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, 284 (35), 23272-85

Identification of Caspase-Mediated Decay of Interferon Regulatory factor-3, Exploited by a Kaposi Sarcoma-Associated Herpesvirus Immunoregulatory Protein

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Identification of Caspase-Mediated Decay of Interferon Regulatory factor-3, Exploited by a Kaposi Sarcoma-Associated Herpesvirus Immunoregulatory Protein

Cristina Aresté et al. J Biol Chem.

Abstract

Upon virus infection, the cell mounts an innate type I interferon (IFN) response to limit the spread. This response is orchestrated by the constitutively expressed IFN regulatory factor (IRF)-3 protein, which becomes post-translationally activated. Although the activation events are understood in detail, the negative regulation of this innate response is less well understood. Many viruses, including Kaposi sarcoma-associated herpesvirus (KSHV), have evolved defense strategies against this IFN response. Thus, KSHV encodes a viral IRF (vIRF)-2 protein, sharing homology with cellular IRFs and is a known inhibitor of the innate IFN response. Here, we show that vIRF-2 mediates IRF-3 inactivation by a mechanism involving caspase-3, although vIRF-2 itself is not pro-apoptotic. Importantly, we also show that caspase-3 participates in normal IRF-3 turnover in the absence of vIRF-2, during the antiviral response induced by poly(I:C) transfection. These data provide unprecedented insight into negative regulation of IRF-3 following activation of the type I IFN antiviral response and the mechanism by which KSHV vIRF-2 inhibits this innate response.

Figures

FIGURE 1.
FIGURE 1.
KSHV vIRF-2 expression represses IFN-β promoter transactivation and reduces wild type IRF-3 protein levels. In A: upper panel: transcriptional activity of the full-length IFN-β promoter (p125-luc) reporter vector. HEK293 cells were transiently co-transfected with p125-luc and an expression plasmid of either FLAG epitope-tagged IRF-3 wild type (IRF-3WT), or the mutants IRF-3(2A), IRF-3(5A), and IRF-3(2A5A). Each transfection also included the Xpress epitope-tagged vIRF-2-expressing plasmid, pcDNA4/vIRF-2 (vIRF-2), or the empty parental plasmid backbone, pcDNA4. The pRLSV40 plasmid constitutively expressing Renilla luciferase was added as an internal control to which firefly luciferase levels were normalized. Twenty-four hours after plasmid transfection, the cells were transfected with poly(I:C) (10 μg/ml), and luciferase activity was measured 18 h later. All reporter assays were repeated at least three times and mean values (± S.D.) are presented from one representative experiment (**, p < 0.01, Student's t test). White bars, cells lacking ectopic IRF-3 or vIRF-2; gray bars, cells expressing ectopic IRF-3; black bars, cells co-transfected to express ectopic vIRF-2. Lower panel: the IRF-3 C-terminal phosphorylation sites 1 and 2 (amino acids 382 to 414) are presented with the identities of the serine/threonine substitutes. B, vIRF-2 expression reduces total phosphorylated IRF-3 levels. Lysates were prepared from HEK293 cells that had been transfected with the IRF-3WT expression vector and either the vIRF-2 expression vector or the empty vector control (pcDNA4). Twenty-four hours after plasmid transfection, the cells were transfected with poly(I:C). They were then harvested at the times indicated and separated by SDS-PAGE and immunoblotted with the following antibodies: anti-phospho-IRF-3 (α-p-IRF-3; Ser396), anti-Xpress (vIRF-2), anti-FLAG (total ectopic IRF-3) and anti-β-actin. C, vIRF-2 expression reduces both monomeric and dimeric IRF-3 levels. Lysates prepared as described in B were separated by native PAGE to detect the IRF-3 dimeric (D) and monomeric (M) forms with anti-FLAG antibody. D, interaction of vIRF-2 with IRF-3. Co-immunoprecipitation studies were performed with lysates of HEK293 cells that had been transfected with the expression plasmids and poly(I:C) as described in A. vIRF-2 was immunoprecipitated with anti-Xpress monoclonal antibody. Immunoprecipitates and input extracts were separated by SDS-PAGE before immunoblotting with anti-FLAG polyclonal antibody to detect IRF-3 or anti-Xpress to detect vIRF-2. WT, IRF-3 wild type; 2A, IRF-3(2A) mutant; 5A, IRF-3(5A) mutant; 2A5A, IRF-3(2A5A) mutant; IP, antibody used for immunoprecipitation; IB, antibody used for immunoblot. IgG, provides a measure ensuring equal loading of immunoprecipitate between lanes; heavy chain is shown. Input, these lanes demonstrate the expression levels for vIRF-2 and IRF-3 in the lysates before immunoprecipitation. E, IRF-3(5A) accumulates in the presence of vIRF-2. HEK293 cells were transfected with FLAG-tagged IRF-3(5A)-expression vector in the presence and absence of the Xpress-tagged vIRF-2 expression vector and transfected with poly(I:C) 24 h later. After incubating for the times indicated, protein extracts were prepared and analyzed by SDS-PAGE and immunoblot.
