Activation of TBK1 and IKKvarepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity.
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.
tenOever
Benjamin R
BR
Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, McGill University, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T 1E2.
Sharma
Sonia
S
Zou
Wen
W
Sun
Qiang
Q
Grandvaux
Nathalie
N
Julkunen
Ilkka
I
Hemmi
Hiroaki
H
Yamamoto
M
M
Akira
Shizuo
S
Yeh
Wen-Chen
WC
Lin
Rongtuan
R
Hiscott
John
J
D045744:Cell Line, Tumor; D018946:Chemokine CCL5 / Q000032:analysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000821:virology; D017353:Gene Deletion; D006801:Humans; D051550:I-kappa B Kinase; D007113:Immunity, Innate; D050838:Interferon Regulatory Factor-3; D050839:Interferon Regulatory Factor-7; D007370:Interferon Type I / Q000032:analysis; D016898:Interferon-alpha; D007372:Interferons / Q000096:biosynthesis* / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000502:physiology; D012367:RNA, Viral / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins; D012261:Ribonucleoproteins / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction; D051193:Toll-Like Receptors; D014157:Transcription Factors / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014721:Vesicular stomatitis Indiana virus / Q000276:immunology*; D014764:Viral Proteins / Q000378:metabolism
D018946:Chemokine CCL5; D004268:DNA-Binding Proteins; C494232:IRF3 protein, human; C494234:IRF7 protein, human; D050838:Interferon Regulatory Factor-3; D050839:Interferon Regulatory Factor-7; D007370:Interferon Type I; D016898:Interferon-alpha; D008562:Membrane Glycoproteins; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D051193:Toll-Like Receptors; D014157:Transcription Factors; D014764:Viral Proteins; C068567:interferon alpha 4A; D007372:Interferons; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human