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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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Functional equivalence of vIRF-1 BBD and Bid BH3-B.(A) Bid expression constructions were generated in which the BH3-B domain (BidL residues 33–48) was mutated [GHE41VLA (mBH3-B), BH3 refractory] or replaced with vIRF-1 BBD (residues 170–185) or substitution variant of BBD [GK179AA (mBBD)]. (B) These plasmids were individually cotransfected with GFP expression vector into HEK293T cells and after 24 h GFP fluorescence was quantified by fluorometry, providing a readout of cell viability. Relative fluorescence unit (RFU) values indicate the percentage of fluorescence relative to empty vector transfected cells (-Bid), with background, non-specific signal from untransfected cells (-GFP) subtracted. Error bars represent standard deviations from the average values derived from triplicate samples. (C) The functional equivalence of BH3-B and BBD in respect of apoptotic inhibition, specifically, was tested by using annexin V-Cy3 staining to detect HEK293T cells undergoing apoptosis at 7 h post-transfection. The inclusion of tBid expression plasmid and untransfected cultures in this experiment provided, respectively, a positive (BH3-B-deleted) control for induced apoptosis and a control for effects of transfection (by comparison to empty vector-transfected cultures). Cy3+ cells were counted from three random fields for each condition to generate the presented data; error bars show standard deviations from mean values obtained from individual fields. (D) Apoptotic inhibitory activity of vIRF-1 BBD in the context of BidL was further confirmed using a cytochrome c release assay. HEK293T cells were transfected with the indicated plasmids and harvested after 18 h. Dounce-derived extracts were either untreated or processed by centrifugation to derive total or S100 (soluble, sol.) samples for SDS-PAGE and immoblotting. Soluble sample blots were probed with antibodies specific for cytochrome c (cytC) or β-actin (loading control), and total cell extracts were probed with Flag antibody to detect and confirm expression of Flag-tagged Bid proteins.
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ppat-1002748-g003: Functional equivalence of vIRF-1 BBD and Bid BH3-B.(A) Bid expression constructions were generated in which the BH3-B domain (BidL residues 33–48) was mutated [GHE41VLA (mBH3-B), BH3 refractory] or replaced with vIRF-1 BBD (residues 170–185) or substitution variant of BBD [GK179AA (mBBD)]. (B) These plasmids were individually cotransfected with GFP expression vector into HEK293T cells and after 24 h GFP fluorescence was quantified by fluorometry, providing a readout of cell viability. Relative fluorescence unit (RFU) values indicate the percentage of fluorescence relative to empty vector transfected cells (-Bid), with background, non-specific signal from untransfected cells (-GFP) subtracted. Error bars represent standard deviations from the average values derived from triplicate samples. (C) The functional equivalence of BH3-B and BBD in respect of apoptotic inhibition, specifically, was tested by using annexin V-Cy3 staining to detect HEK293T cells undergoing apoptosis at 7 h post-transfection. The inclusion of tBid expression plasmid and untransfected cultures in this experiment provided, respectively, a positive (BH3-B-deleted) control for induced apoptosis and a control for effects of transfection (by comparison to empty vector-transfected cultures). Cy3+ cells were counted from three random fields for each condition to generate the presented data; error bars show standard deviations from mean values obtained from individual fields. (D) Apoptotic inhibitory activity of vIRF-1 BBD in the context of BidL was further confirmed using a cytochrome c release assay. HEK293T cells were transfected with the indicated plasmids and harvested after 18 h. Dounce-derived extracts were either untreated or processed by centrifugation to derive total or S100 (soluble, sol.) samples for SDS-PAGE and immoblotting. Soluble sample blots were probed with antibodies specific for cytochrome c (cytC) or β-actin (loading control), and total cell extracts were probed with Flag antibody to detect and confirm expression of Flag-tagged Bid proteins.

Mentions: The relationship between vIRF-1 BBD and Bid BH3-B was tested by substitution of the latter with the former in the context of full-length, uncleaved Bid (BidL) and testing the constructions (Fig. 3A) for pro-apoptotic activities in appropriately transfected cells. Apoptotic activity of Bid was measured by a GFP-based assay, in which loss of GFP fluorescence in GFP vector-cotransfected cells correlates with loss of cell viability and corresponds to rates of apoptosis, e.g. as measured by TUNEL assay [23]. Transfection of BidL and GFP expression vectors into HEK293T cells led to substantially reduced GFP fluorescence (∼39%) relative to empty vector plus GFP control, set at 100% (Fig. 3B), consistent with previously reported apoptotic activity of uncleaved Bid [45], [40]. However, this activity was increased substantially by introduction of Bid-BH3 binding-abrogating mutations (GHE41VLA [40]) into BH3-B, suppressing GFP fluorescence to ∼20% [Fig. 3B, Bid(mBH3-B)]. Importantly, BBD could substitute fully for BH3-B in this assay, inhibiting BH3-mediated Bid apoptotic activity more effectively than native BH3-B (75% GFP fluorescence relative to 39%). Increased inhibition by BBD indicates that it may bind with higher affinity than BH3-B to Bid BH3. Although it is more likely that BBD and BH3-B mediated inhibition of BidL activity occur via intramolecular interactions with Bid BH3, it is also possible that trans-inhibition may occur. Introduction of mutation GK179AA (previously shown to abrogate Bim interaction [23]) into BBD (mBBD) abolished its inhibition of BidL activity, leading to GFP fluorescence (cell viability) levels similar to those obtained upon mutation of BH3-B.


