PMC

WT, Trif^−/−, MyD88^−/−, DKO and CD118^−/− mice were infected with 1,000 PFU HSV-1/eye. At the indicated time pi, mice were euthanized and corneas harvested. Viral levels in the cornea were then determined by plaque assay at (A) 120 (B), 24, and (C), 48 hr pi. Bars represent 2–4 experiments of 2–4 corneas per group/experiment and are expressed as the mean log PFU/cornea ± SEM. **, p < 0.01; *, p < 0.05 when comparing all groups.

(A) WT and CD118^−/− corneal sensitivity to VSV was compared 48 hours pi following infection with 1,000 PFU/eye VSV by RT-PCR. *, p < 0.05 comparing WT to CD118^−/−. (B) WT mice were transfected with either nonspecific siRNA or targeted to p204. Twenty four hr post transfection, corneas were scarified and infected with VSV. Corneas were isolated 48 hr pi and VSV levels were then evaluated by RT-PCR. Expression was normalized to β-actin levels. Bars represent 2 independent experiments of 2–4 corneas per group.

(A) WT, Trif^−/−, MyD88^−/−, and CD118^−/− mice were infected with 1,000 PFU HSV-1/eye. Forty-four to forty-eight hr pi, IFN production (green) was examined by confocal imaging relative to HSV-1 lesions (red) in the cornea (n = 3–6 corneas/group). Images are representative of 2 independent experiments acquired with a 400x objective with a digital zoom of 10x. White arrows depict uninfected IFN-α expressing cells; yellow arrows depict HSV-infected IFN-α expressing cells; white bars, 50 μm. (B) Mice were infected with HSV-1 and 24 or 48 hrs pi, corneas were evaluated for mRNA expression of ISG54 and OAS1a by RT-PCR. Bars are normalized to uninfected controls and represent 2 independent experiments of 2–4 corneas per group plotted as relative expression ± SEM.

To evaluate the role of p204/IFI-16 in other epithelial tissues, WT mice were transfected and infected with 2,000 PFU/vagina. Viral shedding into the vaginal lumen (A) was evaluated by plaque assay 48 hr pi. Results represent 2 independent experiments of 2 mice per group and are expressed as the mean log PFU/tissue ± SEM. *, p < 0.05 when comparing si-DAI to si-p204; black line represents levels seen in control-transfected mice. (B–C) RT-PCR or Western blot analysis was used to confirm knockdown efficiency of DAI and p204 in the vaginal tissue respectively. (D) To identify the presence of IFI-16 (green) in human skin, adult primary keratinocytes were stained and imaged at 200x with a digital zoom of 2.5. Images represent 2 independent experiments of 1–2 samples per group. White bars, 10 μm. KD, knockdown.

(A) To assess siRNA knockdown of p204, WT mice were transfected with either nonspecific siRNA or to p204. Twenty-four hr post-transfection, knockdown was evaluated by confocal microscopy. White bar, 100 μm. (B) WT mice were infected with HSV-1 (1,000 pfu/cornea) and subsequently given a subconjunctival injection of isotype or anti-mouse p204 polyclonal antibody (10 μg) to neutralize cytosolic protein. Forty-eight hr pi corneas were harvested and evaluated for viral titer by plaque assay. Bars represent 2 experiments of 4 corneas per group and are expressed as the mean log PFU/cornea ± SEM. (C, left panels) To confirm the ability of the p204 antibody (red) to access cellular cytoplasm, WT mice were infected with HSV-1 and given a subconjunctival p204 injection (lower panel) or PBS (upper panel). The corneas were then fixed and stained with a DyLight549-conjugated anti-rabbit secondary and DAPI (blue, nuclei). Images represent two independent experiments of 2–4 corneas per group and were imaged at a magnification of 200x. White bars, 100 μm. (C, right panels) To establish cellular location of the injected antibody, infected corneas were given a subconjunctival injection of p204 antibody or PBS and were then stained with phalloidin (green, cell perimeters), p204 (red), and DAPI (blue) and imaged at 600x with a digital zoom of 10. White bar, 10 μm; N, nuclei, yellow arrows, cytoplasmic location of p204 antibody.

