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.