Intracellular ROS produced following HSV infection is essential for cytokine expression.

<p>(A) RAW264.7 cells were seeded in chambered-cover slides, either pre-treated with 6.4mM LNAC or complete media for 30 min, and subsequently infected with HSV-2 for 1, 2, or 3 h. CM-H₂DCFDA (5 µM) was added immediately before imaging. ROS production was monitored in z-stack images (1 µm) and images were acquired with a 40x objective every 30 seconds by confocal microscopy. Images represent maximum intensity projections. Amount of ROS was determined and represented as mean fluorescence per cell with a minimum of 100 cells counted per condition. Data is presented as mean of 3 experiments for the untreated samples and of 2 experiments for the LNAC samples +/- sem. (B–D) Peritoneal macrophages were seeded and either left uninfected or infected with HSV-2 (3×10⁶ pfu/ml, MOI 3); each in the absence or presence of 10 µM hydrogen peroxide. Supernatants were harvested after 16 h for measurement of IFN-α/β, CXCL10, and CCL5. (E, F) Peritoneal macrophages were pre-incubated with increasing concentrations of PDTC or LNAC and subsequently infected with HSV-2 (3×10⁶ pfu/ml, MOI 3). Supernatants were harvested 12 h post infection and CCL5 protein was measured by ELISA. The data is shown as mean of 3 replicates +/- st. dev. and is representative of 3 experiments. CCL5 levels significantly different from those induced by HSV-2 alone (p<0.05) are indicated with *.</p

Redox-sensitive signaling following HSV recognition.

<p>(A) pDCs and (B) BMMs were treated with LNAC (3.2 mM) 30 min prior to infection with HSV-1 or 2 (MOI 3). Culture supernatants were harvested 16 h post infection, and levels of CCL5 were measured by ELISA. All data are shown as means of 3–5 replicates +/- st.dev. (C) MEFs were treated with LNAC (3.2 mM) 30 min prior to infection with HSV-1 or 2 (MOI 3). Total RNA was harvested after 6 h post-infection and IFN-β mRNA measured by real time PCR. Data is presented as means of triplicate measurements +/- st.dev. RU, relative units. (D, E) Mice were treated i.p. with LNAC (1.1 mmol/kg body weight) in saline or with saline alone followed 4 h later by infection with HSV-2 (5×10⁶ pfu). Eight and 24 hours post infection, serum and livers were harvested for measurement of type I IFN and viral load, respectively (n = 5). Data represents mean of 3–6 replicates +/- st.dev. (F, G) RAW264.7 macrophage-like cells were treated with LNAC (6.4 mM) for 30 min before infection with HSV-2 (3×10⁶ pfu/ml, MOI 3). (F) Total cell lysates were harvested at the indicated time points post infection, and phospho-IκBα was measured by Luminex. (G) The cells were fixed 3 h post infection, stained with anti-IRF-3 antibody and DAPI, and visualised by confocal microscopy. Cells were scored for nuclear IRF-3 staining. Data represent mean +/- st.dev. (H, I) Peritoneal macrophages from C57BL/6 WT and ASK1^-/- mice were cultured <i>in vitro</i> and infected with HSV-2 (3×10⁶ pfu/ml, MOI 3). Supernatants were harvested 16 h post infection, and the levels of CCL5 and IFN-α/β were measured. (J) C57BL/6 WT and ASK1^-/- mice were infected i.p. with 5×10⁶ pfu of HSV-2. Livers were isolated 3 days post infection and viral load in the organs was determined (n = 8). The data represent mean of multiple measurements +/- st.dev.</p

ROS and innate antiviral response: Cell-type and PRR dependence.

<p>(A, B) Macrophages were isolated from C57BL/6 WT, MAVS^-/- or double knock-out TLR2/9^-/- mice and cultured <i>in vitro</i>. Cells were treated with (A) HSV-1 KOS or Sendai Virus, or (B) HSV-1 KOS or ODN1826. Supernatants were harvested after 16 h and presence of type I IFNs were measured by bioassay. (C) pDCs were isolated from C57BL/6 WT or TLR9^-/- mice. Cells were treated with HSV-1 KOS or ODN1826. Supernatants were harvested after 16 h and presence of type I IFNs were measured by bioassay. (D) RAW264.7 macrophage-like cells were transfected with si-p204 or control siRNA and infected with HSV-1 KOS. Total RNA was harvested after 6 h post-infection and CCL5 mRNA measured by real time PCR. Data is presented as mean of duplicate measurements +/- st.dev. RU, relative units. (E) RAW264.7 cells were transfected with HSV1-60mer (2 µg/ml) following treatment with 6.4 mM LNAC or complete media. Culture supernatants were harvested 12 h p.i. and production of CCL5 was measured by ELISA. (F) RAW264.7 cells were seeded and treated with vehicle or LNAC (3.2 mM) 30 min prior to stimulation with Pam₃Csk₄ (200 ng/ml), ODN1826 (1 µM), and poly(IC):LyoVec (25 µg/ml). Culture supernatants were harvested 16 h post-stimulation, and levels of CCL5 were measured by ELISA. (G) RAW264.7 cells were incubated with poly(I∶C) (25 µg/ml) following treatment with 6.4 mM LNAC or complete media. Culture supernatants were harvested 16 h p.i. and production of CCL5 was measured by ELISA. Data represents mean of measurements from triplicate cultures +/- st.dev.</p

Identification of a conserved redox-sensitive surface-exposed cysteine in the TRAF C-terminal domain of TRAF2, 3, and 6.

