Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) is an immediate-early and tegument protein that plays critical roles in antagonizing host antiviral responses. We have previously shown (Zhu et al, Proc. Natl. Acad. Sci. U. S. A., 99:5573–5578, 2002) that ORF45 suppresses activation of interferon regulatory factor 7 (IRF7), a crucial regulator of type I interferon gene expression, by blocking its virus-induced phosphorylation and nuclear accumulation. We report here further characterization of the mechanisms by which ORF45 inhibits IRF7 phosphorylation. In most cell types, IRF7 is phosphorylated and activated by IKKε and TBK1 after viral infection. We found that phosphorylation of IRF7 on Ser477 and Ser479 by IKKε or TBK1 is inhibited by ORF45. The inhibition is specific to IRF7 because phosphorylation of its close relative IRF3 is not affected by ORF45, implying that ORF45 does not inactivate the kinases directly. In fact, we found that ORF45 is phosphorylated efficiently on Ser41 and Ser162 by IKKε and TBK1. We demonstrated that ORF45 competes with the associated IRF7 and inhibits its phosphorylation by IKKε or TBK1 by acting as an alternative substrate.