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. 2011 Feb 18;6(2):e17192.
doi: 10.1371/journal.pone.0017192.

PLP2 of mouse hepatitis virus A59 (MHV-A59) targets TBK1 to negatively regulate cellular type I interferon signaling pathway

Gang Wang  1 , Gang Chen, Dahai Zheng, Genhong Cheng, Hong Tang
Affiliations

PLP2 of mouse hepatitis virus A59 (MHV-A59) targets TBK1 to negatively regulate cellular type I interferon signaling pathway

Gang Wang et al. PLoS One. .

Abstract

Background: Coronaviruses such as severe acute respiratory syndrome (SARS) coronavirus (SCoV) and mouse hepatitis virus A59 (MHV-A59) have evolved strategies to disable the innate immune system for productive replication and spread of infection. We have previously shown that papain-like protease domain 2 (PLP2), a catalytic domain of the nonstructural protein 3 (nsp3) of MHV-A59, encodes a deubiquitinase (DUB) and inactivates IFN regulatory factor 3 (IRF3) thereby the type I interferon (IFN) response.

Principal findings: Here we provide further evidence that PLP2 may also target TANK-binding kinase-1 (TBK1), the upstream kinase of IRF3 in the IFN signaling pathway. Overexpression experiments showed that PLP2 deubiquitinated TBK1 and reduced its kinase activity, hence inhibited IFN-β reporter activity. Albeit promiscuous in deubiquitinating cellular proteins, PLP2 inactivated TBK1 and IFN-β response in TNF receptor associated factor 3 (TRAF3) deficient cells, suggesting that targeting TBK1 would be sufficient for PLP2 to inhibit IRF3 activation. This notion was further supported by in vitro kinase assays, in which prior treatment of TBK1 with PLP2 inhibited its kinase activity to phosphorylate IRF3. Intriguing enough, results of PLP2 overexpression system and MHV-A59 infection system proved that PLP2 formed an inactive complex with TBK1 and IRF3 in the cytoplasm and the presence of PLP2 stabilized the hypo-phosphorylated IRF3-TBK1 complex in a dose-dependent manner.

