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Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1 - PubMed

  • ️Tue Jan 01 2008

Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1

Lenette L Lu et al. J Biol Chem. 2008.

Abstract

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFkappaB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of kappaB kinase epsilon (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.

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Figures

FIGURE 1.
FIGURE 1.

Select paramyxoviral V proteins inhibit TLR3-mediated gene induction. A, paramyxoviral V proteins were transiently expressed in TLR3 293 561-TK cells, which were then treated with dsRNA and GCV for 4 days (top panel) or GCV alone (bottom panel). Estimated cell survival is shown. Error bars represent standard error from two experiments. Lane 1, no V protein; lane 2, HeV protein; lane 3, MeV protein; lane 4, hPIV2 V protein/VH (H); lane 5, MuV V protein/VM (M); lane 6, PIV5 V protein/VP (P). B, HA immunoblotting of extracts from the above cells was performed to determine the levels of HA-tagged V proteins. Lanes are as described above. Approximate molecular weights are marked. C, P56 mRNA levels were measured by quantitative reverse transcription-PCR in untreated (lanes 1–4) or dsRNA-treated (lanes 5–8) HT1080 cells stably expressing V proteins. Error bars represent standard error from triplicate samples. D, P56 protein levels were measured by immunoblotting in untreated (lanes 1–4) or dsRNA-treated (lanes 5–8) cells described in C. Immunoblotting for actin and FLAG-V served as controls.

FIGURE 2.
FIGURE 2.

V proteins from hPIV2, MuV, and PIV5 inhibit IRF3 activation. A, pools of TLR3 293 561-TK cells expressing V proteins were transfected with expression vectors for different components of the TLR3 pathway; the specific signaling protein expressed is shown on the left. Induced p56 levels were determined by immunoblotting. Actin served as a loading control. Lane 1, noV; lane 2, VH (H); lane 3, VM (M); lane 4, VP (P). B, nuclear extracts from control cells (lanes 1 and 2) or cells stably expressing VM (lanes 3 and 4), that were untreated (lanes 1 and 3) or treated with dsRNA (lanes 2 and 4) were immunoblotted for IRF3, with histone as loading control. C, control and VM-expressing HT1080 cells were treated with dsRNA (rows 2 and 4) and LMB (rows 3 and 4). Subcellular location of IRF3 was determined by immunofluorescence. D, cell extracts from samples, as described for C, rows 3 and 4, were used for immunoblotting to detect p56, FLAG-V, and actin. E, IRF3 phosphorylation at serine 396 (P396) was detected by immunoblotting cell extracts with a phospho-IRF3-specific antiserum, with total IRF3 as a control. Whole cell extracts were prepared from control cells (lanes 1 and 3) or cells stably expressing VM (lanes 2 and 4) that were untreated (lanes 1 and 2) or treated with dsRNA (lanes 3 and 4).

FIGURE 3.
FIGURE 3.

Interaction of V with IKKe is essential for inhibition of TLR3 signaling. A, TRIF and V proteins were transiently expressed in TLR3 293 cells. V proteins were immunoprecipitated (IP), and co-immunoprecipitated proteins were immunoblotted to detect TRIF (indicated by arrow), HA-tagged V, stably expressed FLAG-tagged TLR3 and endogenous STAT2, phosphatidylinositol 3-kinase p85 (PI3K P85), c-Src (indicated by arrow), and IRF3 (indicated by arrow). Cell extracts (W) served as controls for expression levels. B, the same as A except that immunoblotting to detect endogenous IKK α, β, and γ was used in place of other TLR3 signaling mediators. C, IKKe and V proteins were transiently expressed in TLR3 293 cells. V proteins were immunoprecipitated, and co-immunoprecipitated proteins were immunoblotted to detect Myc-tagged IKKe, STAT2, and HA-tagged V; whole cell extracts served as controls for expression levels. Lanes 1 and 2, noV; lanes 3 and 4, VH (H); lanes 5 and 6, VM (M); lanes 7 and 8, VP (P). D, IKKe, transiently expressed with tagged ubiquitin (Ub) and VM or empty vector control, was immunoprecipitated. Samples were immunoblotted to detect HA-tagged ubiquitin (top panel, IKKeUb). Membranes were stripped and reprobed to detect Myc-tagged IKKe (bottom panel, IKKe) at the same molecular weight. E, FLAG-tagged WT and VM mutants M-AAA (W174A/W178A/W188A), E95D, E95R, C189A, C214A, and C217A were immunoprecipitated (top two panels), and co-immunoprecipitated proteins were immunoblotted to detect Myc-tagged IKKe and FLAG-tagged V. The bottom two panels show corresponding analyses of whole cell extracts. F, FLAG-tagged WT and VM C189A mutant were immunoprecipitated (lanes 2 and 4) as in D. Samples were then electrophoresed alongside whole cell extracts to show shifted as compared with unshifted mobilities of Myc-tagged IKKe. G, P56 protein levels were measured by immunoblotting in untreated (lanes 1, 3, 5, and 7) or dsRNA-treated (lanes 2, 4, 6, and 8) HT1080 cells stably expressing WT or VM mutants C189A or M-AAA. Immunoblotting for actin and FLAG-V served as controls.

FIGURE 4.
FIGURE 4.

MuV and PIV5 V proteins are phosphorylated by IKKe and TBK1. A, VM was expressed by itself (lane 1) or co-expressed with kinase active (lanes 2–3) or kinase inactive (KI)(lanes 4–5) IKKe, immunoprecipitated, and treated with calf intestine phosphate (CIP)(lanes 3 and 5). HA immunoblotting was used to detect V. B, the same as A except that VP was used in place of VM. C, in vitro [γ-32P]ATP kinase assays were conducted with GST·IKKe and V protein or IRF3. Radiolabeled proteins were visualized by autoradiography (top two panels) and immunoblotted (bottom two panels) to determine total amounts of IRF3 or V in the loaded samples. Lane 1, VM + IKKe; lane 2, IRF3 + IKKe; lane 3, IKKe; lane 4, VM. D, in vitro [γ-32P]ATP kinase assays were performed using purified Myc-TBK1. The top two panels are autoradiographs, and the bottom two panels are immunoblots. Lane 1, TBK1; lane 2, VM; lane 3, TBK1 + VM; lane 4, IRF3; lane 5, TBK1 + IRF3.

FIGURE 5.
FIGURE 5.

Phosphorylated MuV V protein is degraded. A, VM was expressed alone (lane 1), or along with WT kinase active (lane 2) or kinase inactive (KI) (lane 3) IKKe. Levels of HA-tagged V and Myc-tagged IKKe were determined by immunoblotting of cell extracts. B, TLR3 293 cells transiently expressing VM were treated with DMSO as a control (lane 1), dsRNA and DMSO (lane 2), or dsRNA and MG132 (lane 3). FLAG immunoblotting was used to detect FLAG-tagged V with actin as control. C, VM WT (lanes 1 and 2) and mutant M-AAA (lanes 3 and 4) were expressed with WT kinase active (lanes 1 and 3) or kinase inactive (lanes 2 and 4) IKKe. Levels of FLAG-tagged V and Myc-tagged IKKe were determined by immunoblotting of cell extracts.

FIGURE 6.
FIGURE 6.

Effects of dsRNA signaling on WT and mutant PIV5 replication. A, PIV5 WT (lane 1) or mutant C-terminally truncated (VDC) (lane 2) V proteins were co-expressed with IKKe and immunoprecipitated. V5 immunoblotting was used to detect V and Myc to detect IKKe. B, virus yields 6 days after infection were measured for WT or VDC PIV5 replication in 293 cells with and without TLR3 and pre- or mock-treated with dsRNA. Bars represent standard error derived from two independent experiments. PFU, plaque-forming units.

FIGURE 7.
FIGURE 7.

A negative feedback loop between rubulaviral V proteins and IKKe/TBK1. Although rubulaviral V proteins block TBK1/IKKe kinase activity by acting as an alternative substrate to IRF3/7, the resulting phosphorylated V protein (VPO4) is degraded. V proteins also mediate ubiquitination of TBK1/IKKe (Ub), leading to their degradation.

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References

    1. Bowie, A. G., and Fitzgerald, K. A. (2007) Trends Immunol. 28 147–150 - PubMed
    1. Hovanessian, A. G. (2007) Cytokine Growth Factor Rev. 18 351–361 - PubMed
    1. Medzhitov, R. (2007) Nature 449 819–826 - PubMed
    1. Platanias, L. C. (2005) Nat. Rev. Immunol. 5 375–386 - PubMed
    1. Garcia-Sastre, A. (2004) Curr. Top. Microbiol. Immunol. 283 249–280 - PubMed

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