Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: De novo HSV-2 infection in HFF cells induces the biphasic production of IFN-β. (A) Interferon-β levels in culture supernatants (CS) of HFF cells uninfected or HSV-2 (1 MOI) infected at 4, 8 and 24 h.p.i. was quantitated by ELISA. (B) Interferon-β levels in CS of HFF cells electroporated with SiCT, SicGAS or SiIFI16 and left uninfected or infected with HSV-2 (1 MOI) for 4, 8 and 24h was quantitated by ELISA. The data represent the mean of three determinants ± S.D. and are representative of three independent experiments with similar observation. *p<0.05, **p<0.01 and ****p<0.0001. NS, Non-significant. Control (C) or IFI16 knockdown (D) HFF cells were infected for 2, 4 or 8h with HSV-2 (1 MOI), processed for IFA, reacted with anti-IFI16 and anti-pIRF3 antibodies followed by Alexa Fluor-594 and Alexa Fluor-488 secondary antibodies and DAPI (blue) ( Table 1 ). The boxed areas are enlarged. Translocation of pIRF3 from cytoplasm to nucleus is suggested. 40X magnification. (E) Total pIRF3 foci in nuclei of the cells counted over the entire image as a measurement of amount of pIRF3 localized in the nuclei.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Control, Knockdown, Translocation Assay

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: De novo HSV-2 infection induces IFI16-cGAS and IFI16-STING-dependent IRF3 activation, and the crosstalk of type I interferon pathway with inflammasome and autophagy pathways, as well as dynamic ubiquitination and acetylation of cGAS. HFF cells were left uninfected or infected with HSV-2 at 1 MOI for 4, 8 or 24h. (A) Equal amounts of whole cell lysate proteins were IPed with anti-cGAS antibody and western blotted for NLRP3, IFI16, K-63 linkage-specific polyubiquitin, Beclin-1 and Caspase-1 (pro and cleaved) ( Table 1 ). Fold changes are included at the bottom of the respective blots. (B) Equal amounts of whole cell lysate proteins were IPed with anti-STING antibody and western blotted for NLRP3, IFI16, cGAS, ASC, pro-Caspase-1 and LC3 IIA/B. Equal amounts of whole cell lysate proteins were IPed with anti-Acetylated Lysine antibody and western blotted for IFI16 and cGAS. Red boxes are used to identify bands. (C) Equal amounts of whole cell lysate proteins were IPed with antibodies against the proteins found to interact with STING and western blotted for STING or IPed with antibodies against cGAS or IFI16 and western blotted for Acetylated Lysine. (D) Levels of total NLRP3, IFI16, cGAS, AIM2, ASC, Caspase-1 (pro and cleaved), IL-1β (pro and cleaved), IL-18 (pro- and cleaved), Gasdermin-D (pro and cleaved), STING, pSTING, IRF3, pIRF3, ATG5, Beclin-1, LC3 IIA/B and Tubulin (as loading control) were detected with their respective antibodies. Fold changes are included at the bottom of the respective blots.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection, Activation Assay, Ubiquitin Proteomics, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: Cytoplasmic and nuclear distribution of cGAS and other innate molecules. (A) HFF cells were left uninfected or infected with HSV-2 at 1 MOI for 4 or 24h. Equal amounts of cytoplasmic and nuclear fractions were immuno-precipitated with anti-cGAS antibody and western blotted for IFI16, K-63 linked polyubiquitin, Beclin-1, ASC, Caspase-1 and γH2AX. Equal amounts of cytoplasmic and nuclear fractions were immuno-precipitated with antibodies against the proteins found to interact with cGAS and western blotted for cGAS. Red boxes are used to identify bands. Fold changes are included at the bottom of the respective blots. (B) Input controls are represented, with Tubulin and TBP as purity and loading controls. Fold changes are included at the bottom of the respective blots.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection, Western Blot

