Mounting an immune response to a viral pathogen involves the initial recognition of
viral antigens through
Toll-like receptor-dependent and -independent pathways and the subsequent triggering of signal transduction cascades. Among the many cellular
kinases stimulated in response to
virus infection, the noncanonical IKK-related
kinases TBK1 and
IKKepsilon have been shown to phosphorylate and activate
interferon regulatory factor 3 (IRF-3) and IRF-7, leading to the production of alpha/beta
interferons and the development of a cellular
antiviral state. In the present study, we examine the activation of TBK1 and
IKKepsilon kinases by
vesicular stomatitis virus (VSV)
infection in human lung epithelial A549 cells. We demonstrate that replication-competent VSV is required to induce activation of the IKK-related
kinases and provide evidence that
ribonucleoprotein (RNP) complex of VSV generated intracellularly during virus replication can activate TBK1 and
IKKepsilon activity. In TBK1-deficient cells, IRF-3 and IRF-7 activation is significantly reduced, although transcriptional upregulation of
IKKepsilon following treatment with VSV,
double-stranded RNA, or RNP partially compensates for the loss of TBK1. Biochemical analyses with purified TBK1 and
IKKepsilon kinases in vitro demonstrate that the two
kinases exhibit similar specificities with respect to IRF-3 and IRF-7 substrates and both
kinases target
serine residues that are important for full transcriptional activation of IRF-3 and IRF-7. These data suggest that intracellular RNP formation contributes to the early recognition of VSV
infection, activates the catalytic activity of TBK1, and induces transcriptional upregulation of
IKKepsilon in epithelial cells. Induction of
IKKepsilon potentially functions as a component of the amplification mechanism involved in the establishment of the
antiviral state.