(A) Uninfected WT mice were evaluated for p204 expression (red) by confocal microscopy and the sensor was found in the nuclear (blue), perinuclear, and cytoplasmic regions of corneal epithelium using a 400x objective with a digital zoom of 10x. The lower panels are 3-D reconstructions of the cornea. S, stroma; E, epithelium; white bars, 50 μm; UI, uninfected. (B) In vivo siRNA knockdown of p204 was then performed in infected WT mice and consistently greater than 50% of levels of infected mice transfected with non-specific siRNA. Following siRNA knockdown of p204 and HSV-1 infection, IRF-3 cytoplasmic levels were diminished in siRNA transfected groups (C); however, nuclear translocation of IRF-3 (D) was severely diminished 48 hours pi in the nuclear extract of WT corneas treated exclusively with si-p204 as compared to those of control siRNA-treated or uninfected mice. n, nuclear. (E) A loss of p204 or STING following siRNA knockdown resulted in significantly more infectious virus in the cornea compared to WT mice treated with non-specific siRNA or siRNA to DAI (n = 4–8 corneas) as determined by plaque assay. *, p<.05 comparing STING or p204 to DAI or control, values are representative of 2–3 independent experiments. (F) WT, STING^−/− and CD118^−/− mice were infected with HSV-1 (1,000 pfu/cornea) and viral load determined by plaque assay 48 hrs pi. Results represent 2 experiments of 2–4 corneas/group/experiment and are expressed as the mean log PFU/cornea ± SEM. **, p<.01 compared to WT. (G) Trif^−/− mice were transfected with non-specific or anti-p204 siRNA and subsequently infected with HSV-1 (1,000 pfu/cornea). Forty-eight hr pi IFN production (green) was analyzed by confocal microscopy in relation to herpetic lesions (red) in the cornea. White bars, 50 μm.

(A) The human homologue, IFI-16 (green), was localized to the nucleus (blue) of uninfected THCE cells by confocal imaging using a 400x objective and digital zoom of 10x. White bars, 200 μm. (B) Imaging was confirmed by Western blot of uninfected THCE cells indicating the presence of the approximately 90 kD protein as highlighted by the red arrow. (C) THCE cells were transfected with 2 pmols of control siRNA or siRNA to IFI-16, TLR9, or RIG-I and infected 4 hr later. Twenty-four hr pi, infectious content was evaluated by plaque assay (n = 4–6/group, 2 independent experiments). **, p < 0.01 comparing siRNA IFI-16 to siRNA control-treated groups. (D) To confirm a direct interaction between the DNA sensor (IFI-16) and HSV-1, ChIP pulldown of a biotinylated 50mer of DNA (single or double stranded, GC or non-GC rich) with IFI-16 antibody was performed in THCE cells and then subsequently identified by western blot analysis. (E) IFI-16 knockdown was performed in THCE cells with varying concentrations of control and anti-IFI-16 siRNA (2 or 4 pmols). Knockdown was confirmed by Western blot. (F–H) Following IFI-16 knockdown, IRF-3 nuclear translocation (G) was severely diminished 8 hr pi as seen by confocal microscopy (quantified in F) and western blot analysis (H). n, nuclear; white bar, 50 μm. Images and blots are representative of 2–4 independent experiments. (I) However, IRF-7 nuclear translocation was no different (levels 98% of control) comparing IFI-16 knockdown to control transfected THCE cells. Image represents 2 independent experiments. (J) THCE cells were transfected and infected. Twenty-four hours pi, CXCL10 expression was evaluated by RT-PCR. Results are representative of 2 independent experiments of 2–3 samples/group and expressed as the relative expression ± SEM.