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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 localization to mitochondria.(A) Homogenates of HEK293T cells transfected with wild-type or BBD-mutated (GK179AA) vIRF-1 (or empty vector control, vIRF-1-negative) were subjected to differential and Optiprep gradient centrifugation to isolate enriched mitochondrial fractions (see Materials and Methods). These were analyzed by immunoblotting with vIRF-1-specific rabbit antiserum for the presence of vIRF-1 protein, and also with antibody to mitochondrial protein VDAC1 [voltage-dependent anion-selective channel protein 1; integral mitochondrial outer membrane (int. OM) protein] to provide a positive control. Normalization of vIRF-1 amounts in lysates versus mitochondrial fractions from these transfected cells was achieved using a ratio of 1∶50 (bottom). (B) In vitro mitochondrial binding assays using enriched mitochondria, untreated or pre-treated with proteinase K (Prot. K), and recombinant T7-tagged vIRF-1 for assessment of the requirement for surface protein integrity for mitochondrial binding by vIRF-1. Total protein released from mitochondria was precipitated with trichloroacetic acid (TCA) for direct quantitative comparison with protein from mitochondrial pellets. Blots were probed with antibodies to mitochondrial outer membrane (OM)-associated Bax and inner membrane (IM)-localized prohibitin to provide controls for appropriate fractionation, mitochondrial integrity, and proteinase K activity. (C) Generation and Western analysis of mitochondrial preparations from HHV-8+ BCBL-1-TRE/RTA cells [70] revealed mitochondrial association of endogenous vIRF-1, both in resting (latent) and lytically reactivated (+Dox) cultures; the latter, as expected, expressed higher levels of vIRF-1. In both cases, mitochondrial-associated vIRF-1 was susceptible to protease digestion, consistent with peripheral binding to mitochondria. Immunodetection of Bim verified peripheral protein susceptibility to proteinase K digestion. (D) Mitochondrial localization of vIRF-1 as determined using immunoflourescence assay for detection of vIRF-1 and mitochondrial marker TOM20 in TIME-TRE/vIRF-1 endothelial cells, +Dox. Arrows indicate examples of vIRF-1/TOM20 co-localization. (E) Western analyses of total cell and mitochondrial extracts of HHV-8+ TIME-TRE/RTA and BCBL1-TRE/RTA cells, untreated or treated with Dox for 2 days, were undertaken to quantify the relative amounts of mitochondrial-localized vIRF-1 in these latently and lytically infected cells. For TIME and BCBL-1 cells, 10- and 50-fold excess of mitochondrial extract over total extract, respectively, was loaded onto the gels to achieve near normalization. Relative signal intensities of bands were obtained from digitally captured images and calculated values of mitochondrial relative to total vIRF-1 levels are shown under the vIRF-1 blots. vIRF-1 was detected in latency (−Dox) only in the BCBL-1 (PEL) cells, and mitochondrial∶total vIRF-1 was increased upon Dox addition. Immunoblotting for VDAC1 (mitochondrial), histone deacetylase-1 (HDAC1, nuclear), calreticulin (endoplasmic reticulum) and β-actin (cytoplasmic) provided quality controls for mitochondrial and total cell extracts. (Arrowhead, vIRF-1; *non-specific). (F) A similar experiment in HHV-8+ TIME-TRE/RTA cells, demonstrating mitochondrial localization of Bid, in addition to vIRF-1, in lytically reactivated cultures.
