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Data supplements

  • Supplemental materials for: Daubeuf et al

    Files in this Data Supplement:

    • Figure S1 (JPG, 65.4 KB) -




    • Figure S2 (JPG, 79.6 KB) -
      To confirm the role of helper virus derived ICP0 in TLR inhibition, the macrophage cell line J774 was transduced with both H+-HSV and HF-HSV amplicon stocks at varying MOI and the innate response (TNF-α, IP10 and RANTES) elicited by both amplicons was compared and correlated to helper virus-derived ICP0 mRNA and protein expression. As (A) shows, at higher MOI, the effect of HF-HSV and H+-HSV diverged, with H+-HSV suppressing innate response. We correlated the cytokine/chemokine response engendered by H+-HSV to levels of ICP0 mRNA (B) and protein (C) expressed in transduced cells. As (B) and (C) show, the innate response to H+-HSV inversely correlates with ICP0 mRNA and protein levels. Although this result does not necessarily rule out the contribution of another HSV-encoded TLR inhibitor besides ICP0, previous experiments ruled out TLR inhibition by the remaining IE proteins, ICP22, ICP27, and ICP47.





    • Figure S3. USP7 knock-down augments the TLR-mediated NF-κB response in 293-TLR2/6 (JPG, 45.8 KB) -
      Previous studies identified two DUB, A20, and Cyld capable of inhibiting TLR signaling. In order to investigate whether endogenous USP7 played a similar role, we compared cytokine/chemokine response to Pgn stimulation in 293-TLR2/6 transfected with shRNA targeting USP7, A20, and Cyld. Knock-down of USP7-enhanced innate cytokine/chemokine gene induction comparable to that seen in A20- and Cyld-depleted cells. 293-TLR2/6 cells were transfected with nonsilencing shRNA or shRNA against A20, Cyld, or USP7, and knock-down of individual mRNA/protein were confirmed 72 hours later. TLR2/6 was stimulated with Pgn for 6 hours before TNF-α, and IP10 mRNA were assayed by qRT-PCR.





    • Figure S4. USP7 synergize with ICP0 to suppress TLR mediated NF-κB response (JPG, 54 KB) -
      HEK293-TLR2/6, HEK293-TLR9 and 293T transiently expressing MyD88 were transfected with ICP0, USP7, USP7.C223S, a combination of USP7, and ICP0 at a 1:1 ratio plus NF-κB�Luc reporter plasmid. 24 hours later NF-κB response to Pgn or CpG ODN stimulation or over-expression of MyD88 was assessed. Co-expression USP7 synergized with ICP0 and augmented its capacity to inhibit NF-κB response.

Article Figures & Data

Figures

  • Figure 1

    Comparison of innate immune response initiated by HF-HSV and H+-HSV in CLL B cells and THP1 cells. (A-C) CLL B cells (A,B) and THP1 cells (C) were transduced with HF-HSV or H+-HSV amplicons at the indicated MOI; 8 hours later (A,C) innate response was assayed by qRT-PCR for TNF-α, IL6, IP10, and IFN-β mRNA. AU indicates arbitrary units. Data are representative of 3 experiments. *P < .05. In panel B, CLL B cells were transduced with HF-HSV or H+-HSV amplicons at MOI of 0.5, and TNF-α and IP10 response were assayed by qRT-PCR at multiple time points as indicated on the x-axis. (D) Comparison of innate response to HF-HSV, H+-HSV, and the HSV helper virus component of H+-HSV in THP1 cells at MOI of 0.5, 8 hours after transduction, shows that HF-HSV generates more potent innate response than either H+-HSV or HSV helper virus alone. Data are representative of 2 experiments. Error bars represent SEM.

  • Figure 2

    HSV helper virus encodes an inhibitor of TLR signaling that suppresses NF-κB and MAPK. (A) THP1 cells were transduced with HF-HSV and HSV helper virus and immunoblotted at 15, 30, and60 minutes with the indicated antibodies; blots are representative of 3 experiments. (B) Nuclear extracts from mock-transduced THP1 cells or THP1 cells transduced with HF-HSV or HSV helper virus were studied by NF-κB EMSA. P indicates probe alone; M, mock; HF, HF-HSV amplicon; and H, HSV helper virus. (C) Nuclear and cytoplasmic fractions of THP1 cells mock transduced (M) or transduced with HF-HSV or HSV helper virus were immunoblotted with anti-RelA/p65. (D) THP1 cells were left untreated or preincubated with 50 μM cyclohexamide (CHX) for 30 minutes before transduction with HF-HSV, H+-HSV amplicon, or HSV helper virus at MOI of 0.5. Six hours later, TNF-α and IP10 mRNA was assayed by qRT-PCR. Inhibition of HSV protein expression restored innate response to both H+-HSV amplicon and HSV helper virus. *P < .05. Data are representative of 2 experiments. (E) Expression of ICP0, ICP22, ICP27, and ICP47 was verified by Western blot using anti-FLAG for ICP22, ICP27, and ICP47 and specific anti-ICP0 for ICP0. (F) HEK293-TLR2/6 were transfected by expression vectors for ICP0, ICP22, ICP27, ICP47, or empty vector (EV) as a control and 24 hours later stimulated with Pgn. TNF-α expression was assayed by qRT-PCR 6 hours later. Error bars represent SEM.

