Figure 7.

UL35 interacts with TBK1 and impairs the phosphorylation events within the TBK1-STING-IRF3 axis. (A) HEK293T cells were co-transfected with IRES-GFP (unstimulated), TBK1, or IRF3-5D together with either empty vector (ev) or UL35-HA. 20 h later, cells were lysed and whole cell lysates analyzed by immunoblotting for phosphorylated TBK1 (pTBK1), total TBK1, phosphorylated IRF3 (pIRF3), total IRF3, anti-HA for UL35, and Actin. (B) HEK293T cells were transfected with TBK1 together with either ev or UL35-HA. 20 h later, cells were lysed and 10% of the whole cell lysate was used for the input control. The remainder was subjected to immunoprecipitation with an anti-HA antibody to precipitate UL35. Input and IP fractions were analyzed by immunoblotting with anti-TBK1 and anti-HA antibodies. (C) HFF-1 stably expressing ev or UL35-HA were infected with HCMV UL35stop at an MOI of 0.1. Cells were lysed 0, 2, 4 and 6 h later and analyzed by immunoblotting with anti-pTBK1, anti-TBK1, anti-pSTING, anti-pIRF3, anti-IRF3, anti-pSTAT1, anti-HA, and anti-Actin antibodies. (D) HFF-1 stably expressing ev or UL35-HA were stimulated by transfection of ISD and immunoblotting was performed as described in (C). (A–D) One representative experiment out of two independent experiments is shown. Quantification of band intensities relative to the corresponding Actin band was performed using ImageJ and is shown below the corresponding immunoblot.