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ppat-1002748-g006: vIRF-1 localization to mitochondria.(A) Homogenates of HEK293T cells transfected with wild-type or BBD-mutated (GK179AA) vIRF-1 (or empty vector control, vIRF-1-negative) were subjected to differential and Optiprep gradient centrifugation to isolate enriched mitochondrial fractions (see Materials and Methods). These were analyzed by immunoblotting with vIRF-1-specific rabbit antiserum for the presence of vIRF-1 protein, and also with antibody to mitochondrial protein VDAC1 [voltage-dependent anion-selective channel protein 1; integral mitochondrial outer membrane (int. OM) protein] to provide a positive control. Normalization of vIRF-1 amounts in lysates versus mitochondrial fractions from these transfected cells was achieved using a ratio of 1∶50 (bottom). (B) In vitro mitochondrial binding assays using enriched mitochondria, untreated or pre-treated with proteinase K (Prot. K), and recombinant T7-tagged vIRF-1 for assessment of the requirement for surface protein integrity for mitochondrial binding by vIRF-1. Total protein released from mitochondria was precipitated with trichloroacetic acid (TCA) for direct quantitative comparison with protein from mitochondrial pellets. Blots were probed with antibodies to mitochondrial outer membrane (OM)-associated Bax and inner membrane (IM)-localized prohibitin to provide controls for appropriate fractionation, mitochondrial integrity, and proteinase K activity. (C) Generation and Western analysis of mitochondrial preparations from HHV-8+ BCBL-1-TRE/RTA cells [70] revealed mitochondrial association of endogenous vIRF-1, both in resting (latent) and lytically reactivated (+Dox) cultures; the latter, as expected, expressed higher levels of vIRF-1. In both cases, mitochondrial-associated vIRF-1 was susceptible to protease digestion, consistent with peripheral binding to mitochondria. Immunodetection of Bim verified peripheral protein susceptibility to proteinase K digestion. (D) Mitochondrial localization of vIRF-1 as determined using immunoflourescence assay for detection of vIRF-1 and mitochondrial marker TOM20 in TIME-TRE/vIRF-1 endothelial cells, +Dox. Arrows indicate examples of vIRF-1/TOM20 co-localization. (E) Western analyses of total cell and mitochondrial extracts of HHV-8+ TIME-TRE/RTA and BCBL1-TRE/RTA cells, untreated or treated with Dox for 2 days, were undertaken to quantify the relative amounts of mitochondrial-localized vIRF-1 in these latently and lytically infected cells. For TIME and BCBL-1 cells, 10- and 50-fold excess of mitochondrial extract over total extract, respectively, was loaded onto the gels to achieve near normalization. Relative signal intensities of bands were obtained from digitally captured images and calculated values of mitochondrial relative to total vIRF-1 levels are shown under the vIRF-1 blots. vIRF-1 was detected in latency (−Dox) only in the BCBL-1 (PEL) cells, and mitochondrial∶total vIRF-1 was increased upon Dox addition. Immunoblotting for VDAC1 (mitochondrial), histone deacetylase-1 (HDAC1, nuclear), calreticulin (endoplasmic reticulum) and β-actin (cytoplasmic) provided quality controls for mitochondrial and total cell extracts. (Arrowhead, vIRF-1; *non-specific). (F) A similar experiment in HHV-8+ TIME-TRE/RTA cells, demonstrating mitochondrial localization of Bid, in addition to vIRF-1, in lytically reactivated cultures.

Mentions: To address the hypothesis that vIRF-1 may act directly at the mitochondrion to suppress BOP-induced apoptosis, we isolated mitochondria from vIRF-1 vector-transfected HEK293T cells and undertook SDS-PAGE and Western analysis for detection of vIRF-1 in the mitochondrial fraction. Both wild-type and Bim-refractory (GK179AA) vIRF-1 proteins were present in mitochondrial fractions, representing approximately 2% of total vIRF-1 present in the transfected cell lysates (Fig. 6A). To assess whether this vIRF-1 was likely to be membrane inserted/associated or mitochondrial outer membrane (OM) protein associated, in vitro mitochondrial binding assays were used. These assays employed purified recombinant vIRF-1 (T7 epitope-tagged) added to isolated mitochondria prior to and after proteinase K treatment. Binding of vIRF-1 to mitochondria, apparent absent treatment, was completely abrogated by proteinase K pre-treatment (Fig. 6B), indicating mitochondrial association of vIRF-1 via interaction with a cytoplasmic-exposed mitochondrial protein. Furthermore, endogenously-expressed vIRF-1 was present in mitochondrial fractions prepared from HHV-8+ primary effusion lymphoma (PEL) cells, BCBL1-TRE/RTA [50], with or without lytic induction (+Dox), and this vIRF-1 was susceptible to proteinase K digestion, demonstrating peripheral association of vIRF-1 with mitochondria in HHV-8 infected cells (Fig. 6C). vIRF-1 has been reported previously to be expressed during latency in PEL cells but to be induced during lytic reactivation [51], consistent with our detected patterns of vIRF-1 expression in BCBL1-TRE/RTA cells. Mitochondrial localization of vIRF-1 was verified by IFA in TIME-TRE/vIRF-1 cells [23] in which vIRF-1 expression is inducible upon addition of doxycycline to culture medium. These results demonstrated localization of detectable levels of vIRF-1 to a subset of loci staining positively for mitochondrial marker TOM20 (Fig. 6D). In both Dox-inducible BCBL1-TRE/RTA PEL cells and HHV-8-infected TIME-TRE/RTA endothelial cells (see Materials and Methods and Fig.S1), vIRF-1 was found by immunoblotting of mitochondrial fractions to localize in part to mitochondria during productive replication (Fig. 6E). The proportion of vIRF-1 localizing to mitochondria in the BCBL-1 cells was comparable with that measured in transfected cells (Fig. 6A); the level in the TIME cells (+Dox) was >4 times higher at 9%. For the PEL cells, which express vIRF-1 also during latency but at reduced levels, the proportion of mitochondrial-localized vIRF-1 was increased from 0.9% to 2% in Dox-treated, lytically-induced cultures, possibly reflecting biological significance of vIRF-1 activity at this site during productive replication. It should be noted that as only a subset of these cells support lytic reactivation, the proportion of vIRF-1 localized to mitochondria in lytically infected cells is likely to be substantially greater than the 2% level observed for the culture as a whole. Similar fractionation experiments in HHV-8+ TIME-TRE/RTA cells treated with Dox verified Bid, as well as vIRF-1, localization to mitochondria during lytic reactivation (Fig. 6F). Confocal immunofluorescence microscopy detected at least some colocalization of vIRF-1 and Bid in these cells (Fig.S2). Combined, our data provide evidence of mitochondrial association of vIRF-1, in both transduced and infected cells, and indicate that vIRF-1 may target BOPs for inhibition at this site.


