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. 2012 Sep;86(18):10162–10172. doi: 10.1128/JVI.05224-11

Fig 6.

Fig 6

Ser41 and Ser162 of KSHV ORF45 contribute to inhibition of IRF7 phosphorylation and transactivation activity. (A) Ser41 and Ser162 of KSHV ORF45 are required for inhibition of IKKε- or TBK1-induced IRF7 phosphorylation. HEK293T cells were transfected with 1 μg of Flag-IRF7, 100 ng of HA-IKKε, and 2 μg of wild-type ORF45 or ORF45-S41/162A mutant expression plasmids. At 48 h after transfection, cell lysates were prepared and analyzed by Western blotting with the indicated antibodies. Similar results were obtained with TBK1 (data not shown). (B) Mutation of Ser41 and Ser162 of ORF45 does not affect IRF7/ORF45 or IRF7/IKKε interaction. HEK293T cells were transfected with 5 μg of Flag-IRF7, 500 ng of HA-IKKε, and 10 μg of wild-type ORF45 or ORF45-S41/162A mutant expression vectors. At 48 h after transfection, cell lysates were prepared and immunoprecipitated with anti-Flag M2 affinity beads. The input WCL and IP complexes were analyzed by immunoblotting with antibodies as indicated. (C) Mutation of both Ser41 and Ser162 of ORF45 does not affect interactions between ORF45 and IKKε or TBK1. HEK293T cells were transfected with IKKε (500 ng) or TBK1 (500 ng) with wild-type ORF45 or ORF45-S41/162A mutant expression vectors. At 48 h after transfection, cell lysates were prepared and immunoprecipitated with anti-Flag M2 affinity beads. The input WCL and IP complexes were analyzed by immunoblotting with antibodies as indicated. (D) Mutation of both Ser41 and Ser162 of ORF45 impairs the inhibition of virus-induced IRF7 transactivation activity. HEK293T cells were transfected with IFN-α1 promoter reporter, pRL-TK, IRF7, and increasing amounts of ORF45 expression plasmids as indicated. Eight hours after transfection, each well was infected with 80 hemagglutinin units of Sendai viruses (SV). At 24 h after transfection, cell lysates were prepared and used for dual-luciferase assays. The relative luciferase activity was expressed in arbitrary units by normalization of firefly luciferase activity to Renilla luciferase activity. Values are averages ± standard deviations from three experiments. (E) Mutation of both Ser41 and Ser162 of ORF45 impairs the inhibition of IKKε/TBK1-induced IRF7 transactivation activity. HEK293T cells were transfected with the IFN-α1 promoter reporter, IRF7, and 60 ng ORF45 with or without 2 ng IKKε expression plasmids. At 24 h after transfection, cell lysates were prepared and used for dual-luciferase assays. (F) ORF45 contributes to inhibition of IRF7 during KSHV lytic replication. The iSLK/BAC36-wt and iSLK/BAC-stop45 cells seeded in 24-well plates were transfected with IFN-α1 promoter reporter, pRL-TK, and IRF7 plasmids using the Fugene 6 reagent. Six hours after transfection, cells were treated with 2 μg/ml doxycycline (Dox) and 1 mM sodium butyrate. At 24 h after transfection, cell lysates were prepared and used for dual-luciferase assays and for Western blot analysis. (G) Ser41 and Ser162 are required for inhibition of IRF7 by ORF45 during KSHV lytic replication. The iSLK/BAC36-wt and iSLK/BAC-stop45 cells were transfected with IFN-α1 promoter reporter, pRL-TK, IRF7, and increasing amounts of the wild-type ORF45 or the S41/162A mutant plasmids as indicated. Six hours after transfection, the cells were treated and assayed as described above.