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Human herpesvirus 8 interferon regulatory factor-mediated BH3-only protein inhibition via Bid BH3-B mimicry.

Choi YB, Sandford G, Nicholas J - PLoS Pathog. (2012)

Bottom Line: Consistent with this finding, direct BBD-dependent inactivation by vIRF-1 of Bid-induced mitochondrial permeabilization was demonstrable in vitro and isolated BBD sequences were also active in this assay.In addition to Bim and Bid BH3 domains, BH3s of BOPs Bik, Bmf, Hrk, and Noxa also were found to bind BBD, while those of both pro- and anti-apoptotic multi-BH domain Bcl-2 proteins were not.Together, our data demonstrate and characterize BH3 targeting and associated inhibition of BOP pro-apoptotic activity by vIRF-1 via Bid BH3-B mimicry, identifying a novel mechanism of viral evasion from host cell defenses.

View Article: PubMed Central - PubMed

Affiliation: Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of host cells. For HHV-8, viral interferon regulatory factor-1 (vIRF-1) contributes to this process in part via inhibitory interactions with BH3-only protein (BOP) Bim, recently identified as an interaction partner of vIRF-1. Here we recognize that the Bim-binding domain (BBD) of vIRF-1 resembles a region (BH3-B) of Bid, another BOP, which interacts intramolecularly with the functional BH3 domain of Bid to inhibit it pro-apoptotic activity. Indeed, vIRF-1 was found to target Bid in addition to Bim and to interact, via its BBD region, with the BH3 domain of each. In functional assays, BBD could substitute for BH3-B in the context of Bid, to suppress Bid-induced apoptosis in a BH3-binding-dependent manner, and vIRF-1 was able to protect transfected cells from apoptosis induced by Bid. While vIRF-1 can mediate nuclear sequestration of Bim, this was not the case for Bid, and inhibition of Bid and Bim by vIRF-1 could occur independently of nuclear localization of the viral protein. Consistent with this finding, direct BBD-dependent inactivation by vIRF-1 of Bid-induced mitochondrial permeabilization was demonstrable in vitro and isolated BBD sequences were also active in this assay. In addition to Bim and Bid BH3 domains, BH3s of BOPs Bik, Bmf, Hrk, and Noxa also were found to bind BBD, while those of both pro- and anti-apoptotic multi-BH domain Bcl-2 proteins were not. Finally, the significance of Bid to virus replication was demonstrated via Bid-depletion in HHV-8 infected cells, which enhanced virus production. Together, our data demonstrate and characterize BH3 targeting and associated inhibition of BOP pro-apoptotic activity by vIRF-1 via Bid BH3-B mimicry, identifying a novel mechanism of viral evasion from host cell defenses.

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Direct functional inhibition of Bid by vIRF-1.(A) T7-fused vIRF-1, vIRF-1ΔBBD and GFP (negative control) and S-tag-fused tBid recombinant proteins (Materials and Methods) were checked for purity by SDS-PAGE and Coomassie staining. (B) Recombinant proteins were utilized in an in vitro mitochondrial permeabilization assay to determine the ability of vIRF-1 to suppress tBid-induced cytochrome c release from sucrose gradient-purified mitochondria (Materials and Methods). After 30 min. incubation, relative amounts of cytochrome c present in and released from mitochondria were determined by Western analysis of pellets and supernatants. Wild-type vIRF-1 (100 nM) completely blocked activity of tBid (applied at 100 nM) in this assay, whereas vIRF-1ΔBBD was inactive. (supe, supernatant; BSA, bovine serum albumin). (C) T7-vIRF-1 recombinant protein containing BH3-B in place of the BBD region was generated (shown diagrammatically) along with recombinant GST, GST-BBD and GST-BH3-B proteins. The purity and concentrations of these proteins were checked by SDS-PAGE and Coomassie staining. (D) As before, the proteins were utilized in in vitro mitochondrial permeabilization assays to determine their abilities to inhibit tBid-induced cytochrome c release. T7-vIRF-1.BH3-B and GST-fused BH3-B and BBD were all functional in this assay. Applied concentrations of tBid and vIRF-1 proteins were 100 nM (top); for tBid and GST-fusion proteins, the concentrations were 10 nM and 500 nM, respectively (bottom).
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ppat-1002748-g007: Direct functional inhibition of Bid by vIRF-1.(A) T7-fused vIRF-1, vIRF-1ΔBBD and GFP (negative control) and S-tag-fused tBid recombinant proteins (Materials and Methods) were checked for purity by SDS-PAGE and Coomassie staining. (B) Recombinant proteins were utilized in an in vitro mitochondrial permeabilization assay to determine the ability of vIRF-1 to suppress tBid-induced cytochrome c release from sucrose gradient-purified mitochondria (Materials and Methods). After 30 min. incubation, relative amounts of cytochrome c present in and released from mitochondria were determined by Western analysis of pellets and supernatants. Wild-type vIRF-1 (100 nM) completely blocked activity of tBid (applied at 100 nM) in this assay, whereas vIRF-1ΔBBD was inactive. (supe, supernatant; BSA, bovine serum albumin). (C) T7-vIRF-1 recombinant protein containing BH3-B in place of the BBD region was generated (shown diagrammatically) along with recombinant GST, GST-BBD and GST-BH3-B proteins. The purity and concentrations of these proteins were checked by SDS-PAGE and Coomassie staining. (D) As before, the proteins were utilized in in vitro mitochondrial permeabilization assays to determine their abilities to inhibit tBid-induced cytochrome c release. T7-vIRF-1.BH3-B and GST-fused BH3-B and BBD were all functional in this assay. Applied concentrations of tBid and vIRF-1 proteins were 100 nM (top); for tBid and GST-fusion proteins, the concentrations were 10 nM and 500 nM, respectively (bottom).

