Activation of TBK1 and IKKvarepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity

Abstract

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.

FIG. 1.

VSV-induced activation of the antiviral state. (A) WCE (50 μg) prepared from A549 cells infected with VSV (MOI of 10.0) from 0 to 48 hpi was resolved by SDS-7.5% PAGE and transferred to nitrocellulose. IRF-3, IRF-3 S396, IRF-7, IRF-1, ISG56, VSV proteins, and actin were detected by immunoblotting with specific antisera. VSV nucleocapsid protein is denoted by N. Additionally, A549 whole-cell RNA extract was obtained from a duplicate experiment for RT-PCR analysis of RANTES and actin as indicated. (B) WCE as outlined above was used to analyze IRF-3 binding activity by electrophoretic mobility shift assay with the ISRE of the ISG15 probe. Arrows indicate complexes of IRF-3 and IRF-3/CBP.

FIG. 2.

VSV-induced IRF-3 phosphorylation is MOI dependent and requires virus replication. (A) A549 cells were treated with VSV at MOIs of 0.1, 1.0, 10, and 100 for 0, 2, 4, and 6 hpi as indicated. WCE (50 μg) was analyzed by SDS-7.5% PAGE, transferred to nitrocellulose, and immunoblotted for IRF-3, IRF-3 S396, and antisera to VSV. (B) A549 cells were pretreated with ddH₂O, DMSO, cycloheximide (CHX; 100 μg/ml), or ribavirin (500 μg/ml) 30 min prior to VSV infection (MOI of 10). WCE was analyzed by SDS-7.5% PAGE, transferred to nitrocellulose, and immunoblotted for IRF-3 and VSV antisera (N denotes nucleocapsid protein). RNA isolated from duplicate samples was subjected to RT-PCR and analyzed using specific primers for VSV N and actin.

FIG. 3.

VSV infection, dsRNA, or RNP induces an antiviral response. (A) A549 cells were treated with either vector alone, vector and VSV (MOI of 10), dsRNA, or RNP for 6 h. WCE (50 μg) was analyzed by SDS-7.5% PAGE, transferred to nitrocellulose, and immunoblotted for IRF-3, IRF-3 S396, IRF-1, ISG56, VSV antisera, or actin as indicated. (B) WCE (5 μg) as outlined was used to analyze IRF-3 binding activity by electrophoretic mobility shift assay with the ISRE of the ISG15 probe. Arrows indicate the protein-DNA complexes of IRF-3 and IRF-3/CBP. (C) A549 cells were pretreated with vehicle (ddH₂O or DMSO), ribavirin (500 μg/ml), or cycloheximide (100 μg/ml) 30 min prior to RNP treatment as indicated. WCE (30 μg) was analyzed by SDS-7.5% PAGE, transferred to nitrocellulose, and immunoblotted for IRF-3 and ISG56. (D) In vitro transcriptions either alone or coupled to in vitro translation of VSV N cDNA and VSV genome cDNA were transfected into A549 cells and compared to de novo isolated RNP transfection for 6 h. WCE (60 μg) was analyzed by SDS-7.5% PAGE, transferred to nitrocellulose, and immunoblotted for IRF-3 and ISG56 as indicated.

FIG. 4.

Activation of TBK1 kinase activity by VSV infection, dsRNA, or RNP complex. A549 cells were treated with vehicle (Control), VSV (MOI of 100), dsRNA, or VSV RNP for the time points indicated. WCE (1 mg) was immunoprecipitated with anti-TBK1 antisera, and following washing, the immunoprecipitate was assayed for IRF-3 kinase activity (top panels) or transferred to nitrocellulose and immunoblotted for TBK1 expression to ensure equal binding (middle panels). Additionally, duplicate WCE (50 μg) were analyzed by Western blotting to detect IRF-3 phosphorylation (bottom panel). hpt, hours posttreatment.

FIG. 5.

