Murine Gamma-Herpesvirus 68 Hijacks MAVS and IKKβ to Initiate Lytic Replication
Figure 5
IKKβ phosphorylates and potentiates the transcription activity of γHV68 RTA.
(A) IKKβ or IKKβΔKD purified from 293T cells (left) were incubated with [32P]γATP and bacterial GST fusion proteins containing RTA fragments (middle), and examined by autoradiography (right). (B) MEFs of indicated genotype were infected with γHV68 (MOI = 2) for 4 h, labeled with [32P]-orthophosphoric acid for 8 h. Whole cell lysates were precipitated with anti-RTA antibody and analyzed by autoradiography (top) or immunoblot with anti-RTA antibody (bottom). (C) 293T cells were transfected with reporter plasmids and plasmids containing RTA, TRAF6, IKKβ, and IKKβΔKD. Luciferase activity normalized against β-galactosidase activity was shown. (D) Phosphorylation of GST fusion proteins, containing the C-terminal 127 amino acids of wild-type RTA, STS/A, or TTS/A variants, by IKKβ was analyzed similarly as in (A). (E) 293T transfection and luciferase reporter assays were carried out as in (C). (F) Whole cell lysates of 293T transfected with plasmids containing wild-type RTA, STS/A, or TTS/A variants were analyzed by immunoblot (top) and used to normalize the basal transcriptional activity of wild-type RTA, the STS/A and TTS/A variants (bottom). Data in (C), (E), and (F) represent the mean ± SEM with indicated P values (*, P<0.05; **, P<0.02) of at least three independent experiments.