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: Cytoplasmic and nuclear distribution of cGAS-IFI16 and IFI16-STING complexes in HSV-2 infected HFF cells. (A) HFF cells were infected for 4h with HSV-2 (1 MOI), processed for IFA, reacted with anti-IFI16 and anti-cGAS antibodies followed by Alexa Fluor-594/488 secondary antibodies and DAPI (blue). The boxed areas are enlarged. Co-localization of IFI16 with cGAS in the cytoplasm (yellow spots, white arrows) and in the nucleus (white spots, yellow arrow). 40X magnification. (B) Pearson’s correlation coefficient among the red and green channels over the entire field as a measurement of co-localization of IFI16 and cGAS in the nucleus and the cytoplasm. (C) HFF cells were infected for 4h with HSV-2 (1 MOI), processed for IFA, reacted with anti-IFI16 and anti-STING antibodies followed by Alexa Fluor-594/488 secondary antibodies and DAPI (blue). The boxed areas are enlarged. Co-localization of IFI16 with STING in the cytoplasm (yellow spots, white arrows). 40X magnification. (D) Pearson’s correlation coefficient among the red and green channels over the entire field as a measurement of co-localization of IFI16 and STING in the cytoplasm.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: Cytoplasmic and nuclear distribution of cGAS having K-63 polyubiquitin and acetylation in HSV-2 infected HFF cells. (A) HFF cells were infected for 4h with HSV-2 (1 MOI), processed for IFA, reacted with anti-cGAS and anti-K-63 linkage-specific polyubiquitin antibodies followed by Alexa Fluor-594/488 secondary antibodies and DAPI (blue). The boxed areas are enlarged. Co-localization of cGAS with K-63 linkage-specific polyubiquitin in the cytoplasm (yellow spots, white arrows) and in the nucleus (white spots, yellow arrows). 40X magnification. (B) Pearson’s correlation coefficient among the red and green channels over the entire field as a measurement of co-localization of cGAS and K-63 linkage-specific polyubiquitin in the nucleus and the cytoplasm. (C) HFF cells were infected for 4h with HSV-2 (1 MOI), processed for IFA, reacted with anti-cGAS and anti-acetylated lysine antibodies followed by Alexa Fluor-594/488 secondary antibodies and DAPI (blue). The boxed areas are enlarged. Co-localization of cGAS with lysine acetylation in cytoplasm (yellow spots, white arrows) and in the nucleus (white spots, yellow arrows). 40X magnification. (D) Pearson’s correlation coefficient among the red and green channels over the entire field as a measurement of co-localization of cGAS and lysine acetylation in the nucleus and the cytoplasm.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: Cytoplasmic and nuclear distribution of cGAS in complex with Beclin-1 and amount of γH2AX in the nucleus post HSV-2 infection. (A) HFF cells were infected for 2, 4 or 8h with HSV-2 (1 MOI), processed for IFA, reacted with anti-cGAS and anti-Beclin-1 antibodies followed by Alexa Fluor-594/488 secondary antibodies and DAPI (blue). The boxed areas are enlarged. Co-localization of cGAS with Beclin-1 in the nucleus (white spots, yellow arrows), at the nuclear membrane, perinuclear space and in the cytoplasm (yellow spots, white arrows). 40X magnification. (B) Pearson’s correlation coefficient among the red and green channels over the entire field as a measurement of co-localization of cGAS and Beclin-1 in the nucleus and the cytoplasm. (C) HFF cells were infected for 2, 4 and 8h with HSV-2 (1 MOI), processed for IFA, reacted with anti-γH2AX antibody followed by Alexa Fluor-594 secondary antibody and DAPI (blue). The boxed areas are enlarged. 40X magnification. (D) Total γH2AX foci in nuclei of the cells counted over the entire image as a measurement of amount of γH2AX present in the nuclei.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection, Membrane