<p>(A) Structure-based sequence alignment of a region of the TRAF domain of human and murine TRAF2, 3, 4, and 6. Secondary structures in the region and TRAF-motif interacting residues are indicated in the figure. (B) <i>Traf6-/-</i> MEFs were left untreated or treated with HSV-2 (3×10⁶ pfu/ml, MOI 3, 5 h) or poly(I∶C) (25 µg/ml, 2 h). Total cellular lysates we generated and phospho-IκBα was measured by Luminex. (C–F) Traf6-/- MEFs were transfected with empty vector (pCMV), WT TRAF6, or C390S TRAF6. The cells were treated with HSV-2 (3×10⁶ pfu/ml, MOI 3, 5 h), poly(I∶C) (25 µg/ml, 2 h), IL-1β (10 ng/ml), and LNAC (3.2 mM, 30 min prior to further treatment) as indicated. Total RNA and total cell lysates were isolated and analyzed for IFN-β mRNA and P-IκBα by RT-qPCR and Luminex, respectively. Data is shown as means of triplicates +/- st.dev. (G) <i>Traf3-/-</i> MEFs were transfected with empty vector (pRK5), WT TRAF3, or C455S TRAF3. The cells were treated with HSV-2 (3×10⁶ pfu/ml, MOI 3) and LNAC (3.2 mM, 30 min prior to further treatment) as indicated. Total RNA was isolated 5 h post-infection and analyzed for IFN-β mRNA by RT-qPCR. Data is shown as means of triplicates +/- st.dev. RU, relative units; NR, normalized ratio.</p

Genomic HIV RNA induces innate immune responses dominated by ISGs.

<p>PBMCs were stimulated for 16 h with HIV genomic RNA in increasing doses (from 0.3 to 3.0 µg/ml). Positive and negative controls included ssRNA40 (2 µg/ml) and ssRNA41 (2 µg/ml), respectively. Supernatants were harvested for measurement of (A) CXCL10, (B) IFN-β, (C) IFN-α, (D) TNF-α, and (E) IL-6. Data are shown as means of triplicates +/− st.dev. Similar results were obtained in two or three independent experiments. Mock, Lipofectamine 2000 alone.</p

HIV RNA activates the NF-κB, p38, and IRF signaling pathways.

<p>PBMCs were stimulated with HIV Tar (oligo 2, 2 µg/ml) and in panel A also with oligo 4 (2 µg/ml). (A–B) Whole-cell lysates were isolated 2 h post treatment, and (C–E) nuclear extracts were isolated at the indicated time points (IFN-γ, 10 ng/ml, 6 h; SeV, MOI 1, 6 h; R848, 500 ng/ml, 6 h). (A–B) P-IκBα and P-p38 were measured by Luminex technology and (C–E) DNA binding of IRF-1, 3, and 7 to an ISRE consensus sequence was measured by TransAM. Data are shown as means of duplicates or triplicates +/− st.dev. Similar results were obtained in two or three independent experiments. RU, relative units. Mock, Lipofectamine 2000 alone. *, p<0.05.</p

The innate immune response induced by HIV genomic RNA or RNA oligos is dependent on RIG-I and MAVS.

<p>(A) PBMCs were treated with bafilomycin A1 (0.5 µM) as indicated 15 min prior to stimulation with genomic HIV RNA, RNA oligos, or ssRNA40 (all 2 µg/ml). IFN-α was included as a positive control (10 ng/ml). Supernatants were harvested 18 h post stimulation for measurement of CXCL10. (B) BMMs from C57BL/6 wildtype and MAVS−/− mice were stimulated with genomic HIV RNA (2 µg/ml), Tar (2 µg/ml), Sendai virus (MOI 1), IFN-α (10 ng/ml), ssRNA40 (2 µg/ml), or R848 (2 µg/ml). Supernatants were harvested after 16 h and CXCL10 was measured by ELISA. UT, untreated cells. Data are shown as means of triplicates +/− st.dev. (C) Huh7, Huh7.5 (RIG-I mutant), Huh7.5 EV (empty vector), and Huh7.5 RIG-I cells were transfected with Tar RNA (2 µg/ml), or subjected to mock transfection with Lipofectamine 2000. Total RNA was harvested 6 h later and CXCL10 mRNA levels were analysed by qPCR. Data are shown as means of triplicates +/− st.dev. Similar results were obtained in two or three independent experiments. Mock, Lipofectamine 2000 alone. RU, relative units *, p<0.05.</p