Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses

Abstract

The IL-1β and type I interferon-β (IFN-β) molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16) involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1) episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the increased nuclear acetylation of IFI16 as a dynamic essential post-genome recognition event in the nucleus that is common to the IFI16-mediated innate responses of inflammasome induction and IFN-β production during herpesvirus (KSHV, EBV, HSV-1) infections.

Fig 1. Colocalization of IFI16 with BrdU genome labeled KSHV in the nucleus, acetylation of IFI16 in the nucleus and cytoplasmic redistribution during de novo KSHV infection of HMVEC-d cells.

(A) HMVEC-d cells were infected for 15 and 30 min with BrdU genome labeled KSHV (30 DNA copies/cell), processed for IFA, reacted with anti-IFI16 and anti-BrdU antibodies followed by Alexa Fluor-594/488 secondary antibodies and DAPI (blue). The boxed areas are enlarged. Red arrows: KSHV genome in the cytoplasm (green dots); yellow arrows: cytoplasmic IFI16; white arrows: colocalization of IFI16 with KSHV genome (yellow spots) in the nucleus. 60X magnification. (B) HMVEC-d cells were uninfected (UI) or infected with KSHV for different time points, nuclear and cytoplasmic fractions isolated and western blotted for IFI16. Tubulin and TBP were used as purity markers for cytoplasmic and nuclear fractions, respectively, and as loading controls. (C and D) Equal quantities of whole cell lysate (WCL) proteins from uninfected and KSHV infected (24 h) cells in NETN buffer were immunoprecipitated (IP-ed) with anti-acetylated lysine antibody and western blotted for IFI16 and tubulin (C), and cytoplasmic and nuclear proteins (D) were IP-ed with anti-acetylated lysine antibody and western blotted for IFI16, tubulin and H3. Cytoplasmic and nuclear proteins were western blotted for total H3, tubulin and TBP. (E) HMVEC-d cells were serum-starved without (UT = untreated) or with 1μM p300 inhibitor C-646 for 2 h, uninfected or infected with KSHV for 2 h, washed and incubated in complete medium in the presence or absence of inhibitor for 4 and 24 h. WCL in NETN buffer were IP-ed with anti-acetylated lysine or IFI16 antibodies and western blotted for IFI16. (F) 24 h lysates from (E) were IP-ed with anti-IFI16 and western blotted for acetylated lysine. (G) HMVEC-d cells treated or untreated with C-646 for 2 h were either left uninfected (UI) or infected with KSHV for various time points, nuclear and cytoplasmic fractions isolated and western blotted for IFI16.

Fig 2. Proximity Ligation Assay (PLA) of IFI16’s acetylation and cytoplasmic redistribution during de novo KSHV infection of HMVEC-d cells.

(A and B) HMVEC-d cells were preincubated with C-646 for 2 h, washed, infected with KSHV for 2 h, washed and incubated in complete medium with or without C-646. (A) Cells were reacted with anti-IFI16 mouse and rabbit antibodies, washed, and subjected to PLA by incubating with species specific PLA oligo probe tagged secondary antibodies, washed, incubated with ligation mixture containing ligase, amplification solution with polymerase, and fluorescently labeled oligonucleotides. The signal, detected as a fluorescent dot at 594 nm wave length, was visualized by fluorescence microscopy. Nuclei were stained with DAPI, boxed areas are enlarged and the red dots represent IFI16. The yellow and white arrows indicate the nuclear and cytoplasmic IFI16, respectively. (B) PLA with anti-IFI16 and anti-acetylated lysine antibodies. White arrows and yellow arrows indicate cytoplasmic and nuclear acetylated IFI16, respectively. (C) HMVEC-d cells uninfected or infected with live or UV-KSHV (30 DNA copies/cell) for 30 min or 2 h. The 2 h infected cells were further incubated for 4 or 24 h, and WCL proteins in NETN buffer were IP-ed with anti-acetylated lysine antibody and western blotted for IFI16. The bands were scanned and quantitated using FluorChemFC2 software and an AlphaImager system. Total IFI16 and tubulin were immunoblotted as input and loading controls, respectively.

Fig 3. Effect of p300 inhibitor C-646 on IFI16 and Ran-GTPase association and effect of nuclear export blockage by Leptomycin B and protein synthesis inhibitor cycloheximide on the redistribution of IFI16 during de novo KSHV infection of HMVEC-d cells.