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: HSV-2 ICP27 targets STING. (A) HFF cells were infected for 4h with HSV-2 (1 MOI), processed for IFA, reacted with anti-HSV-2 ICP27 and anti-STING antibodies followed by Alexa Fluor-594/488 secondary antibodies and DAPI (blue). The boxed areas are enlarged. Colocalization of HSV-2 ICP27 with STING at the nuclear membrane and perinclear region (yellow spots, white arrows). 40X magnification. (B) Pearson’s correlation coefficient among the red and green channels over the entire field as a measurement of co-localization of HSV-2 ICP27 and STING in the cytoplasm.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection, Membrane

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: HSV-2 infection in HFF cells induces cGAS-independent IFI16 acetylation, inflammasome formation and initial IFI16-STING association for IRF3 activation. (A) HFF cells treated with SiCT or SicGAS were left uninfected or infected with HSV-2 at 1MOI for 4 or 24h. Equal amounts of whole cell lysate proteins were immuno-precipitated with anti-STING, ASC or Acetylated lysine antibody and western blotted for IFI16. Input controls of cGAS, IFI16, STING and ASC are represented, with Actin as loading control. Fold changes are included at the bottom of the respective blots. (B) HFF cells treated with SiCT or SicGAS were left uninfected or infected with HSV-2 at 1 MOI for 4, 8 or 24h. whole cell lysate proteins were western blotted for cGAS and cleaved-Caspase-1, with Actin as loading control. (C) Mock or (D) cGAS knockdown HFF cells were infected for 2, 4 or 8h with HSV-2 (1 MOI), processed for IFA, reacted with anti-IFI16 and anti-STING antibodies followed by Alexa Fluor-594/488 secondary antibodies and DAPI (blue). The boxed areas are enlarged. Co-localization of IFI16 with STING in the cytoplasm (yellow spots). 40X magnification.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection, Activation Assay, Western Blot, Control, Knockdown

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: Genome pull down demonstrates the association of IFI16 and cGAS with viral genomes in the nucleus. (A) Model depicting the process and major steps involved in the genome pull down assay carried out. After preparation of EdU genome-labelled HSV-2 (i-ii), HFF cells either left uninfected or infected with WT-HSV-2 (10 PFU/cell) or EdU genome-labeled HSV-2 (10 PFU/cell) for 2h (iii), protein-DNA cross-linking was done using formaldehyde. Then biotin-TEG azide was selectively linked to the reactive alkyne group of EdU containing DNA by Click reaction (iv). DNA shearing was performed and the small chromatin fragments captured by streptavidin beads (v). Streptavidin captured samples were used to isolate proteins and DNA. Proteins and DNA from the input materials were analyzed by western blotting (vi) and agarose gel electrophoresis (vii), respectively. HFF cells treated with SiCT, SiIFI16 or SicGAS were infected with WT or EdU-labelled HSV-2 (B) or HSV-1 (C) for 2h. The proteins were IPed and western blotted. An agarose gel was used to analyze DNA in the samples. Top panels represent the host proteins pulled down with EdU-labelled viral genome. Lower panels show the DNA fragments pulled down with EdU. Input control panels: show the presence of IFI16 and cGAS proteins in the samples. The agarose gel shows the presence of DNA in SiCT, SiIFI16 or SicGAS treated HFF cells.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Pull Down Assay, Infection, Labeling, Western Blot, Agarose Gel Electrophoresis, Control