Conclusions: These results suggest that PLP2 not only inactivates TBK1, but also prevents IRF3 nuclear translocation hence inhibits IFN transcription activation. Identification of the conserved DUB activity of PLP2 in suppression of IFN signaling would provide a useful clue to the development of therapeutics against coronaviruses infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. K63-linked ubiquitination involved in the process of TBK1 activation could be inhibited by MHV-A59 PLP2.
(A) SeV infection induces K63-linked polyubiquitination of TBK1. HEK293T cells in 60 mm plates were transiently transfected with 3.6 µg HA-tagged ubiquitin K63 (HA-Ub-K63) expressing plasmids. At 24 h post transfection, cells were infected with SeV (HA titer 1∶25). At indicated time post infection, ubiquitination status of the endogenous TBK1 was immunoblotted with anti-HA antibody after immunoprecipitated by anti-TBK1 antibody (3 µg, IP: TBK1). TBK1 was not apparently degraded after viral infection as similar amounts of TBK1 were immunoabsorbed on beads (IB: TBK1). The whole cell lysates (WCL) was immunoblotted with anti-HA antibody for ubiquitin expression and massive cellular ubiquitination (HA), and anti-β-actin antibody for input. Immunobloting with phosphor-IRF3 and phosphor-STAT1 specific antibodies showed activation of TBK1 after viral infection for time indicated (p-IRF3, p-STAT1). (B) PLP2 associates with TBK1. HEK293T cells transiently expressing Flag-tagged TBK1 (Flag-TBK1) and Myc-tagged PLP2 (Myc-PLP2, WT or C106A) were lysed and immuoprecipitated with anti-Flag or -Myc antibodies. The immunoprecipitates were SDS-PAGE resolved and immunoblotted with antibody indicated. Mouse IgG was used as IP controls for Myc or Flag antibodies. (C) PLP2 deconjugates K63-linked polyubiquitin chains on TBK1. HEK293T cells (in 35 mm plates) transiently transfected with plasmids (800 ng each) encoding Flag-TBK1, HA-Ub-K63 or Myc-PLP2 (WT or C106A) for 24 h. Whole cell lysates were immunoprecipitated with anti-Flag antibody (1 µg) and SDS-PAGE resolved precipitates were immunoblotted with anti-HA or -Flag antibodies, respectively (IP: Flag). The expression of the epitope-tagged exogenous proteins was verified with the indicated antibodies (WCL). (D) PLP2 inhibits TBK1-driven IFN-β promoter activities. IFN-β-Luc promoter reporter (50 ng) and pCMV-Renilla internal control (15 ng) plasmids were co-transfected with Myc-TBK1 (100 ng) and Myc-PLP2 (WT or C106A, in three doses of 50, 100 and 200 ng) into HEK293T cells (24 well plates). Dual luciferase activities were measured and normalized to Renilla luciferase activities 24 h post transfection. Fold activation over the sham vector (pCMV-Myc) was averaged from three independent experiments (mean±SD). Expression of the exogenous epitope-tagged proteins was verified with the indicated antibodies (WCL). Data are representative of at least three independent experiments.
Figure 2
Figure 2. PLP2 inhibits IFN-β signaling by deubiquitinating both TBK1 and IRF3.
(A) PLP2 inhibits TBK1-driven IFN-β promoter activities in Traf3 −/− MEF cells. Luciferase assays were performed as in Fig. 1D except that Traf3 −/− MEF cells (in 24-well plates) were transfected with different amount of each plasmids: 150 ng for IFN-β-Luc reporter, 50 ng for Renilla, 200 ng for Myc-TBK1 and increasing doses (100, 200 and 400 ng) for Myc-PLP2 (WT or C106A). Fold activation over the sham vector (pCMV-Myc) was averaged from three independent experiments (mean±SD). (B) PLP2 deubiquitinates TBK1 in Traf3 −/− MEF cells. Experiments were performed as in Fig. 1C except that Traf3 −/− MEF cells (in 10 cm plates) were transfected with 8 µg of each plasmid for Flag-TBK1, HA-Ub or Myc-PLP2 (WT or C106A) for 36 h before immunoprecipitation. (C) PLP2 inhibits IRF3-driven IFN-β promoter activities in Tbk1 −/− cells. Experiments were carried out as in (A) except that plasmids expressing Flag-IRF3 and Myc-PLP2 (WT or C106A) were co-transfected into Tbk1 −/− cells. (D) PLP2 deubiquitinates IRF3 in Tbk1 −/− cells. Experiments were performed as in (B) except that Tbk1 −/− MEF cells were transfected with Flag-IRF3, HA-Ub and Myc-PLP2 (WT or C106A). Data are representative of at least three independent experiments.
Figure 3
Figure 3. PLP2 inhibits IFN signaling by inactivating the kinase activity of TBK1.
