Additional file 1: Figure S1. of Type I Interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by IPS-1

IPS-1 −/− mice have lower IFN-α levels in serum after LGTV infection compared to WT mice. WT and IPS-1 −/− mice were infected intraperitoneally with 104 FFU of LGTV, and serum samples were collected at indicated time points (n = 5–10). The amount of IFN-α in the mouse serum was determined by enzyme linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (PBL). Significance was calculated with student’s T test, *p < 0.05. (PDF 53.6 KB

Additional file 2: Figure S2. of Type I Interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by IPS-1

Effect of IPS-1 signaling on BBB permeability upon LGTV infection. WT and IPS-1 −/− mice were mock or intraperitoneally infected with 104 FFU of LGTV (n = 3). Mice were intravenously injected with 100 μl 2 % Evans blue (Sigma) in PBS 4 and 7 dpi. After 1 h, animals were transcardially perfused with 20 ml PBS and the brains were removed. The brains were weighted and homogenized in 50 % TCA, and absorbance was measured at 610 nm. The absorbance was divided by the weight of the sample and normalized to mock infected samples. (PDF 42.5 KB

Additional file 5: Figure S4. of Type I Interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by IPS-1

Enhanced viral replication in olfactory bulb of IPS-1 −/− mice 4 dpi. WT and IPS-1 deficient mice were infected intraperitoneal with LGTV and olfactory bulb, cerebrum, cerebellum and brain stem was collected 4 dpi. LGTV RNA levels were quantified with the NS3 real-time qPCR assay (detection limit 10 copies). Asterisks indicates statistical significance between IPS-1 −/− olfactory bulb compared to other brain regions and calculated by Mann-Whitney test. Number sign indicates not detectable. (PDF 45.6 KB

Additional file 4: of Type I Interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by IPS-1

Method S1. Virus neutralization assay. Neutralization titer of antibody solution was determined by modified method of plaque reduction neutralization test (PRNT) called as rapid fluorescent focus inhibition test (RFFIT) [68, 69]. Briefly, mice sera were diluted 1:10 in serum-free maintenance media and heat-inactivated. The sera were serially diluted and incubated with 50 FFU LGTV for 1 h, and focus forming assay was performed. Neutralizing antibody titer was calculated as the reciprocal of serum dilution that gave 50 % (NT50) reduction of the number of FFU as compared to virus control. The test was accepted if virus dose was in the range of 30–90 FFU. (DOC 21.5 KB

HCMV infected monocyte-derived DC and MΦ mount IFN-α responses in a cGAS-dependent manner.

<p>(A) moDC, GM-CSF MΦ, and M-CSF MΦ were transfected with siRNA directed against cGAS or control siRNA and cGAS or IFI16 expression was monitored by western blot analysis, while actin was used as loading control. Untreated or siRNA-mediated cGAS knock-down moDC, GM-CSF MΦ, and M-CSF MΦ were infected with HCMV-GFP at MOI 3 for 24 h and (B) IFN-α contents of cell-free supernatants were monitored by ELISA or (C) IFN-α⁺ and/or HCMV-GFP⁺ cells were analyzed by flow cytometry. (D) cGAS knock-down moDC, GM-CSF MΦ, and M-CSF MΦ were mock transfected or transfected with synthetic cGAMP and analyzed for IFN-β mRNA expression relative to HPRT1 mRNA by qPCR. Mean ± SEM of 6 (B) and 5 (D) or representative for 6 (A, C) different donors from 3 independent experiments. ns = not significant, *: p ≤ 0.04 one-tailed Wilcoxon signed rank test.</p

HCMV infection induces IFN-I responses in pDC as well as monocyte-derived DC and MΦ.

<p>pDC, moDC, GM-CSF MΦ, and M-CSF MΦ were infected with HCMV at MOI 3 and 24 hours post infection (hpi) (A) IFN-α and (B) IFN-β mRNA expression was determined relative to HPRT1 mRNA by qPCR (mean ± SEM of 7–9 different donors). (C, D) IFN-α expressing cells were analyzed by flow cytometry and (E) the IFN-α content in cell-free supernatants was determined by an ELISA method upon infection with three independently produced HCMV preparations (#1, #2, and #3) at MOI 3. (F) Upon HCMV infection with varying MOI between 0.1 to 30 IFN-α concentrations in cell-free supernatants (y-axis) were blotted against percentages of IFN-α⁺ cells (x-axis) of the same culture (Values from 12–28 different donors. Black line: confidence interval of 95%). Median of 9–26 (D) and 12–23 (E) different donors. ns = not significant, *: p ≤ 0.031, **: p ≤ 0.0078, ***: p ≤ 0.0002 one-tailed paired Wilcoxon signed rank test was used for statistical analyses between monocyte-derived cell subsets, because the subsets were derived from monocytes of the same donors. In contrast, pDC were derived from different donors. Therefore, statistical analysis between pDC and one of the monocyte-derived cell subsets was performed using one-tailed unpaired Mann-Whitney test.</p

Upon HCMV stimulation PMA-matured THP-1 cells mount IFN-β responses in a cGAS-dependent manner.