FIGURE 2.
FIGURE 2.
The accelerated decay of activated wild-type IRF-3 by vIRF-2 is independent of the proteasome, but inhibited by a general caspase inhibitor. A, vIRF-2-accelerated decay of wild-type IRF-3 is independent of the proteasome. HEK293 cells were transiently co-transfected with expression vectors for either FLAG epitope-tagged wild type IRF-3 (IRF-3WT) (left panel) or IRF-3(5A) (right panel) and either Xpress epitope-tagged vIRF-2, or the empty parental plasmid backbone, pcDNA4. Twenty-four hours later, cells were treated for 30 min with either DMSO (as the vehicle negative control) or MG132 (10 μm) and then transfected with poly(I:C) (10 μg/ml). Lysates were prepared at various times thereafter, and protein samples were separated by SDS-PAGE and immunoblotted with anti-FLAG (to detect IRF-3), anti-Xpress (to detect vIRF-2), and anti-β-actin antibodies. This experiment is representative of more than three performed independently. A longer exposure of the anti-FLAG (IRF-3) (top) panel is presented in supplemental Fig. S1, to confirm IRF-3 can be visualized in lanes 12-18. B, vIRF-2-accelerated decay of wild-type IRF-3 is repressed by Z-VAD-FMK (Z-VAD) treatment, which inhibits caspase activity. This experiment was repeated essentially as described for A, with the exception that the cells were treated with Z-VAD-FMK (10 μm) in place of MG132 (left panel) and the IRF-3(5A) expression plasmid was not used. Alternatively, cells were treated with DMSO, MG132 (10 μm), or both Z-VAD-FMK (10 μm) and MG132 (10 μm) (right panel). Immunoblotting was performed with anti-FLAG (IRF-3), anti-Xpress (vIRF-2), anti-PARP, and anti-β-actin antibodies. The anti-PARP antibody recognizes uncleaved PARP (Un-Cl, 116 kDa) and one cleaved fragment (Cleaved, 83 kDa). This experiment is representative of more than three performed independently.
FIGURE 3.
FIGURE 3.
The accelerated decay of activated wild-type IRF-3 by vIRF-2 depends on caspase-3 activity. A, siRNA knockdown of caspase-3. Functional levels of caspase-3 protein were analyzed with the Caspase-Glo®-3/7 assay kit (Promega). HEK293 cells were transfected with either Lit28i Polylinker ShortCut® siRNA mix (Control siRNA) or caspase-3 ShortCut® siRNA (CAS-3 siRNA) for 24 h. They were then transfected with poly(I:C) for 18 h before harvesting for caspase activity assay. Activity assays were repeated at least twice, and average values (± S.D.) are presented from one representative experiment (*, p < 0.05, Student's t test). B, knockdown of caspase-3 elevates IFN-β promoter-driven reporter gene activity. HEK293 cells were transiently co-transfected with the full-length IFN-β promoter reporter plasmid (p125-luc) and an expression vector of FLAG-tagged IRF-3 wild-type (IRF-3WT), or Xpress-tagged vIRF-2-expression vector (vIRF-2), as indicated. The pRLSV40 plasmid constitutively expressing Renilla luciferase was added as an internal control to which firefly luciferase levels were normalized. Co-transfection with control (Control siRNA) and caspase-3 (CAS-3) siRNA was performed as indicated. Twenty-four hours after plasmid transfection, the cells were transfected with poly(I:C) (10 μg/ml), and luciferase activity was measured 18 h later. Average values (± S.D.) are presented from one representative experiment of at least three performed independently (*, #, and + indicate p < 0.05, Student's t test). Gray bars, cells expressing ectopic IRF-3; black bars, cells co-transfected to express ectopic vIRF-2. C, knockdown of caspase-3 elevates phospho-IRF-3 levels. HEK293 cells were transiently co-transfected with an expression vector for FLAG-tagged IRF-3 wild-type (IRF-3WT), and Xpress-tagged vIRF-2 (vIRF-2) or empty vector parental plasmid (pcDNA4), as indicated. Co-transfection with control (Control siRNA) and caspase-3 (CAS-3) siRNA was performed as indicated. Twenty-four hours after plasmid transfection, the cells were transfected with poly(I:C) (10 μg/ml). Protein extracts were prepared at the indicated times thereafter and analyzed by immunoblot with the following antibodies: anti-PARP, anti-Xpress (vIRF-2), anti-phospho-IRF-3 (α-p-IRF-3; Ser-396) and anti-β-actin. The