(A) HMVEC-d cells preincubated with 1 μM C-646 were uninfected or infected with KSHV for 2 h, washed and incubated for 4 h without (UT) or with 1 μM C-646. WCL proteins in NETN lysis buffer were IP-ed with anti-Ran antibodies and immunoblotted for IFI16 and Ran, or IP-ed with IFI16 and WB for IFI16, samples were also immunoblotted for total IFI16, Ran and tubulin. (B) HMVEC-d cells preincubated with 1 μM C-646 were uninfected or infected for 2 h, washed, incubated in the absence or presence of 1 μM C-646 for 2 h, and subjected to PLA with anti-Ran-GTPase and IFI16 antibodies. White and yellow arrows denote the cytoplasmic and nuclear association of Ran and IFI16, respectively. (C and D) HMVEC-d cells preincubated in the presence or absence of 50 nM Leptomycin B (LPT) for 2 h were uninfected (UI) or infected with KSHV for 2 h, washed, incubated for 24 h with or without LPT. (C) Nuclear or cytoplasmic fraction proteins were western blotted for IFI16, cyclin B1, caspase-1, TBP and tubulin. (D) WCL proteins were IP-ed with anti-acetylated lysine or anti-ASC antibodies and western blotted for IFI16 and tubulin. Proteins were also IP-ed for ASC or IFI16 and WB with ASC or IFI16 antibodies, respectively. Levels of total IFI16, ASC and tubulin were detected with their respective antibodies. (E) HMVEC-d cells were starved for 2 h with 200 μg/ml cycloheximide (CHX), washed, infected with KSHV for 2 h, washed, incubated for 4 h with or without CHX. Cytoplasmic and nuclear fractions were subjected to western blot for IFI16, TBP and tubulin. (F and G) HMVEC-d cells in (C) were subjected to PLA. (F) Anti-IFI16 mouse and rabbit antibodies. Red dots: IFI16; yellow arrows: nuclear IFI16; white arrows: cytoplasmic IFI16. (G) Anti-IFI16 and anti-ASC antibodies. Red dots: IFI16-ASC association. Yellow arrows: nuclear IFI16-ASC interaction. White arrow: cytoplasmic IFI16-ASC interaction. Magnification: 60X.

Fig 4. IFI16-ASC inflammasome formation, IFI16 aggregation and consequences during de novo KSHV infection in the presence of C-646.

(A) HMVEC-d cells preincubated with or without 1 μM C-646 for 2 h were uninfected or infected with KSHV for 2 h, washed, incubated in complete medium for 24 h with or without C-646 and analyzed by PLA with anti-IFI16 and anti-ASC antibodies. The boxed areas are enlarged. White arrows and yellow arrows indicate cytoplasmic and nuclear IFI16+ASC complexes, respectively. (B) BJAB and BCBL-1 cells untreated or treated with 1 μM C-646 for 24 h were analyzed by PLA with anti-IFI16 and anti-ASC antibodies. White arrows and yellow arrows indicate cytoplasmic and nuclear IFI16+ASC complexes, respectively. (C) HMVEC-d cells preincubated with or without 1 μM C-646 for 2 h were washed, uninfected or infected with KSHV for 2 h, washed and incubated in complete medium with or without C-646 for 4 and 24 h. WCL proteins were IP-ed with anti-ASC antibodies and western blotted for IFI16 and ASC. Total IFI16 and ASC were used as input controls and tubulin was used as loading control. (D) WCL proteins from BJAB and BCBL-1 cells, untreated or treated with 1 μM C-646 for 24 h, were IP-ed with anti-ASC antibodies and western blotted for IFI16. (E and F) HMVEC-d cells starved with or without 1μM C-646 for 2 h, washed, infected with KSHV for 2 h, washed, incubated with complete medium for 24 h in the presence or absence of 1 μM C-646 (E). BJAB or BCBL-1 cells were untreated or treated with 1 μM C-646 for 24 h (F). Whole cell lysates were prepared in HEPES-lysis buffer, equal amount of proteins were cross-linked using glutaraldehyde and immunoblotted for IFI16. Tubulin was used as loading control. (G) The protein samples from panel C experiments were western blotted for caspase-1, IL-1β and IL-33. (H) The protein samples from panel D experiments were western blotted for caspase-1 and IL-1β. (I) Culture supernatants of HMVEC-d cells infected with KSHV in the presence or absence of C-646 were evaluated for IL-1β by ELISA.

Fig 5. Effect of ASC knockdown on IFI16 acetylation and translocation.

(A) HMVEC-d cells electroporated with Si-ASC and Si-control RNAs were uninfected or infected with KSHV for 30 min or 2 h, washed, the 2 h infected cells incubated for 4 and 24 h, and WCL prepared in NETN buffer. The knockdown efficiency was checked by immunoblotting for total ASC. WCL proteins were IP-ed with anti-acetylated lysine antibodies and western blotted for IFI16 or, IP-ed with anti-caspase-1 antibodies and WB for IFI16 and casapse-1. Total IFI16, caspase-1 and tubulin were tested as input and loading controls. (B) HMVEC-d cells uninfected or infected with KSHV for 2 h, washed, incubated with complete medium for 24 h, nuclear and cytoplasmic proteins western blotted for ASC and IFI16 or IP-ed with anti-acetylated lysine antibodies and western blotted for IFI16. TBP and tubulin were used as loading control and purity markers.

