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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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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.
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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.

Mentions: Because IRF-3 stability in the presence of vIRF-2 protein expression was higher in caspase-3-deficient MCF-7 cells than in HEK293 cells, the distribution of these proteins in the breast adenocarcinoma cell line was investigated (Fig. 5). The studies were performed 16 h following poly(I:C) transfection, because after longer times in the presence of caspase-3, IRF-3 levels become undetectable (see Fig. 4B). The vIRF-2 protein assumed a diffuse cytoplasmic and nuclear distribution, with some concentration around the inner nuclear membrane (Fig. 5A). Upon activation of the antiviral response by poly(I:C) transfection, this distribution became more punctate with expression emphasized in the cytoplasm and not the nucleus (Fig. 5A). Conversely, IRF-3 redistributed to both the nuclear and cytoplasmic compartments from a predominantly cytoplasmic location upon poly(I:C) transfection (Fig. 5B), as described previously (23). When vIRF-2 and IRF-3 were co-expressed, IRF-3 was sufficiently abundant to be detected readily, consistent with the Western blot data (Fig. 4B, lanes 7–9), but its redistribution to the nucleus was minimized (Fig. 5C). These data are consistent with caspase-3-independent inhibition of IRF-3 transactivation by vIRF-2, as suggested previously (Figs. 3B and 4A). Both vIRF-2 and IRF-3, when expressed independently in MCF-7 cells, were sufficiently abundant to be identified as colocalizing with ectopic caspase-3, predominantly in the cytoplasm (Fig. 5, D and E, respectively).


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)

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.
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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.
Mentions: Because IRF-3 stability in the presence of vIRF-2 protein expression was higher in caspase-3-deficient MCF-7 cells than in HEK293 cells, the distribution of these proteins in the breast adenocarcinoma cell line was investigated (Fig. 5). The studies were performed 16 h following poly(I:C) transfection, because after longer times in the presence of caspase-3, IRF-3 levels become undetectable (see Fig. 4B). The vIRF-2 protein assumed a diffuse cytoplasmic and nuclear distribution, with some concentration around the inner nuclear membrane (Fig. 5A). Upon activation of the antiviral response by poly(I:C) transfection, this distribution became more punctate with expression emphasized in the cytoplasm and not the nucleus (Fig. 5A). Conversely, IRF-3 redistributed to both the nuclear and cytoplasmic compartments from a predominantly cytoplasmic location upon poly(I:C) transfection (Fig. 5B), as described previously (23). When vIRF-2 and IRF-3 were co-expressed, IRF-3 was sufficiently abundant to be detected readily, consistent with the Western blot data (Fig. 4B, lanes 7–9), but its redistribution to the nucleus was minimized (Fig. 5C). These data are consistent with caspase-3-independent inhibition of IRF-3 transactivation by vIRF-2, as suggested previously (Figs. 3B and 4A). Both vIRF-2 and IRF-3, when expressed independently in MCF-7 cells, were sufficiently abundant to be identified as colocalizing with ectopic caspase-3, predominantly in the cytoplasm (Fig. 5, D and E, respectively).

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