anti-PARP antibody recognizes uncleaved PARP (Un-Cl, 116 kDa) and one cleaved fragment (Cleaved, 83 kDa). This experiment is representative of more than three performed independently. D, expression of vIRF-2 influences caspase-3/-7 activity. HEK293 cells were transiently transfected with an expression vector for FLAG-tagged IRF-3 wild type (IRF-3WT), and/or Xpress-tagged vIRF-2 (vIRF-2), as indicated. Twenty-four hours after plasmid transfection, the cells were treated with etoposide (50 μm) or DMSO and 30 min later transfected with poly(I:C) (10 μg/ml). Caspase-3/-7 activities were measured 18 h later. Average values (± S.D.) are presented from one representative experiment of three performed independently (*, p < 0.05, Student's t test). Black bars, cells co-transfected to express ectopic vIRF-2. E, pro-caspase-3 interacts with IRF-3 and vIRF-2. Upper panel: co-immunoprecipitation studies were performed with lysates of HEK293 cells that had been transfected with an expression vector for FLAG-tagged IRF-3 wild-type (IRF-3WT), or Xpress-tagged vIRF-2 (vIRF-2), as indicated. Twenty-four hours after plasmid transfection, the cells were transfected with poly(I:C) (10 μg/ml), and immunoprecipitation assays were performed 18 h later. Wild-type IRF-3 was immunoprecipitated with anti-FLAG polyclonal antibody and vIRF-2 with anti-Xpress monoclonal antibody. Immunoprecipitates and input extracts (lower panel) were separated by SDS-PAGE before immunoblotting with anti-Pro-caspase-3 antibody (α-Pro-cas-3). PE, protein extract; NT, non-transfected cells. Plasmid pCEP-4 expresses a negative control FLAG-tagged protein that did not immunoprecipitate pro-caspase-3. Note that all seven lanes were separated on the same gel and transferred to the same membrane, but because the vIRF-2 lanes were not contiguous with the others, intervening, irrelevant lanes were removed from the image. IP, antibody used for immunoprecipitation; IB, antibody used for immunoblot. Right panel, Input: these lanes demonstrate the expression levels for vIRF-2, IRF-3 and pro-caspase-3 in the lysates before immunoprecipitation.
FIGURE 4.
FIGURE 4.
IRF-3WT accumulates in a caspase-3-deficient cell line unless ectopic caspase-3 is introduced, when vIRF-2 accelerates the decay. A, IFN-β promoter-driven reporter gene activity is elevated in the absence of caspase-3. Caspase-3-deficient MCF-7 cells were transiently co-transfected with the full-length IFN-β promoter reporter plasmid (p125-luc) and an expression vector for FLAG-tagged IRF-3 wild type (IRF-3WT), or Xpress-tagged vIRF-2 (vIRF-2) or pcDNA3/caspase-3, a caspase-3 expression vector (CAS-3), as indicated in the figure. The pRLSV40 plasmid constitutively expressing Renilla luciferase was added as an internal control to which firefly luciferase levels were normalized. Twenty-four hours after plasmid transfection, the cells were transfected with poly(I:C) (10 μg/ml), and luciferase activity was measured 18 h later. Average values (± S.D.) are presented from one representative experiment (●, *, #, and + p < 0.05, Student's t test) of at least three performed independently. White bars, cells lacking ectopic IRF-3 or vIRF-2; gray bars, cells expressing ectopic IRF-3; black bars, cells co-transfected to express ectopic vIRF-2. B, IRF-3WT accumulates in a caspase-3-deficient cell line. MCF-7 cells were transiently co-transfected with an expression vector for FLAG-tagged IRF-3 wild-type (IRF-3WT), or Xpress-tagged vIRF-2 (vIRF-2) or pcDNA3/Caspase-3, a caspase-3 expression vector (CAS-3) or the empty vector pcDNA4, as indicated in the figure. Protein extracts were analyzed by immunoblot. C, vIRF-2 sequesters caspase-3 activity. Caspase-3/7 activity was measured in MCF-7 cells transfected with plasmids described above, as indicated in the figure. Twenty-four hours after plasmid transfection, the cells were transfected with poly(I:C) (10 μg/ml). Caspase-3/-7 activities were measured 18 h later. Average values (±S.D.) are presented from one representative experiment of three performed independently (* and #, p < 0.05, Student's t test). Black bars, cells co-transfected to express ectopic vIRF-2.
