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

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

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

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

ORF20 interacting partners identified by q-AP-MS analysis.

<p>ORF20 interacting partners identified by q-AP-MS analysis.</p

ORF20 and OASL copurify with ribosomal subunits and polysomes.

<p>293T cells were cotransfected with myc-tagged ORF20 (A-B), OASL (C-D), or ORF20 and OASL together (E-F), then ribosomes were purified by 5–45% sucrose gradient fractionation in the presence of EDTA (A, C, E) or MgCl₂ (B, D, F). Fractionation profiles were determined by absorbance at 254 nm. 40S and 60S indicate the small and large ribosomal subunits, respectively, and 80S indicates the monosome peak. Fractions were collected and analyzed for 18S and 28S rRNA by denaturing gel electrophoresis and for OASL and ORF20 expression by anti-myc immunoblotting. Results are representative of two independent experiments.</p

Quantitative proteomics identified nucleolar and ribosomal interaction partners of OASL.

<p>(A) q-AP-MS was performed as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006937#ppat.1006937.g003" target="_blank">Fig 3A</a>. For the forward experiment, heavy-labeled HeLa S3 cells were transfected with OASL-myc and light-labeled cells were transfected with LacZ-myc. In the crossover experiment, OASL-myc was labeled with light amino acids and LacZ-myc with heavy amino acids. Cell lysates were immunoprecipitated with anti-myc magnetic beads and analyzed by LC-MS/MS. Specific interacting partners of OASL identified in both the forward and crossover experiments are located in the lower right quadrant. 40S and 60S ribosomal proteins are shown in blue and nucleolar proteins are indicated in green. (B) The interacting partners shown in Tables <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006937#ppat.1006937.t001" target="_blank">1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006937#ppat.1006937.t002" target="_blank">2</a> were compared using VennDis to determine the number of specific and shared interacting partners for ORF20 and OASL (related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006937#ppat.1006937.s010" target="_blank">S1 Supporting Information</a>).</p

OASL expression is beneficial for MHV68 and KSHV replication.

<p>(A-F, H) 293T OASL^-/- cells were reconstituted with empty vector (EV), mOASL1, or hOASL as indicated. (A-B) 24 h later, cells were infected with VSV-GFP for 16 h. The GFP intensity in fixed cells was determined by flow cytometry. (A) Histograms of the VSV-GFP signal for duplicates from one representative experiment are shown. (B) The number of highly-GFP positive cells was determined and scaled to EV. Means + SD of duplicates from two independent experiments are shown. (C-D) 24 h post transfection, cells were infected with MHV68-GFP at an MOI of 0.05 for 2 h at 37°C. Supernatant was harvested at the indicated times and the viral titer was determined by TCID₅₀ on M2-10B4 cells. Means ± SD of triplicates from two independent experiments are shown. (E, F, H) 24 h post transfection, cells were infected with (E) MHV68-GFP, (F) KSHV_LYT wildtype, or (H) KSHV_LYT ORF20stop. 20–24 h later, cells were detached, fixed, and the number of GFP-positive cells was determined by flow cytometry. Means + SD of 4–6 replicates total from two independent experiments are shown. (G) In KSHV_LYT ORF20stop, the ORF20 GAG codon for E69 is replaced with the stop codon TAG. For all subfigures: ns, not significant, * P<0.05, **P<0.01, *** P<0.001.</p

OASL interacting partners identified by q-AP-MS.

<p>OASL interacting partners identified by q-AP-MS.</p

OASL RNA binding and ubiquitin-like domains are not required for interaction with ORF20.

<p>(A) Multiple OASL constructs were utilized, including WT OASL and OASL mutants, including ΔUBL lacking the ubiquitin-like domain, the P-loop mutants V67G and N72K, the RNA binding mutants R45E/K66E/R196E/K200E (RKRK), K63E, and K66E, and the catalytic triad mutants, E81A, E83A, and T152A. (B) 293T cells were transfected with myc-tagged ORF20WT or LacZ, V5-tagged OAS1, WT or mutant OASL, and/or empty vector (EV) as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of at least three independent experiments. Similar results were obtained with simultaneous and sequential antibody exposures. (C) The amino acid sequence for ORF20B is shown, with the predicted nuclear localization sequence indicated in red, the predicted nucleolar localization sequence indicated in green, and predicted disordered regions underlined. The predictions formed the basis for three ORF20B C-terminal truncation mutants: ORF20B 1–186, 1–220, and 1–235. (D) 293T cells were transfected with myc-tagged ORF20 isoforms, ORF20B mutants, or LacZ, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed. Input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of three independent experiments.</p