  • Figure 3

    ICP0 inhibits TLR response to HSV, and this activity depends on its association with the deubiquitinating enzyme USP7. (A) 293-TLR2/6 cells were transfected with the empty vector (EV), pCI-ICP0, or pcDNA3.1-DN-MyD88 encoding MyD88 TIR domain. Twenty-four hours later, cells were left untreated or stimulated with PGN or HF-HSV for 6 hours. TNF-α, IP10, and IL6 mRNA was assayed by qRT-PCR.(B) 293-TLR4 cells transduced with empty vector (EV) or ICP0 (ICP0) were stimulated with LPS and immunoblotted at the indicated time points with antibodies against I-κBα, phosphorylated-I-κBα, JNK, and phosphorylated JNK. (C) 293T or 293 stably expressing indicated TLRs were transfected with pCI-ICP0, ICP22, ICP27, and ICP47 or empty vector (EV), pcDNA3-NIK, together with NF-κB luciferase reporter. Twenty-four hours later, cells were activated by corresponding TLR ligands, TNF-α (10 ng/mL), IL-1β (10 ng/mL), or PMA/ionomycin (5 ng/mL and 1 μg/mL, respectively) for 6 hours before luciferase activity was measured. To rule out nonspecific interference with mRNA transcription or protein expression, 293T cells were cotransfected with pCI-ICP0, ICP22, ICP27, and ICP47 and heat-shock response element HSE-Luc, and 24 hours later incubated for 30 minutes at 42°C before luciferase activity was measured 6 hours later. None of the ICP proteins interfered with heat-shock response. (D) ICP0 inhibits NF-κB and IRF3 signaling pathways through different mechanisms: HEK293-TLR4 or HEK293-RIG-I cells were transfected with empty vector, ICP0, ICP0-FXE, ICP0-M4, or ICP0.NLS-mut and stimulated with their respective ligands, and innate response (TNF-α and IL8 for HEK293-TLR4 or IFN-β and IP10 for HEK293-RIG-I) was assayed by qRT-PCR. An ICP0 mutant that did not bind USP7 (ICP0-M4) lacked the ability to inhibit NF-κB response to TLR4 activation. Deletion of the E3 ligase RING domain (FXE) attenuated ICP0 inhibition of IFN-β promoter response to RIG-I. ICP0 with mutated NLS motif (ICP0-NLS-mut) failed to inhibit either NF-κB or IRF3 response. Error bars represent SEM.

  • Figure 4

    ICP0 translocates USP7 from a nuclear to cytoplasmic protein. (A) 293T cells were transfected with GFP-tagged wt-ICP0 or ICP0.NLS-mut alone or in combination with DsRed-tagged USP7 at a ratio of at 1:1. Wt-ICP0 was detectable both within the nucleus and cytoplasm (i). On the other hand, wt-USP7 was an entirely nuclear protein (iii). Modification of USP7 by attaching a NES motif allowed it to be expressed both in the nucleus and cytoplasm (iv). In cells coexpressing USP7 and wt-ICP0, USP7 was a predominantly cytoplasmic protein (v) that colocalized with ICP0. As expected, mutation of ICP0 NLS motif excluded it from the nucleus (ii) and abolished its colocalization with USP7 (vi). In these cells, USP7 behaved as in non–ICP0-expressing cells, remaining a nuclear protein. (B) HEK293-TLR4 cells were transfected with DsRed-USP7 and stimulated by LPS. DsRed-tagged USP7 cellular localization was traced over 1 hour after LPS stimulation. USP7 was detectable within the cytoplasm starting 20 minutes after LPS stimulation and lasting for up to an hour. Images were obtained with a Zeiss LSM 510 confocal microscope using 63× water-immersion objective lens.

  • Figure 5

    Nuclear USP7 migrates to cytoplasm to inhibit TLR signal. Nuclear and cytoplasmic fractions of LPS-stimulated 293-TLR4 (A) and LPS and CpG ODN-stimulated THP1 (B) cells were collected at the indicated time points and immunoblotted with anti-USP7. Nucleolin and α-tubulin were blotted as nuclear and cytoplasmic fraction controls, respectively. (C) 293-TLR4 cells were transfected with nonsilencing shRNA or shRNA against A20, Cyld, or USP7 and knockdown of individual proteins was confirmed. Seventy-two hours later, TLR4 was stimulated with LPS for 6 hours before TNF-α and IL8 mRNA were assayed by qRT-PCR. (D) 293-TLR2/6 cells were transfected with empty vector (EV), full-length USP7 (USP7.FL), USP7.TD (aa's 1-210), USP7-ΔTD (aa's 210-1102), and USP7.C223S, and 24 hours later, TLR2/6 was stimulated with Pgn for 6 hours before TNF-α mRNA was assayed by qRT-PCR. Error bars represent SEM.