FIGURE 5.
FIGURE 5.
IRF-3WT, vIRF-2, and caspase-3 co-localize in the cytoplasm. A, vIRF-2 redistributes from the nucleus and the cytoplasm (panel: vIRF-2) to the cytoplasm following activation of the antiviral response by poly(I:C) transfection (panel: vIRF-2+IC). MCF-7 cells were transfected with the expression vector for Xpress-tagged vIRF-2. After 24 h they either were (+IC) or were not transfected with poly(I:C), fixed, and stained a further 16 h later. B, IRF-3 redistributes from the cytoplasm (panel: IRF-3) to the nucleus and the cytoplasm following activation of the antiviral response by poly(I:C) transfection (panel: IRF-3+IC). MCF-7 cells were transfected with the expression vector for FLAG-tagged IRF-3. After 24 h they either were (+IC) or were not transfected with poly(I:C), fixed, and stained a further 16 h later. C, vIRF-2 and IRF-3 co-localize in the cytoplasm. MCF-7 cells were co-transfected with the expression vectors for Xpress-tagged vIRF-2 and FLAG-tagged IRF-3. After 24 h they were (+IC) or were not transfected with poly(I:C) and fixed and stained a further 16 h later. D, vIRF-2 and caspase-3 co-localize in the cytoplasm. MCF-7 cells were co-transfected with the expression vectors for Xpress-tagged vIRF-2 and caspase-3 (CAS-3). After 24 h they were (+IC) or were not transfected with poly(I:C) and fixed and stained a further 16 h later. E, IRF-3 and caspase-3 co-localize in the cytoplasm. MCF-7 cells were cotransfected with the expression vectors for FLAG-tagged IRF-3 and caspase-3 (CAS-3). After 24 h they were (+IC) or were not transfected with poly(I:C) and fixed and stained a further 16 h later. F, vIRF-2, IRF-3, and caspase-3 co-localize in the cytoplasm. MCF-7 cells were co-transfected with the expression vectors for Xpress-tagged vIRF-2, FLAG-tagged IRF-3, and caspase-3 (CAS-3). After 24 h they were (+IC) or were not transfected with poly(I:C) and fixed and stained a further 16 h later. A region of co-localization in the merge panel (white arrow) was magnified, and the images are presented in the bottom row of panels.
FIGURE 5.
FIGURE 5.
IRF-3WT, vIRF-2, and caspase-3 co-localize in the cytoplasm. A, vIRF-2 redistributes from the nucleus and the cytoplasm (panel: vIRF-2) to the cytoplasm following activation of the antiviral response by poly(I:C) transfection (panel: vIRF-2+IC). MCF-7 cells were transfected with the expression vector for Xpress-tagged vIRF-2. After 24 h they either were (+IC) or were not transfected with poly(I:C), fixed, and stained a further 16 h later. B, IRF-3 redistributes from the cytoplasm (panel: IRF-3) to the nucleus and the cytoplasm following activation of the antiviral response by poly(I:C) transfection (panel: IRF-3+IC). MCF-7 cells were transfected with the expression vector for FLAG-tagged IRF-3. After 24 h they either were (+IC) or were not transfected with poly(I:C), fixed, and stained a further 16 h later. C, vIRF-2 and IRF-3 co-localize in the cytoplasm. MCF-7 cells were co-transfected with the expression vectors for Xpress-tagged vIRF-2 and FLAG-tagged IRF-3. After 24 h they were (+IC) or were not transfected with poly(I:C) and fixed and stained a further 16 h later. D, vIRF-2 and caspase-3 co-localize in the cytoplasm. MCF-7 cells were co-transfected with the expression vectors for Xpress-tagged vIRF-2 and caspase-3 (CAS-3). After 24 h they were (+IC) or were not transfected with poly(I:C) and fixed and stained a further 16 h later. E, IRF-3 and caspase-3 co-localize in the cytoplasm. MCF-7 cells were cotransfected with the expression vectors for FLAG-tagged IRF-3 and caspase-3 (CAS-3). After 24 h they were (+IC) or were not transfected with poly(I:C) and fixed and stained a further 16 h later. F, vIRF-2, IRF-3, and caspase-3 co-localize in the cytoplasm. MCF-7 cells were co-transfected with the expression vectors for Xpress-tagged vIRF-2, FLAG-tagged IRF-3, and caspase-3 (CAS-3). After 24 h they were (+IC) or were not transfected with poly(I:C) and fixed and stained a further 16 h later. A region of co-localization in the merge panel (white arrow) was magnified, and the images are presented in the bottom row of panels.
