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Blood, Vol. 113, Issue 14, 3264-3275, April 2, 2009
HSV ICP0 recruits USP7 to modulate TLR-mediated innate response
Blood Daubeuf et al.
113: 3264
Supplemental materials for: Daubeuf et al
Files in this Data Supplement:
- Figure S1 (JPG, 65.4 KB)
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293T cells were transfected with expression vectors for MyD88, TIRAP, TRIF, TRAM, IRAK-1, IRAK-4, TRAF-6, TAK1/TAB1, or constitutively active IKKβ (EE) together with pCI-ICP0 plasmid (0, 0.05, 0.1 and 0.2 µg/well), and NF-κB-luciferase reporter. Total amount of transfected DNA was kept constant by adding appropriate amount of pCI-Neo. 24 hours later, luciferase activity was assayed and data plotted as fold induction, error bars represent standard deviation.
- Figure S2 (JPG, 79.6 KB)
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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)
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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)
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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.
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