<p>WT (wild-type), and 2 clones (#1 and #2) of cGAS, IFI16, or STING deficient THP-1 cells differentiated with PMA for 3 days were stimulated with (A) HCMV at MOI 10, (B) MVA at MOI 1, or (C) VSV at MOI 1 for 24 h. Cell-free supernatant was tested for IFN-β by an ELISA method (A, B), or cell lysates were tested for IFN-β mRNA expression relative to HPRT1 mRNA by qPCR (C). Lysates of virus infected cells were analyzed for phosphorylated IRF3 (P-IRF3) and IRF3 by western blot (A, B, C). Mean ± SEM of 3–6 (A), 3 (B), and 4–5 (C) data points from 3 (A, C) and 2 (B) independent experiments. *: p ≤ 0.05, **: p ≤ 0.0076 one-tailed Mann-Whitney test.</p

pDC and monocyte-derived cells show constitutive cGAS expression that is enhanced upon IFN-α or HCMV stimulation.

<p>Primary human pDC (yellow bars), moDC (green bars), GM-CSF MΦ (GM-MΦ, red bars), and M-CSF MΦ (M-MΦ, blue bars) were (A) stimulated for 6 h with recombinant IFN-α2b and analyzed for relative MxA, cGAS, and STING mRNA expression. Furthermore, the cells were infected with HCMV at MOI 3 for 24 h and analyzed for (B) cGAS and (C) STING mRNA expression relative to HPRT1 mRNA by qPCR. (D) Protein levels of cGAS and STING upon HCMV infection for 24 h or recombinant IFN-α2b treatment for 6 h (upper panel) or 24 h (lower panel) were determined by western blot analysis, while actin was used as loading control. Mean ± SEM of 5–14 (A), 5–15 (B, C) or representative for 3–4 (D) different donors. ns = not significant, *: p ≤ 0.032, **: p ≤ 0.0078 one-tailed Wilcoxon signed rank test.</p

HCMV infection induces cGAMP formation in monocyte-derived DC and MΦ, but not in pDC.

<p>cGAMP synthesis was analyzed and quantified using a HPLC-MS/MS method. Chromatograms (blue line: quantifier, green/red lines: identifiers) of (A) synthetic cGAS-derived cGAMP (2´-5´/3´-5´) and bacterial cGAMP (3´-5´/3´-5´) (upper panel) or lysed, HCMV stimulated M-CSF MΦ (lower panel) as well as (B) lysates of 24 h HCMV infected pDC, moDC, GM-CSF MΦ, and M-CSF MΦ (enlarged visualization of grey area shown in (A)). (C) Quantification of the detected cGAMP shown in (B). (D) cGAMP (filled circles) synthesis and IFN-α contents in cell-free supernatants (open triangles) were monitored in unstimulated (0 h) moDC, GM-CSF MΦ, and M-CSF MΦ or at indicated time points after HCMV treatment. pDC, moDC, GM-CSF MΦ, and M-CSF MΦ were infected with MVA at MOI 1 and 24 hpi (E) cGAMP synthesis as well as (F) IFN-α contents of cell-free supernatants were quantified. moDC, GM-CSF MΦ, and M-CSF MΦ were infected with VSV-M2 at MOI 1 and 24 hpi (G) cGAMP synthesis as well as (H) IFN-α contents of cell-free supernatants were quantified. Mean ± SEM of 4–7 (C), 4 (D), 3–7 (E, F), or 4 (G, H) different donors. ns = not significant, *: p ≤ 0.032, **: p ≤ 0.0078 one-tailed Wilcoxon signed rank test.</p

HCMV-derived genomic DNA efficiently activates recombinant human cGAS.

<p>Recombinant human cGAS was incubated for 2 h in the presence or absence of ATP and GTP with (A) a 50 bp long control dsDNA, (B) purified HCMV containing approximately 6.5 x 10⁶ infectious virus particles, or purified HCMV treated for 10 minutes at 95°C, or (C, D) genomic DNA isolated from purified HCMV. All samples were tested with and without DNA digestion prior to incubation with recombinant cGAS. cGAMP formation was quantified using a HPLC-MS/MS method. Mean ± SEM of 5–6 (A) and 5–7 (B, C, D) data points from 3 independent experiments. **: p ≤ 0.005, ***: p ≤ 0.0003 one-tailed Mann-Whitney test.</p