(A) PLP2 inhibits IRF3 phosphorylation by inactivating TBK1. Plasmids (800 ng) expressing Flag-TBK1 (WT or kinase dead mutant K38A) and Myc-PLP2 (WT or C106A) were co-expressed in HEK293T cells (in 35 mm plates). At 36 h post transfection, cells were lysed and Flag-TBK1 was immunoprecipitated with anti-Flag antibody. Immunoabsorbed Flag-TBK1 was incubation with recombinant GST-IRF3131–426 (1 µg) and γ-32P-ATP at 25°C for 30 min. Phosphorylation of proteins was resolved by SDS-PAGE and autoradiography. Expression of the exogenous proteins was verified with the indicated antibodies (WCL). (B) PLP2 inhibits TBK1 kinase activity in Traf3 −/− MEF cells. The similar in vitro kinase assays were carried out as in (A) except that 8 µg of each plasmid expressing Flag-TBK1(WT or K38A) and Myc-PLP2 (WT or C106A) were co-transfected into Traf3 −/− MEF cells (in 10 cm plates). (C) PLP2 inactivates the recombinant TBK1 by deubiquitination. An equal amount of recombinant TBK1 (500 ng) was incubated with Myc-PLP2 (WT or C106A) immunopurified from HEK293T cells at 37°C for 2 h. The kinase activities were measured as in (A). The deubiquitination efficiency of TBK1 was examined with anti-ubiquitin antibody and the amount of Myc-PLP2 (WT or C106A) used in each reaction was measured by anti-Myc antibody. Data are representative of at least three independent experiments.
Figure 4
Figure 4. PLP2 stabilizes TBK1-IRF3 complex.
(A) PLP2 inhibits the phosphorylation of IRF3 by inactivating TBK1 but stabilizes TBK1-IRF3 complex. A fixed amount of plasmids (800 ng each) for expressing Flag-IRF3 and Myc-TBK1, and an increasing amount (200, 400 and 800 ng) of Myc-PLP2 were co-transfected into HEK293T cells (in 35 mm plates). At 24 h post transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. TBK1 and PLP2 associated with IRF3 were detected with anti-Myc antibody. Expression levels of the exogenous proteins were verified with the indicated antibodies. Anti-phosphor-IRF3 antibody was used to detect the activation status of IRF3 (WCL). (B) Hypo-ubiquitinated TBK1 bounds recombinant IRF3 more efficiently. Flag-TBK1 co-expressed with Myc-PLP2 (WT or C106A) was immuno-purified as in Fig. 3A and incubated with recombinant GST-IRF3131–426 (2 µg) in 1 mL lysis buffer at 4°C for 4 h. The formed TBK-IRF3 complex was then separated by centrifugation and SDS-PAGE resolved. The amount of IRF3 and TBK1 was immunoblotted with anti-GST and anti-Flag antibodies, respectively (IP: Flag). Expression of the exogenous proteins was verified with the indicated antibodies (WCL). (C) Hypo-ubiquitinated IRF3 interacts with TBK1 more efficiently. Flag-IRF3 in the presence of Myc-PLP2 (WT or C106A) was immunoprecipitated with anti-Flag antibody and each precipitate was incubated with recombinant human TBK1 as in (B). Pulled-down TBK1 by IRF3 was immunoblotted with anti-TBK1 antibody. Expression of the exogenous proteins was verified with the indicated antibodies (WCL). Data are representative of at least three independent experiments.
Figure 5
Figure 5. TBK1, IRF3 and PLP2 domain containing protein co-localize in one complex in MHV-A59 infected cells.
(A) PLP2 domain containing protein is detected at 2 h post MHV-A59 infection. HEK293T-mCEACAM-1 cells or 17Cl-1 cells (in 35 mm plates) were infected with MHV-A59 (MOI = 5) for the time indicated, and cell lysates were immunoblotted with antiserum directed against MHV-A59 PLP2. Cell lysates of HEK293T ectopically expressing Myc-PLP2 was used as a positive control (lanes 6 and 12). (B) PLP2 containing protein stabilizes TBK1-IRF3 complex in MHV-A59 infected HEK293T-mCEACAM-1 cells. Plasmid encoding Flag-IRF3 or empty vector (1.2 µg) was transfected into HEK293T-mCEACAM-1 cells (in 35 mm plates). At 24 h post transfection, cells were simultaneously mock infected or infected with MHV-A59 for additional 10 h (MOI = 5) before lysed and immunoprecipitated with anti-Flag antibody. Endogenous TBK1 and PLP2 domain containing protein associated with Flag-IRF3 were immunoblotted with anti-TBK1 antibody or antiserum against PLP2 domain (IP: Flag). Expression levels of the exogenous proteins were verified with the indicated antibodies. (C) PLP2 containing protein stabilizes TBK1-IRF3 complex in MHV-A59 infected 17Cl-1 cells. Experiments were carried out as in (B) except with 17Cl-1 cells that expressing Flag-IRF3. Data are representative of at least three independent experiments.
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