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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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vIRF-1 BBD targets Bim and Bid BH3 domains.(A) In vitro co-precipitation assays were undertaken using bacterially-derived and purified recombinant vIRF-1 BBD and Bim BH3 domains fused to T7 epitope-tagged DsRed and GFP-chitin-binding domain (CBD), respectively. T7/DsRed-fused Bid BH3-B (Bid BH3-interacting) was also included, and BBD-AA (GK179AA, Bim BH3-refractory) and GFP-CBD were used as negative controls. Chitin bead-precipitated material was analyzed by immunoblotting to detect co-precipitated T7-tagged protein; wild-type BBD alone could be co-precipitated with Bim BH3-CBD. (B) Analogous experiments carried out using Bid BH3-GFP-CBD as “bait” identified similar interaction between BBD and Bid BH3, as detected also between Bid BH3-B and its cognate BH3 interaction partner (positive control). (C) Immunoprecipitation (IP) assays applied to transfected cell lysates were used to detect interactions between full-length vIRF-1 and Flag-tagged BidEL and BimEL[68], [69]. Co-precipitated vIRF-1 was detected by immoblotting using vIRF-1 antiserum, and α-Bim and α-Bid antibodies were used to confirm Flag antibody-mediated immunoprecipitation of the respective proteins. Immunoblotting for Flag and vIRF-1 verified appropriate expression of proteins in cell lysates.
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ppat-1002748-g002: vIRF-1 BBD targets Bim and Bid BH3 domains.(A) In vitro co-precipitation assays were undertaken using bacterially-derived and purified recombinant vIRF-1 BBD and Bim BH3 domains fused to T7 epitope-tagged DsRed and GFP-chitin-binding domain (CBD), respectively. T7/DsRed-fused Bid BH3-B (Bid BH3-interacting) was also included, and BBD-AA (GK179AA, Bim BH3-refractory) and GFP-CBD were used as negative controls. Chitin bead-precipitated material was analyzed by immunoblotting to detect co-precipitated T7-tagged protein; wild-type BBD alone could be co-precipitated with Bim BH3-CBD. (B) Analogous experiments carried out using Bid BH3-GFP-CBD as “bait” identified similar interaction between BBD and Bid BH3, as detected also between Bid BH3-B and its cognate BH3 interaction partner (positive control). (C) Immunoprecipitation (IP) assays applied to transfected cell lysates were used to detect interactions between full-length vIRF-1 and Flag-tagged BidEL and BimEL[68], [69]. Co-precipitated vIRF-1 was detected by immoblotting using vIRF-1 antiserum, and α-Bim and α-Bid antibodies were used to confirm Flag antibody-mediated immunoprecipitation of the respective proteins. Immunoblotting for Flag and vIRF-1 verified appropriate expression of proteins in cell lysates.

Mentions: To test whether the BBD of vIRF-1 interacted with the BH3 domain of Bim, recombinant fusion proteins were made for co-precipitation binding assays. The proteins comprised T7/DsRed-fused wild-type and Bim-binding-refractory GK179AA versions of BBD (vIRF-1 residues 170–187), and also Bid BH3-B (BidL residues 34–51), and chitin-binding domain (CBD)-tagged GFP-Bim BH3 (BimEL residues 148–161) and GFP (control). Paired T7/DsRed and GFP/CBD fusion proteins were mixed, CBD-tagged proteins precipitated with chitin beads, and precipitated material analyzed by SDS-PAGE and immunoblotting. This experiment revealed binding of BBD, but not BBD(GK179AA) or BH3-B, to Bim BH3, with no detectable background binding to negative control GFP-CBD (Fig. 2A). An analogous experiment using GFP/CBD-fused Bid BH3 (residues 86–99) as the “bait” identified interaction with Bid BH3-B (positive control) and also with vIRF-1 BBD (Fig. 2B), thereby identifying Bid BH3, as well as Bim BH3, as a target of vIRF-1 BBD interaction.