  • Figure 6

    USP7 interacts with TRAF6 and IKKγ. (A) 293-TLR4 cells expressing pcDNA3.2-capTEV-USP7 were LPS-stimulated at different time points (0, 15, and 30 minutes) before they were lysed by freeze-thaw, TAP-purified, and blotted with anti-USP7, anti-TRAF6, and anti-IKKγ. Data are representative of 2 experiments. (B) THP1 cells were LPS-stimulated at different time points (0, 15, 30, and 60 minutes) before endogenous TRAF6 and IKKγ were immunoprecipitated and blotted with anti-USP7. (C) ICP0 enhances USP7 binding to endogenous TRAF6 and IKKγ: 293T cells were transfected with ICP0 and USP7 either alone or in combination and 24 hours later endogenous TRAF6 and IKKγ were immunoprecipitated and blotted with anti-USP7. Expression of ICP0 significantly enhanced USP7 binding to endogenous TRAF6 and IKKγ. (D) HEK293-TLR4 cells were transfected with Myc-tagged USP7-FL (1-1102), USP7-TD (1-210), or USP7.ΔTD (210-1102), stimulated with LPS for 1 hour, and then lysed and endogenous TRAF6 and IKKγ were immunoprecipitated. Immunoprecipitated TRAF6 and IKKγ bound both USP7-FL (1-1102) and USP7-TD (1-210).

  • Figure 7

    Both USP7 and ICPO deubiquitinate TRAF6 and IKKγ. (A,B) 293T cells coexpressing FLAG-tagged TRAF6 or IKKγ, HA-ubiquitin, plus one of the following plasmids: empty vector (EV), wt ICP0, ICP0-FXE, ICP0-NLS-MUT, ICP0-M4 (A) or USP7, USP7-C223S (B). Thirty-six hours later, the cells were lysed and FLAG-tagged proteins were immunoprecipitated and blotted with anti-HA. Whole-cell lysates (WCLs) were immunoblotted for ICP0 and USP7 expression. (C) HEK293-TLR2/6 cells were transfected with nonsilencing shRNA or shRNA targeting USP7 and knockdown was confirmed by WB. Forty-eight hours later, the cells were transfected with ICP0 and NF-κB-Luc reporter and stimulated with Pgn for 6 hours before luciferase activity was assayed. *P < .05. (D) 293T cells were transfected with nontargeting shRNA or shRNA-USP7. Forty-eight hours later, the cells were transfected with FLAG-TRAF6 or IKKγ, HA-ubiquitin, and ICP0 before they were lysed, and FLAG-tagged proteins were immunoprecipitated and blotted with anti-HA. WCLs were immunoblotted with specific antibodies to confirm ICP0 expression and USP7 knockdown. (E) THP1 cells were transduced with HF-HSV amplicon or HSV helper virus or left untransduced. Twenty minutes later the cells were lysed, and endogenous TRAF6 and IKKγ were immunoprecipitated with anti-TRAF6/IKKγ antibodies and blotted with antiubiquitin to assess their ubiquitination status. WCLs were immunoblotted for ICP0 expression. Cell lysates were also fractionated into nuclear and cytoplasmic fractions and blotted for USP7 expression. (F) Overexpression of ICP0 does not deplete endogenous TRAF6 or IKKγ: 293T cells were transfected with increasing concentration of ICP0 together with either FLAG-tagged TRAF6 or IKKγ in the presence or absence of the proteasome inhibitor, MG132 (5 μM added for the last 12 hours), and 24 hours later, cell lysate was blotted using anti-FLAG mAb to assess ICP0 effect on TRAF6 and IKKγ. ICP0 did not deplete either TRAF6 or IKKγ. (G) Comparison of wt-USP7 and USP7.NES ability to deubiquitinate TRAF6 and IKKγ and suppress TLR-induced NF-κB response: 293T cells coexpressing FLAG-tagged TRAF6 or IKKγ, HA-ubiquitin, and USP7 or USP7-NES. Thirty-six hours later, the cells were lysed and FLAG-tagged proteins were immunoprecipitated and blotted with anti-HA. (H) Enhanced deubiquitinating efficacy of USP7-NES against TRAF6/IKKγ compared with wt-USP7 correlated with its ability to suppress TLR-2/6 response to Pgn stimulation, measured as TNF-α mRNA. Error bars represent SEM.

Tables

  • Table 1

    Cytokine secretion in response to HSV viruses by CLL B cells

    TNF-α, pg/mLIL6, pg/mLIP10, pg/mLINF-β, pg/mL
    Mock0.63 ± 0.06242 ± 12NDND
    H+-HSV1.19 ± 0.0265 ± 374 ± 2457.8 ± 2.3
    HF-HSV3.32 ± 0.21035 ± 31341 ± 38200.8 ± 12.7
    • ND indicates nondetectable.

  • Table 2

    ICP0 mutants

    ICP-0 mutantDeletion/mutationImpact on ICP0 function
    ICP0-M4K620ILoss of USP7 binding capacity
    ICP0-FXEDel of aa's 106-149Deletion of E3 ligase RING domain
    ICP0-NLSmut500-VRPRKRR-506 into 500-VRPAARA-506Nuclear exclusion