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 localization to mitochondria.(A) Homogenates of HEK293T cells transfected with wild-type or BBD-mutated (GK179AA) vIRF-1 (or empty vector control, vIRF-1-negative) were subjected to differential and Optiprep gradient centrifugation to isolate enriched mitochondrial fractions (see Materials and Methods). These were analyzed by immunoblotting with vIRF-1-specific rabbit antiserum for the presence of vIRF-1 protein, and also with antibody to mitochondrial protein VDAC1 [voltage-dependent anion-selective channel protein 1; integral mitochondrial outer membrane (int. OM) protein] to provide a positive control. Normalization of vIRF-1 amounts in lysates versus mitochondrial fractions from these transfected cells was achieved using a ratio of 1∶50 (bottom). (B) In vitro mitochondrial binding assays using enriched mitochondria, untreated or pre-treated with proteinase K (Prot. K), and recombinant T7-tagged vIRF-1 for assessment of the requirement for surface protein integrity for mitochondrial binding by vIRF-1. Total protein released from mitochondria was precipitated with trichloroacetic acid (TCA) for direct quantitative comparison with protein from mitochondrial pellets. Blots were probed with antibodies to mitochondrial outer membrane (OM)-associated Bax and inner membrane (IM)-localized prohibitin to provide controls for appropriate fractionation, mitochondrial integrity, and proteinase K activity. (C) Generation and Western analysis of mitochondrial preparations from HHV-8+ BCBL-1-TRE/RTA cells [70] revealed mitochondrial association of endogenous vIRF-1, both in resting (latent) and lytically reactivated (+Dox) cultures; the latter, as expected, expressed higher levels of vIRF-1. In both cases, mitochondrial-associated vIRF-1 was susceptible to protease digestion, consistent with peripheral binding to mitochondria. Immunodetection of Bim verified peripheral protein susceptibility to proteinase K digestion. (D) Mitochondrial localization of vIRF-1 as determined using immunoflourescence assay for detection of vIRF-1 and mitochondrial marker TOM20 in TIME-TRE/vIRF-1 endothelial cells, +Dox. Arrows indicate examples of vIRF-1/TOM20 co-localization. (E) Western analyses of total cell and mitochondrial extracts of HHV-8+ TIME-TRE/RTA and BCBL1-TRE/RTA cells, untreated or treated with Dox for 2 days, were undertaken to quantify the relative amounts of mitochondrial-localized vIRF-1 in these latently and lytically infected cells. For TIME and BCBL-1 cells, 10- and 50-fold excess of mitochondrial extract over total extract, respectively, was loaded onto the gels to achieve near normalization. Relative signal intensities of bands were obtained from digitally captured images and calculated values of mitochondrial relative to total vIRF-1 levels are shown under the vIRF-1 blots. vIRF-1 was detected in latency (−Dox) only in the BCBL-1 (PEL) cells, and mitochondrial∶total vIRF-1 was increased upon Dox addition. Immunoblotting for VDAC1 (mitochondrial), histone deacetylase-1 (HDAC1, nuclear), calreticulin (endoplasmic reticulum) and β-actin (cytoplasmic) provided quality controls for mitochondrial and total cell extracts. (Arrowhead, vIRF-1; *non-specific). (F) A similar experiment in HHV-8+ TIME-TRE/RTA cells, demonstrating mitochondrial localization of Bid, in addition to vIRF-1, in lytically reactivated cultures.