Fig 6. Effect of acetyltransferase p300 knockdown on IFI16 acetylation and IFI16 inflammasomes during de novo KSHV infection of HMVEC-d cells.

(A) HMVEC-d cells pre-incubated with or without 1 μM C-646 for 2 h were uninfected or infected with KSHV for 30 min or 2 h, and the 2 h infected cells incubated with or without C-646 for 4 and 24 h. WCL proteins were IP-ed with anti-p300 antibodies and western blotted for IFI16. Total IFI16, p300 and tubulin were used as controls. (B and C) HMVEC-d cells were starved with or without p300 inhibitor C-646 or HDAC inhibitor tricostatin A (TSA), cytoplasmic and nuclear fractions evaluated for p300 (B) and HDAC (C) activities as in the Material and Methods and the results are presented here as relative fluorescence units (RFU). (D) The HMVEC-d cells electroporated with control or p300 Si-RNA were uninfected or infected with KSHV for 2 h, washed and incubated for various time points. WCL in NETN buffer were western blotted for total p300 and IFI16 or IP-ed with anti-acetylated lysine antibodies and western blotted for IFI16 or IP-ed with anti-caspase-1 antibodies and western blotted for IFI16 and caspase-1. (E, F and G) The control and p300 knockdown HMVEC-d cells were uninfected or infected with KSHV for 2 h, washed, incubated for 24 h and subjected to PLA using (E) anti-IFI16 and anti-acetylated lysine antibodies, (F) anti-IFI16 mouse and rabbit antibodies, and (G) anti-IFI16 and anti-ASC antibodies. The white and red arrows in (E) panels indicate acetylated IFI16 in the cytoplasm or nucleus, respectively. The white and red arrows in (F) panels indicate cytoplasmic and nuclear IFI16, respectively. In (G) panels, the white and red arrows indicate IFI16-ASC colocalization in the cytoplasm and nucleus, respectively.

Fig 7. Effect of IFI16 acetylation on IFN-β production during de novo KSHV infection of HMVEC-d cells.

HMVEC-d cells in the presence or absence of 1 μM C-646 were either left uninfected or infected with KSHV (30 DNA copies/cell) for 2 h, washed and incubated with or without 1 μM C-646 for 6 h. (A) Interferon-β in the culture supernatants was quantitated by ELISA. (B) Cells were examined by IFA with anti-pIRF-3 and Alexa Fluor- 488 secondary antibodies. Insets in the merged panels are enlarged. The yellow arrows indicate the pIRF-3 in the nucleus. (C) Quantitation of nuclear pIRF-3 dots. (D) WCL lysates in NETN buffer were IP-ed with anti-acetylated lysine and STING antibodies and immunoblotted for IFI16. Proteins were also immunoblotted for total IFI16, pIRF-3, total IRF-3, total STING, and tubulin was used as loading control. (E) HMVEC-d cells electroporated with control or STING Si-RNA were either left uninfected or infected with KSHV for 2 h, washed and incubated for 4 and 24 h. WCL prepared in NETN buffer were western blotted for total STING, pIRF3, total IRF3 and tubulin and IP-ed with anti-acetylated lysine antibodies and western blotted for IFI16 and tubulin or IP-ed with anti-IFI16 antibodies and western blotted for IFI16. (F) HMVEC-d cells electroporated with control or STING Si-RNA were uninfected or infected with KSHV for 2 h, washed and incubated for 6 h. Cells culture supernatants were used to measure the levels of IFN-β by ELISA.

Fig 8. Effect of IFI16 acetylation on IFN-β production during de novo HSV-1 infection in HFF cells The HFF cells uninfected or infected with HSV-1 (1 PFU/cell MOI) in the presence or absence of 1 μM C-646 for 30 min or 2 h were washed and incubated with or without 1 μM C-646 for 6 h.

