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Identification of caspase-mediated decay of interferon regulatory factor-3, exploited by a Kaposi sarcoma-associated herpesvirus immunoregulatory protein.

Aresté C, Mutocheluh M, Blackbourn DJ - J. Biol. Chem. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Cancer Centre, School of Cancer Sciences, Vincent Drive, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.

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.

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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.
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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.

Mentions: Proteasome degradation is inhibited by treatment with MG132. As expected, treating cells with MG132 stabilized IRF-3 following poly(I:C) transfection (Fig. 2A, top panel, compare the amount of IRF-3 in lanes 4–6 in the presence of MG132 with that in its absence in lanes 1–3; see also supplemental Figs. S1 and supplemental Fig. S2A(i)). These data confirm that IRF-3 can be the target of a proteasome-dependent degradative process, as previously reported (9).


Identification of caspase-mediated decay of interferon regulatory factor-3, exploited by a Kaposi sarcoma-associated herpesvirus immunoregulatory protein.

Aresté C, Mutocheluh M, Blackbourn DJ - J. Biol. Chem. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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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.
Mentions: Proteasome degradation is inhibited by treatment with MG132. As expected, treating cells with MG132 stabilized IRF-3 following poly(I:C) transfection (Fig. 2A, top panel, compare the amount of IRF-3 in lanes 4–6 in the presence of MG132 with that in its absence in lanes 1–3; see also supplemental Figs. S1 and supplemental Fig. S2A(i)). These data confirm that IRF-3 can be the target of a proteasome-dependent degradative process, as previously reported (9).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Cancer Centre, School of Cancer Sciences, Vincent Drive, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus