged and shown in the rightmost two panels. Green PLA reaction dots indicate IFI16. Arrows (yellow spots) indicate EdU labeled KSHV genome (red spots) and its association with IFI16 (PLA green dots).

Mentions: To investigate the functional implications of BRCA1 in KSHV genome recognition by the BRCA1-IFI16 complex, we verified the consequence of BRCA1 knockdown affecting KSHV genome sensing by IFI16 in HMVEC-d cells at 24 h post-EdU KSHV infection. In control Si-RNA treated cells, we observed the colocalization of nuclear IFI16 molecules (green PLA spots) with EdU KSHV genome (red) (Fig 10D, panels 4 and 5, and Fig 11A, yellow spots, white arrows). As seen before, IFI16 was also detected in the cytoplasm without any colocalization with the viral genome probe (Fig 10D, panels 2, 3 and 4, green spots, red arrows and Fig 11A). These results demonstrated the cytoplasmic redistribution of IFI16 during KSHV infection and the absence of free viral genome in the cytoplasm. Multiple IFI16-DNA colocalization spots indicated interactions with several viral genomes in the presence of functional BRCA1. In contrast, IFI16 was confined to the nucleus of the BRCA1 Si-RNA treated cells with notably decreased colocalization with the EdU KSHV genome at 24 h p.i. (Fig 10E, panels 4 and 5 and Fig 11B and 11F). Consistent with this result, in contrast to Si-Control cells infected with KSHV, very little colocalization of IFI16 PLA spots and EdU KSHV genome was observed in BRCA1 knockdown HMVEC-d cells even at the early time of KSHV infection (Fig 11D and 11E vs Fig 11C). Taken together, these results convincingly demonstrated that in the dynamic nuclear environment, IFI16 relies on BRCA1 to increase its affinity to foreign KSHV DNA leading into stable inflammasome complex formation and translocation into the cytoplasm of infected 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)

PLA analysis demonstrating abrogation of KSHV genome recognition by IFI16 in the absence of BRCA1 during de novo infection.(A and B) Z-stack analysis of control Si-RNA (Si-control) and BRCA1 (Si-BRCA1) knockdown HMVEC-d cells infected with EdU labeled KSHV for 24 h. Images of ten 1μm thick Z planes were acquired and analyzed. EdU labeled KSHV genome was detected by reaction with Alexa 594 labeled picolylazide (red) and IFI16 was detected using PLA as described above. (A) The presence of a significant association of IFI16 with EdU labeled KSHV genome in Si-Control HMVEC-d cells (white arrows) is indicated by a yellow color in the XZ and YZ planes. (B) The absence of IFI16 association with EdU labeled KSHV genome in Si-BRCA1 HMVEC-d cells is shown by the absence of yellow spots (white arrows) in the XZ and YZ planes. (C-F) PLA analysis of the time course of KSHV de novo infection showing the effect of BRCA1 absence on KSHV genome recognition by IFI16. HMVEC-d cells with Si-Control or Si-BRCA1 (BRCA1 knockdown) were infected with EdU labeled KSHV for the indicated time periods. IFI16 was detected by PLA using mouse and goat anti-IFI16 antibodies (green). Nuclei were stained with DAPI. EdU labeled viral genome was detected using Alexa 594 labeled azide (red). Numbered boxed areas are enlarged and shown in the rightmost two panels. Green PLA reaction dots indicate IFI16. Arrows (yellow spots) indicate EdU labeled KSHV genome (red spots) and its association with IFI16 (PLA green dots).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487893&req=5

ppat.1005030.g011: PLA analysis demonstrating abrogation of KSHV genome recognition by IFI16 in the absence of BRCA1 during de novo infection.(A and B) Z-stack analysis of control Si-RNA (Si-control) and BRCA1 (Si-BRCA1) knockdown HMVEC-d cells infected with EdU labeled KSHV for 24 h. Images of ten 1μm thick Z planes were acquired and analyzed. EdU labeled KSHV genome was detected by reaction with Alexa 594 labeled picolylazide (red) and IFI16 was detected using PLA as described above. (A) The presence of a significant association of IFI16 with EdU labeled KSHV genome in Si-Control HMVEC-d cells (white arrows) is indicated by a yellow color in the XZ and YZ planes. (B) The absence of IFI16 association with EdU labeled KSHV genome in Si-BRCA1 HMVEC-d cells is shown by the absence of yellow spots (white arrows) in the XZ and YZ planes. (C-F) PLA analysis of the time course of KSHV de novo infection showing the effect of BRCA1 absence on KSHV genome recognition by IFI16. HMVEC-d cells with Si-Control or Si-BRCA1 (BRCA1 knockdown) were infected with EdU labeled KSHV for the indicated time periods. IFI16 was detected by PLA using mouse and goat anti-IFI16 antibodies (green). Nuclei were stained with DAPI. EdU labeled viral genome was detected using Alexa 594 labeled azide (red). Numbered boxed areas are enlarged and shown in the rightmost two panels. Green PLA reaction dots indicate IFI16. Arrows (yellow spots) indicate EdU labeled KSHV genome (red spots) and its association with IFI16 (PLA green dots).
Mentions: To investigate the functional implications of BRCA1 in KSHV genome recognition by the BRCA1-IFI16 complex, we verified the consequence of BRCA1 knockdown affecting KSHV genome sensing by IFI16 in HMVEC-d cells at 24 h post-EdU KSHV infection. In control Si-RNA treated cells, we observed the colocalization of nuclear IFI16 molecules (green PLA spots) with EdU KSHV genome (red) (Fig 10D, panels 4 and 5, and Fig 11A, yellow spots, white arrows). As seen before, IFI16 was also detected in the cytoplasm without any colocalization with the viral genome probe (Fig 10D, panels 2, 3 and 4, green spots, red arrows and Fig 11A). These results demonstrated the cytoplasmic redistribution of IFI16 during KSHV infection and the absence of free viral genome in the cytoplasm. Multiple IFI16-DNA colocalization spots indicated interactions with several viral genomes in the presence of functional BRCA1. In contrast, IFI16 was confined to the nucleus of the BRCA1 Si-RNA treated cells with notably decreased colocalization with the EdU KSHV genome at 24 h p.i. (Fig 10E, panels 4 and 5 and Fig 11B and 11F). Consistent with this result, in contrast to Si-Control cells infected with KSHV, very little colocalization of IFI16 PLA spots and EdU KSHV genome was observed in BRCA1 knockdown HMVEC-d cells even at the early time of KSHV infection (Fig 11D and 11E vs Fig 11C). Taken together, these results convincingly demonstrated that in the dynamic nuclear environment, IFI16 relies on BRCA1 to increase its affinity to foreign KSHV DNA leading into stable inflammasome complex formation and translocation into the cytoplasm of infected 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