FIGURE 6.
FIGURE 6.
Endogenous IRF-3 shares the same fate as ectopic IRF-3. A, vIRF-2 inhibition of the transcriptional activity of the full-length IFN-β promoter (p125-luc) reporter vector driven by endogenous IRF-3 is repressed by the caspase inhibitor Z-VAD-FMK. HEK293 cells were transiently co-transfected with p125-luc and the Xpress epitope-tagged vIRF-2-expressing plasmid, pcDNA4/vIRF-2 (vIRF-2; black bars), or the empty parental plasmid backbone, pcDNA4 (light bars). The pRLSV40 plasmid constitutively expressing Renilla luciferase was added as an internal control to which firefly luciferase levels were normalized. Twenty-four hours later, cells were treated for 30 min with either DMSO (as the vehicle negative control) or Z-VAD-FMK (10 μm) and then transfected with poly(I:C) (10 μg/ml). Luciferase activity was measured 12 h later. The data represent the mean (± S.D.) from one representative experiment of three performed independently (*, #, and +, p < 0.005, Student's t test). B, vIRF-2 inhibition of endogenous IRF-3 activity is repressed by Z-VAD-FMK (Z-VAD) treatment. The clone #3–9 cell line (#3–9) harboring doxycycline-inducible vIRF-2 and the negative control empty vector cell line (EV) were both treated with doxycycline (DOX) for 24 h. They were then treated with either DMSO (as the vehicle negative control) or Z-VAD-FMK (Z-VAD; 10 μm) and transfected with poly(I:C) (10 μg/ml) for the times indicated. IRF-3 activity was determined with the TransAMTM IRF-3 ELISA (Active Motif). The data represent the mean (± S.D.) of two independent experiments (* and #, p < 0.005, Student's t test). C, vIRF-2-accelerated decay of endogenous IRF-3 is repressed by Z-VAD-FMK (Z-VAD) treatment. This experiment was performed essentially as described for Fig. 6B, with the exception that IRF-3 levels were measured by Western blot. The clone #3–9 cell line (#3–9) harboring doxycycline-inducible vIRF-2 and the negative control empty vector cell line (EV) were both treated with doxycycline (DOX) for 24 h. They were then treated with either DMSO (as the vehicle negative control) or Z-VAD-FMK (10 μm) and transfected with poly(I:C) (10 μg/ml) for the times indicated. Immunoblotting was performed on 10 μg of lysates separated either by native PAGE (upper panel) or denaturing PAGE (lower three panels). Native PAGE enabled IRF-3 dimeric (D) and monomeric (M) forms to be detected with anti-IRF-3 antibody. Aliquots of the same lysates were analyzed by denaturing PAGE to quantify total IRF-3, c-Myc-tagged vIRF-2, and β-actin. D, vIRF-2 inhibition of the transcriptional activity of the full-length IFN-β promoter (p125-luc) reporter vector driven by endogenous IRF-3 is repressed by caspase-3 siRNA. HEK293 cells were transiently co-transfected with p125-luc and the Xpress epitope-tagged vIRF-2-expressing plasmid, pcDNA4/vIRF-2 (vIRF-2; black bars), or the empty parental plasmid backbone, pcDNA4 (light bars). The pRLSV40 plasmid constitutively expressing Renilla luciferase was added as an internal control to which firefly luciferase levels were normalized. At the same time, either siRNA for caspase-3 (CAS-3 siRNA) or negative control scrambled siRNA (Control siRNA) was transfected. Twenty-four hours later, cells were transfected with poly(I:C) (10 μg/ml), and luciferase activity was measured 12 h later. The data represent the mean (± S.D.) from one representative experiment of three performed independently (* and #, p < 0.005; +, p < 0.05, Student's t test). E, measuring endogenous IRF-3 activity in the presence of vIRF-2 and MG132. The clone #3–9 cell line (#3–9) harboring doxycycline-inducible vIRF-2 and the negative control empty vector cell line (EV) were treated with doxycycline (DOX) for 24 h. They were then treated with either DMSO (as the vehicle negative control) or MG132 (10 μm) and transfected with poly(I:C) (10 μg/ml) for the times indicated. IRF-3 activity was determined with the TransAMTM IRF-3 ELISA (Active Motif). The data represent the mean (± S.D.) of two independent experiments (*, #, and +, p < 0.005, Student's t test).

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