(A) IFN-β in the culture supernatants was quantitated by ELISA. (B and C) Cells were examined by IFA with anti-pIRF-3 and Alexa Fluor-488 secondary antibodies. Insets in the merged panels are enlarged. The white and yellow arrows indicate the pIRF-3 in the cytoplasm or nucleus, respectively. Quantitation of nuclear pIRF-3 dots is shown in (C). (D) PLA with anti-IFI16 and anti-acetylated lysine antibodies. White and yellow arrows indicate the cytoplasmic and nuclear acetylated IFI16, respectively. (E) PLA with mouse and rabbit anti-IFI16 antibodies. White and yellow arrows indicate the cytoplasmic and nuclear IFI16, respectively. (F) WCL lysates in NETN buffer were IP-ed with anti-acetylated lysine and STING antibodies and immunoblotted for IFI16 and IRF-3. Proteins were also immunoblotted for total IFI16, pIRF-3, total IRF-3, and total STING with tubulin used as loading control. (G) HFF cells were transfected with control or STING Si-RNA with electroporation, either left uninfected or infected with HSV-1 (1 PFU/cell MOI) for 30 minutes or 2 h, washed and incubated for 6 h. WCL prepared in NETN buffer were western blotted for total STING, pIRF3, IRF3 and tubulin or IP-ed with anti-acetylated lysine antibodies and western blotted for IFI16 and tubulin, or IP-ed with anti-IFI16 antibodies and western blotted for IFI16. (H) HFF cells electroporated with control or STING Si-RNA were uninfected or infected with HSV-1 for 2 h, washed and incubated for 6h. Cell culture supernatants were used to measure the levels of IFN-β by ELISA.

Fig 9. Effect of acetylation inhibition by C-646 on the ability of IFI16 to recognize and bind the KSHV genome.

(A and B) HMVEC-d cells pre-incubated with or without 1 μM C-646 for 2 h were washed, infected with BrdU genome labeled KSHV (30 DNA copies/cell) for 2 h, washed and incubated with or without 1 μM C-646 for 4 h. Cells were processed, incubated with anti-BrdU antibodies, washed, and reacted with Alexa Fluor-488 (green) secondary antibodies to detect BrdU-KSHV. These slides were subjected to PLA (A) with mouse anti-IFI16 and rabbit-IFI16 antibodies or (B) with anti-IFI16 and anti-acetylated lysine antibodies. Boxed areas are enlarged. The yellow arrows in (A) and (B) indicate the cytoplasmic IFI16 or acetylated IFI16, respectively. The white arrows in (A) indicate colocalization of IFI16 PLA spots with BrdU labeled KSHV genome. The white arrows in (B) indicate colocalization of acetylated IFI16 PLA spots with BrdU labeled KSHV genome. (C and D) Bar graph of quantitation of colocalization of IFI16 or acetylated IFI16 PLA spots with BrdU labeled KSHV genome in (A) and (B) panels. At least ten fields with a minimum of 3–4 cells/field were counted. (E) The above described cells were subjected to PLA using anti-BrdU and anti-IFI16 antibodies to detect the direct association of IFI16 with BrdU-labeled KSHV genome. Boxed areas are enlarged. The PLA dots indicated by white arrows represent the association between IFI16 and BrdU-labeled KSHV genome. (F) PLA spots from 10 fields with at least 3–4 cells/field in (E) were quantitated and presented as a bar graph (G) Chromatin immunoprecipitation was performed using anti-IFI16 antibody as described in the Material and Methods. Untreated BCBL-1 cells (3 x 10⁷) or cells treated with 1 μM C-646 for 24 h were cross-linked with formaldehyde and sonicated to obtain DNA fragments between 200–400 bps. q-PCR was performed with primers for two different regions on the KSHV genome and one control for GAPDH, and the results are presented as fold enrichment of immunoprecipitated DNA. The p values were calculated using Student’s T test. NS (non-significant).

Fig 10. Schematic model depicting herpesvirus infection induced IFI16 acetylation and its role in inflammasome and IFN-β production.

Results presented here demonstrate viral DNA entry into the nucleus (1–2) leads to recognition through IFI16 (3) followed by IFI16 acetylation (4). The acetylated IFI16 forms an inflammasome complex (5) which is translocated to the cytoplasm via RanGTP. Leptomycin B treatment abrogates the acetylated IFI16 translocation (6). The inflammasome complex formation leads into the activation of caspase-1 (7) which in turn cleaves pro-IL-1β and-IL-33 (8), and IL-β is released into the culture supernatant (9). IFI16 acetylation (i) and cytoplasmic redistribution (ii) also activates STING (iii) which phosphorylates IRF-3 (iv) that moves to the nucleus leading into IFN-β gene transcription (v-vi), translation (vii) and IFN-β is secreted into the culture supernatant (viii). Use of p300 inhibitor C-646 and knockdown of p300 does not inhibit the ability of IFI16 to recognize the episomal viral DNA but blocks the acetylation of IFI16 which results in the inhibition of inflammasome formation, IFI16 translocation into the cytoplasm as well as IL-1β and IFN-β production.