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: IFI16 is essential for the association of cGAS with the viral genome in the nucleus but cGAS is dispensable for IFI16-mediated genome recognition. (A, B) HFF cells electroporated with SiCT or SiIFI16 were left uninfected or infected with BrdU genome-labelled HSV-2 (1 MOI) for 2h and associations of IFI16 (A) or cGAS (B) with the BrdU - labelled HSV-2 genome were analyzed by PLA using anti-IFI16 or anti-cGAS and anti-BrdU antibodies. Red dots (white arrows): IFI16-BrdU-HSV-2 (A) or cGAS-BrdU-HSV-2 (B) association in the nuclei. DAPI (blue): nuclei. (C, D) HFF cells electroporated with SiCT or SicGAS were left uninfected or infected with BrdU - genome labelled HSV-2 (1 MOI) for 2h and associations of IFI16 (C) or cGAS (D) with the BrdU - labelled HSV-2 genome were analyzed by PLA using anti-IFI16 or anti-cGAS and anti-BrdU antibodies. Red dots (white arrows): IFI16-BrdU-HSV-2 (A) or cGAS-BrdU-HSV-2 (B) association in the nuclei. DAPI (blue): nuclei. (E–H) Average PLA dots/cell in SiCT and SiIFI16 or SicGAS transfected and HSV-2 infected HFF cells. The data represent the mean of three determinants ± S.D. ****p<0.0001. NS, non-significant.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection, Transfection

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: cGAMP levels in the cytoplasmic and nuclear fractions of HFF cells uninfected or infected with HSV-2 at 1 MOI for 4 or 24h were quantified by ELISA. The data represent the mean of three determinants ± S.D. and are representative of three independent experiments with similar observation. *p<0.05, **p<0.01. NS, non-significant. Equal amounts of cytoplasmic and nuclear fractions were western blotted for Lamin-B1 and Tubulin to validate the purity of both fractions.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: HSV-2 genome recognition by nuclear cGAS instigates IFN-β production and influences inflammasome activation during de novo infection in HFF cells

doi: 10.3389/fimmu.2026.1717787

Figure Lengend Snippet: Infographic: (1–3 black lines) Viral DNA entry into the nucleus and recognition by IFI16 and cGAS. (4–7 red lines) Acetylated IFI16 translocates to the cytoplasm and associates with STING and activates it. (8–11 green lines) cGAS is associated with IFI16, and cGASIFI16 complex induces cGAMP in the nucleus which translocates to the cytoplasm and activates STING. (12–14 violet lines) Genome recognition by IFI16 and cGAS leads to translocation of the cGAS-IFI16 complex to the cytoplasm, where it produces cGAMP. (15–18 blue lines) The newly synthesized viral genomic DNA leaks or translocates to the cytoplasm and is then recognized by the cytoplasmic cGAS independent of IFI16, which produces cGAMP. (19–24 black lines) STING activated by any of the discussed pathways activates IRF3 by phosphorylation. The pIRF3 moves to the nucleus leading into the IFN-β gene transcription, cytoplasmic translocation and translation and secretion of IFN-β into the culture supernatant. (a1-a2-a3 black lines) Reduced inhibition of nuclear cGAS and increased inhibition of cytoplasmic cGAS - Reduction in K-63 linked poly-ubiquitination of nuclear cGAS and its simultaneous increase in the cytoplasm. (b1-b2 black lines) Loss of cGAS-Beclin-1 interaction in the nucleus and its simultaneous increase in the cytoplasm. (25–26 black lines) cGAS suggested to be in complex with IFI16 inflammasome in the nucleus and the cytoplasm (27). Simultaneously, cGAS is also suggested to be in complex with the NLRP3 inflammasome in the cytoplasm. (28a-29) Active Caspase-1 interacts with cGAS in the cytoplasm (30). Activated STING suggested to induce the NLRP3 inflammasome (31). STING also interacts with LC3 (32). HSV-2 ICP27 binds to STING. (33). cGAS interacts with γH2AX in the nucleus.

Article Snippet: To generate 5-ethynyl-2'deoxyuridine (EdU) and 5-bromo-2-deoxyuridine (BrdU)-labelled infectious HSV-2 and EdU-labelled infectious HSV-1, we added EdU or BrdU labelling reagent (Life Technologies) to the culture medium at 8h, 24h and 48h post infection in a 1:100 (v:v) ratio (from the supplied stock).

Techniques: Translocation Assay, Synthesized, Phospho-proteomics, Inhibition, Ubiquitin Proteomics