Figure 3
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.

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.

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