VSV infection induces IRF-3 phosphorylation in the absence of TLR3 or TRIF. (A) C57/BL TLR3^−/− MEFs and wt control MEFs were infected with VSV (MOI of 10) or treated with poly(I) · poly(C) dsRNA for 0 to 6 h. WCE (50 μg) derived from VSV-infected cells were analyzed by SDS-7.5% PAGE; transferred to nitrocellulose; and blotted for IRF-3, TLR3, and actin as indicated. (B) WCE (50 μg) derived from dsRNA-treated TLR3^−/− MEFs and wt control MEFs were analyzed as described for panel A. (C) WCE (40 μg) prepared from TRIF^−/− or TLR3^−/− MEFs were infected with VSV (MOI of 10) for 9 h and immunoblotted for IRF-3 and VSV N as indicated. hpt, hours posttreatment.

FIG. 6.

Defective IRF-3 activation in TBK1^−/− MEFs. (A) C57/BL wt MEFs were treated with VSV (MOI of 10), dsRNA, or RNP for 0, 3, 6, and 9 h. WCE (55 μg) were analyzed by SDS-7.5% PAGE, transferred to nitrocellulose, and immunoblotted for murine IRF-3, IKKɛ, and VSV (ns, G, P, and N denote nonspecific, VSV glycoprotein, VSV phosphoprotein, and VSV nucleocapsid protein, respectively). (B) C57/BL MEFs disrupted in TBK1 gene expression were treated and analyzed as described for panel A.

FIG. 7.

Reduced RANTES and IFNA4 expression in TBK1^−/− MEFs. (A) C57/BL TBK1^+/+ and TBK1^−/− cells were transfected with a luciferase reporter plasmid encoding the κBm RANTES promoter and treated with VSV for 16 h (MOI of 0.1). (B) C57/BL TBK1^+/+ and TBK1^−/− cells were transfected with an IFNA4 promoter luciferase plasmid and an IRF-7 expression plasmid. Luciferase activities were expressed as fold activation relative to the basal level; values represent the averages of two experiments, performed in duplicate.

FIG. 8.

Purified TBK1 and IKKɛ kinases share identical IRF-3 and IRF-7 substrate specificities. (A) Schematic representation of IRF-3. Positions of the DNA binding domain (DBD), nuclear localization sequence (NLS), nuclear export sequence (NES), proline-rich domain (Pro), IRF interaction domain (IAD), and regulatory domain (RD) are indicated. Recombinant TBK1 (0.5 μg, lanes 8 to 14) and IKKɛ (0.5 μg, lanes 1 to 7) purified from baculovirus-infected Sf9 insect cells were used for in vitro kinase analysis of wt and S/T-to-A mutants of GST-IRF-3(aa380-427). Kinase reactions were resolved by SDS-10% PAGE and analyzed by autoradiography; positions of phosphorylated GST-IRF-3 are indicated. (B) Schematic representation of IRF-7. Positions of the DNA binding domain (DBD), constitutive activation domain (CAD), virus activation domain (VAD), regulatory domain (RD), and nuclear export signal (NES) are indicated. GST-IRF-7(aa468-503) peptide substrates are detailed. Recombinant TBK1 (0.5 μg, lanes 10 to 18) and IKKɛ (0.5 μg, lanes 1 to 9) purified from baculovirus-infected Sf9 insect cells were used for in vitro kinase analysis of wt and S/T-to-A mutants of GST-IRF-7(aa468-503) peptide substrates. Kinase reactions were resolved by SDS-10% PAGE and analyzed by autoradiography; positions of phosphorylated GST-IRF-7 are indicated.

FIG. 9.

Activation of the IFNA4 promoter by wt and point mutations of IRF-7. HEK 293 cells were transfected with pRLTK control plasmid; a luciferase reporter plasmid containing the IRF-7-responsive IFNA4 promoter; and either a control plasmid or a plasmid encoding wt (IRF-7 WT), a series of serine-to-alanine (A and B) mutants of IRF-7, or a series of serine-to-aspartic acid (C and D) mutants of IRF-7, in the absence (C) or the presence of either IKKɛ (A) or TBK1 (B and D) as indicated. The point mutation of the IRF-7 plasmid is indicated under each bar. Luciferase activity was analyzed 24 h posttransfection as fold activation relative to the basal level of reporter gene in the presence of control vector (after normalization with cotransfected Renilla relative light units). Values represent the averages of three independent experiments performed in duplicate, with variability shown by error bars.