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)

vIRF-1 BBD targets Bim and Bid BH3 domains.(A) In vitro co-precipitation assays were undertaken using bacterially-derived and purified recombinant vIRF-1 BBD and Bim BH3 domains fused to T7 epitope-tagged DsRed and GFP-chitin-binding domain (CBD), respectively. T7/DsRed-fused Bid BH3-B (Bid BH3-interacting) was also included, and BBD-AA (GK179AA, Bim BH3-refractory) and GFP-CBD were used as negative controls. Chitin bead-precipitated material was analyzed by immunoblotting to detect co-precipitated T7-tagged protein; wild-type BBD alone could be co-precipitated with Bim BH3-CBD. (B) Analogous experiments carried out using Bid BH3-GFP-CBD as “bait” identified similar interaction between BBD and Bid BH3, as detected also between Bid BH3-B and its cognate BH3 interaction partner (positive control). (C) Immunoprecipitation (IP) assays applied to transfected cell lysates were used to detect interactions between full-length vIRF-1 and Flag-tagged BidEL and BimEL[68], [69]. Co-precipitated vIRF-1 was detected by immoblotting using vIRF-1 antiserum, and α-Bim and α-Bid antibodies were used to confirm Flag antibody-mediated immunoprecipitation of the respective proteins. Immunoblotting for Flag and vIRF-1 verified appropriate expression of proteins in cell lysates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369933&req=5

ppat-1002748-g002: vIRF-1 BBD targets Bim and Bid BH3 domains.(A) In vitro co-precipitation assays were undertaken using bacterially-derived and purified recombinant vIRF-1 BBD and Bim BH3 domains fused to T7 epitope-tagged DsRed and GFP-chitin-binding domain (CBD), respectively. T7/DsRed-fused Bid BH3-B (Bid BH3-interacting) was also included, and BBD-AA (GK179AA, Bim BH3-refractory) and GFP-CBD were used as negative controls. Chitin bead-precipitated material was analyzed by immunoblotting to detect co-precipitated T7-tagged protein; wild-type BBD alone could be co-precipitated with Bim BH3-CBD. (B) Analogous experiments carried out using Bid BH3-GFP-CBD as “bait” identified similar interaction between BBD and Bid BH3, as detected also between Bid BH3-B and its cognate BH3 interaction partner (positive control). (C) Immunoprecipitation (IP) assays applied to transfected cell lysates were used to detect interactions between full-length vIRF-1 and Flag-tagged BidEL and BimEL[68], [69]. Co-precipitated vIRF-1 was detected by immoblotting using vIRF-1 antiserum, and α-Bim and α-Bid antibodies were used to confirm Flag antibody-mediated immunoprecipitation of the respective proteins. Immunoblotting for Flag and vIRF-1 verified appropriate expression of proteins in cell lysates.
Mentions: To test whether the BBD of vIRF-1 interacted with the BH3 domain of Bim, recombinant fusion proteins were made for co-precipitation binding assays. The proteins comprised T7/DsRed-fused wild-type and Bim-binding-refractory GK179AA versions of BBD (vIRF-1 residues 170–187), and also Bid BH3-B (BidL residues 34–51), and chitin-binding domain (CBD)-tagged GFP-Bim BH3 (BimEL residues 148–161) and GFP (control). Paired T7/DsRed and GFP/CBD fusion proteins were mixed, CBD-tagged proteins precipitated with chitin beads, and precipitated material analyzed by SDS-PAGE and immunoblotting. This experiment revealed binding of BBD, but not BBD(GK179AA) or BH3-B, to Bim BH3, with no detectable background binding to negative control GFP-CBD (Fig. 2A). An analogous experiment using GFP/CBD-fused Bid BH3 (residues 86–99) as the “bait” identified interaction with Bid BH3-B (positive control) and also with vIRF-1 BBD (Fig. 2B), thereby identifying Bid BH3, as well as Bim BH3, as a target of vIRF-1 BBD interaction.

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