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)

Functional equivalence of vIRF-1 BBD and Bid BH3-B.(A) Bid expression constructions were generated in which the BH3-B domain (BidL residues 33–48) was mutated [GHE41VLA (mBH3-B), BH3 refractory] or replaced with vIRF-1 BBD (residues 170–185) or substitution variant of BBD [GK179AA (mBBD)]. (B) These plasmids were individually cotransfected with GFP expression vector into HEK293T cells and after 24 h GFP fluorescence was quantified by fluorometry, providing a readout of cell viability. Relative fluorescence unit (RFU) values indicate the percentage of fluorescence relative to empty vector transfected cells (-Bid), with background, non-specific signal from untransfected cells (-GFP) subtracted. Error bars represent standard deviations from the average values derived from triplicate samples. (C) The functional equivalence of BH3-B and BBD in respect of apoptotic inhibition, specifically, was tested by using annexin V-Cy3 staining to detect HEK293T cells undergoing apoptosis at 7 h post-transfection. The inclusion of tBid expression plasmid and untransfected cultures in this experiment provided, respectively, a positive (BH3-B-deleted) control for induced apoptosis and a control for effects of transfection (by comparison to empty vector-transfected cultures). Cy3+ cells were counted from three random fields for each condition to generate the presented data; error bars show standard deviations from mean values obtained from individual fields. (D) Apoptotic inhibitory activity of vIRF-1 BBD in the context of BidL was further confirmed using a cytochrome c release assay. HEK293T cells were transfected with the indicated plasmids and harvested after 18 h. Dounce-derived extracts were either untreated or processed by centrifugation to derive total or S100 (soluble, sol.) samples for SDS-PAGE and immoblotting. Soluble sample blots were probed with antibodies specific for cytochrome c (cytC) or β-actin (loading control), and total cell extracts were probed with Flag antibody to detect and confirm expression of Flag-tagged Bid proteins.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369933&req=5

ppat-1002748-g003: Functional equivalence of vIRF-1 BBD and Bid BH3-B.(A) Bid expression constructions were generated in which the BH3-B domain (BidL residues 33–48) was mutated [GHE41VLA (mBH3-B), BH3 refractory] or replaced with vIRF-1 BBD (residues 170–185) or substitution variant of BBD [GK179AA (mBBD)]. (B) These plasmids were individually cotransfected with GFP expression vector into HEK293T cells and after 24 h GFP fluorescence was quantified by fluorometry, providing a readout of cell viability. Relative fluorescence unit (RFU) values indicate the percentage of fluorescence relative to empty vector transfected cells (-Bid), with background, non-specific signal from untransfected cells (-GFP) subtracted. Error bars represent standard deviations from the average values derived from triplicate samples. (C) The functional equivalence of BH3-B and BBD in respect of apoptotic inhibition, specifically, was tested by using annexin V-Cy3 staining to detect HEK293T cells undergoing apoptosis at 7 h post-transfection. The inclusion of tBid expression plasmid and untransfected cultures in this experiment provided, respectively, a positive (BH3-B-deleted) control for induced apoptosis and a control for effects of transfection (by comparison to empty vector-transfected cultures). Cy3+ cells were counted from three random fields for each condition to generate the presented data; error bars show standard deviations from mean values obtained from individual fields. (D) Apoptotic inhibitory activity of vIRF-1 BBD in the context of BidL was further confirmed using a cytochrome c release assay. HEK293T cells were transfected with the indicated plasmids and harvested after 18 h. Dounce-derived extracts were either untreated or processed by centrifugation to derive total or S100 (soluble, sol.) samples for SDS-PAGE and immoblotting. Soluble sample blots were probed with antibodies specific for cytochrome c (cytC) or β-actin (loading control), and total cell extracts were probed with Flag antibody to detect and confirm expression of Flag-tagged Bid proteins.
Mentions: The relationship between vIRF-1 BBD and Bid BH3-B was tested by substitution of the latter with the former in the context of full-length, uncleaved Bid (BidL) and testing the constructions (Fig. 3A) for pro-apoptotic activities in appropriately transfected cells. Apoptotic activity of Bid was measured by a GFP-based assay, in which loss of GFP fluorescence in GFP vector-cotransfected cells correlates with loss of cell viability and corresponds to rates of apoptosis, e.g. as measured by TUNEL assay [23]. Transfection of BidL and GFP expression vectors into HEK293T cells led to substantially reduced GFP fluorescence (∼39%) relative to empty vector plus GFP control, set at 100% (Fig. 3B), consistent with previously reported apoptotic activity of uncleaved Bid [45], [40]. However, this activity was increased substantially by introduction of Bid-BH3 binding-abrogating mutations (GHE41VLA [40]) into BH3-B, suppressing GFP fluorescence to ∼20% [Fig. 3B, Bid(mBH3-B)]. Importantly, BBD could substitute fully for BH3-B in this assay, inhibiting BH3-mediated Bid apoptotic activity more effectively than native BH3-B (75% GFP fluorescence relative to 39%). Increased inhibition by BBD indicates that it may bind with higher affinity than BH3-B to Bid BH3. Although it is more likely that BBD and BH3-B mediated inhibition of BidL activity occur via intramolecular interactions with Bid BH3, it is also possible that trans-inhibition may occur. Introduction of mutation GK179AA (previously shown to abrogate Bim interaction [23]) into BBD (mBBD) abolished its inhibition of BidL activity, leading to GFP fluorescence (cell viability) levels similar to those obtained upon mutation of BH3-B.

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