Mentions: Bid and Bim BH3 domain-targeting by vIRF-1, nuclear localization-independent inhibition of BOP pro-apoptotic activity, and partial mitochondrial localization of vIRF-1 suggested the likelihood of direct BOP inactivation via BBD∶BH3 association. This was tested by using an in vitro mitochondrial permeabilization assay to assess the abilities of wild-type and a ΔBBD (BOP-refractory) variant of vIRF-1 to inhibit tBid-induced cytochrome c release. The vIRF-1 proteins and tBid were expressed as T7/intein/CBD and thioredoxin/His6/S-tag fusion proteins in bacteria and were subsequently purified and cleaved to release the respective T7- and S-tagged proteins (see Materials and Methods). SDS-PAGE and Coomassie staining (Fig. 7A) verified their purity prior to use. Addition of recombinant tBid (1.5 µg/ml, 100 nM) to mitochondrial preparations induced the release of cytochrome c into the soluble fraction of the mitochondrial suspension and led to a corresponding decrease in the level of cytochrome c in the mitochondrial pellet, as determined by Western analysis (Fig. 7B). Inclusion of vIRF-1 (8 µg/ml, 100 nM) blocked all detectable cytochrome c release, but vIRF-1ΔBBD was inactive in respect of tBid inhibition. These data demonstrate that BBD∶BH3 interaction alone is sufficient to inhibit tBid-induced apoptosis.


Human herpesvirus 8 interferon regulatory factor-mediated BH3-only protein inhibition via Bid BH3-B mimicry.

Choi YB, Sandford G, Nicholas J - PLoS Pathog. (2012)

Direct functional inhibition of Bid by vIRF-1.(A) T7-fused vIRF-1, vIRF-1ΔBBD and GFP (negative control) and S-tag-fused tBid recombinant proteins (Materials and Methods) were checked for purity by SDS-PAGE and Coomassie staining. (B) Recombinant proteins were utilized in an in vitro mitochondrial permeabilization assay to determine the ability of vIRF-1 to suppress tBid-induced cytochrome c release from sucrose gradient-purified mitochondria (Materials and Methods). After 30 min. incubation, relative amounts of cytochrome c present in and released from mitochondria were determined by Western analysis of pellets and supernatants. Wild-type vIRF-1 (100 nM) completely blocked activity of tBid (applied at 100 nM) in this assay, whereas vIRF-1ΔBBD was inactive. (supe, supernatant; BSA, bovine serum albumin). (C) T7-vIRF-1 recombinant protein containing BH3-B in place of the BBD region was generated (shown diagrammatically) along with recombinant GST, GST-BBD and GST-BH3-B proteins. The purity and concentrations of these proteins were checked by SDS-PAGE and Coomassie staining. (D) As before, the proteins were utilized in in vitro mitochondrial permeabilization assays to determine their abilities to inhibit tBid-induced cytochrome c release. T7-vIRF-1.BH3-B and GST-fused BH3-B and BBD were all functional in this assay. Applied concentrations of tBid and vIRF-1 proteins were 100 nM (top); for tBid and GST-fusion proteins, the concentrations were 10 nM and 500 nM, respectively (bottom).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369933&req=5

ppat-1002748-g007: Direct functional inhibition of Bid by vIRF-1.(A) T7-fused vIRF-1, vIRF-1ΔBBD and GFP (negative control) and S-tag-fused tBid recombinant proteins (Materials and Methods) were checked for purity by SDS-PAGE and Coomassie staining. (B) Recombinant proteins were utilized in an in vitro mitochondrial permeabilization assay to determine the ability of vIRF-1 to suppress tBid-induced cytochrome c release from sucrose gradient-purified mitochondria (Materials and Methods). After 30 min. incubation, relative amounts of cytochrome c present in and released from mitochondria were determined by Western analysis of pellets and supernatants. Wild-type vIRF-1 (100 nM) completely blocked activity of tBid (applied at 100 nM) in this assay, whereas vIRF-1ΔBBD was inactive. (supe, supernatant; BSA, bovine serum albumin). (C) T7-vIRF-1 recombinant protein containing BH3-B in place of the BBD region was generated (shown diagrammatically) along with recombinant GST, GST-BBD and GST-BH3-B proteins. The purity and concentrations of these proteins were checked by SDS-PAGE and Coomassie staining. (D) As before, the proteins were utilized in in vitro mitochondrial permeabilization assays to determine their abilities to inhibit tBid-induced cytochrome c release. T7-vIRF-1.BH3-B and GST-fused BH3-B and BBD were all functional in this assay. Applied concentrations of tBid and vIRF-1 proteins were 100 nM (top); for tBid and GST-fusion proteins, the concentrations were 10 nM and 500 nM, respectively (bottom).
Mentions: Bid and Bim BH3 domain-targeting by vIRF-1, nuclear localization-independent inhibition of BOP pro-apoptotic activity, and partial mitochondrial localization of vIRF-1 suggested the likelihood of direct BOP inactivation via BBD∶BH3 association. This was tested by using an in vitro mitochondrial permeabilization assay to assess the abilities of wild-type and a ΔBBD (BOP-refractory) variant of vIRF-1 to inhibit tBid-induced cytochrome c release. The vIRF-1 proteins and tBid were expressed as T7/intein/CBD and thioredoxin/His6/S-tag fusion proteins in bacteria and were subsequently purified and cleaved to release the respective T7- and S-tagged proteins (see Materials and Methods). SDS-PAGE and Coomassie staining (Fig. 7A) verified their purity prior to use. Addition of recombinant tBid (1.5 µg/ml, 100 nM) to mitochondrial preparations induced the release of cytochrome c into the soluble fraction of the mitochondrial suspension and led to a corresponding decrease in the level of cytochrome c in the mitochondrial pellet, as determined by Western analysis (Fig. 7B). Inclusion of vIRF-1 (8 µg/ml, 100 nM) blocked all detectable cytochrome c release, but vIRF-1ΔBBD was inactive in respect of tBid inhibition. These data demonstrate that BBD∶BH3 interaction alone is sufficient to inhibit tBid-induced apoptosis.

Bottom Line: Consistent with this finding, direct BBD-dependent inactivation by vIRF-1 of Bid-induced mitochondrial permeabilization was demonstrable in vitro and isolated BBD sequences were also active in this assay.In addition to Bim and Bid BH3 domains, BH3s of BOPs Bik, Bmf, Hrk, and Noxa also were found to bind BBD, while those of both pro- and anti-apoptotic multi-BH domain Bcl-2 proteins were not.Together, our data demonstrate and characterize BH3 targeting and associated inhibition of BOP pro-apoptotic activity by vIRF-1 via Bid BH3-B mimicry, identifying a novel mechanism of viral evasion from host cell defenses.

View Article: PubMed Central - PubMed

Affiliation: Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of host cells. For HHV-8, viral interferon regulatory factor-1 (vIRF-1) contributes to this process in part via inhibitory interactions with BH3-only protein (BOP) Bim, recently identified as an interaction partner of vIRF-1. Here we recognize that the Bim-binding domain (BBD) of vIRF-1 resembles a region (BH3-B) of Bid, another BOP, which interacts intramolecularly with the functional BH3 domain of Bid to inhibit it pro-apoptotic activity. Indeed, vIRF-1 was found to target Bid in addition to Bim and to interact, via its BBD region, with the BH3 domain of each. In functional assays, BBD could substitute for BH3-B in the context of Bid, to suppress Bid-induced apoptosis in a BH3-binding-dependent manner, and vIRF-1 was able to protect transfected cells from apoptosis induced by Bid. While vIRF-1 can mediate nuclear sequestration of Bim, this was not the case for Bid, and inhibition of Bid and Bim by vIRF-1 could occur independently of nuclear localization of the viral protein. Consistent with this finding, direct BBD-dependent inactivation by vIRF-1 of Bid-induced mitochondrial permeabilization was demonstrable in vitro and isolated BBD sequences were also active in this assay. In addition to Bim and Bid BH3 domains, BH3s of BOPs Bik, Bmf, Hrk, and Noxa also were found to bind BBD, while those of both pro- and anti-apoptotic multi-BH domain Bcl-2 proteins were not. Finally, the significance of Bid to virus replication was demonstrated via Bid-depletion in HHV-8 infected cells, which enhanced virus production. Together, our data demonstrate and characterize BH3 targeting and associated inhibition of BOP pro-apoptotic activity by vIRF-1 via Bid BH3-B mimicry, identifying a novel mechanism of viral evasion from host cell defenses.

Show MeSH
Related in: MedlinePlus