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ppat-1002748-g006: vIRF-1 localization to mitochondria.(A) Homogenates of HEK293T cells transfected with wild-type or BBD-mutated (GK179AA) vIRF-1 (or empty vector control, vIRF-1-negative) were subjected to differential and Optiprep gradient centrifugation to isolate enriched mitochondrial fractions (see Materials and Methods). These were analyzed by immunoblotting with vIRF-1-specific rabbit antiserum for the presence of vIRF-1 protein, and also with antibody to mitochondrial protein VDAC1 [voltage-dependent anion-selective channel protein 1; integral mitochondrial outer membrane (int. OM) protein] to provide a positive control. Normalization of vIRF-1 amounts in lysates versus mitochondrial fractions from these transfected cells was achieved using a ratio of 1∶50 (bottom). (B) In vitro mitochondrial binding assays using enriched mitochondria, untreated or pre-treated with proteinase K (Prot. K), and recombinant T7-tagged vIRF-1 for assessment of the requirement for surface protein integrity for mitochondrial binding by vIRF-1. Total protein released from mitochondria was precipitated with trichloroacetic acid (TCA) for direct quantitative comparison with protein from mitochondrial pellets. Blots were probed with antibodies to mitochondrial outer membrane (OM)-associated Bax and inner membrane (IM)-localized prohibitin to provide controls for appropriate fractionation, mitochondrial integrity, and proteinase K activity. (C) Generation and Western analysis of mitochondrial preparations from HHV-8+ BCBL-1-TRE/RTA cells [70] revealed mitochondrial association of endogenous vIRF-1, both in resting (latent) and lytically reactivated (+Dox) cultures; the latter, as expected, expressed higher levels of vIRF-1. In both cases, mitochondrial-associated vIRF-1 was susceptible to protease digestion, consistent with peripheral binding to mitochondria. Immunodetection of Bim verified peripheral protein susceptibility to proteinase K digestion. (D) Mitochondrial localization of vIRF-1 as determined using immunoflourescence assay for detection of vIRF-1 and mitochondrial marker TOM20 in TIME-TRE/vIRF-1 endothelial cells, +Dox. Arrows indicate examples of vIRF-1/TOM20 co-localization. (E) Western analyses of total cell and mitochondrial extracts of HHV-8+ TIME-TRE/RTA and BCBL1-TRE/RTA cells, untreated or treated with Dox for 2 days, were undertaken to quantify the relative amounts of mitochondrial-localized vIRF-1 in these latently and lytically infected cells. For TIME and BCBL-1 cells, 10- and 50-fold excess of mitochondrial extract over total extract, respectively, was loaded onto the gels to achieve near normalization. Relative signal intensities of bands were obtained from digitally captured images and calculated values of mitochondrial relative to total vIRF-1 levels are shown under the vIRF-1 blots. vIRF-1 was detected in latency (−Dox) only in the BCBL-1 (PEL) cells, and mitochondrial∶total vIRF-1 was increased upon Dox addition. Immunoblotting for VDAC1 (mitochondrial), histone deacetylase-1 (HDAC1, nuclear), calreticulin (endoplasmic reticulum) and β-actin (cytoplasmic) provided quality controls for mitochondrial and total cell extracts. (Arrowhead, vIRF-1; *non-specific). (F) A similar experiment in HHV-8+ TIME-TRE/RTA cells, demonstrating mitochondrial localization of Bid, in addition to vIRF-1, in lytically reactivated cultures.
Mentions: To address the hypothesis that vIRF-1 may act directly at the mitochondrion to suppress BOP-induced apoptosis, we isolated mitochondria from vIRF-1 vector-transfected HEK293T cells and undertook SDS-PAGE and Western analysis for detection of vIRF-1 in the mitochondrial fraction. Both wild-type and Bim-refractory (GK179AA) vIRF-1 proteins were present in mitochondrial fractions, representing approximately 2% of total vIRF-1 present in the transfected cell lysates (Fig. 6A). To assess whether this vIRF-1 was likely to be membrane inserted/associated or mitochondrial outer membrane (OM) protein associated, in vitro mitochondrial binding assays were used. These assays employed purified recombinant vIRF-1 (T7 epitope-tagged) added to isolated mitochondria prior to and after proteinase K treatment. Binding of vIRF-1 to mitochondria, apparent absent treatment, was completely abrogated by proteinase K pre-treatment (Fig. 6B), indicating mitochondrial association of vIRF-1 via interaction with a cytoplasmic-exposed mitochondrial protein. Furthermore, endogenously-expressed vIRF-1 was present in mitochondrial fractions prepared from HHV-8+ primary effusion lymphoma (PEL) cells, BCBL1-TRE/RTA [50], with or without lytic induction (+Dox), and this vIRF-1 was susceptible to proteinase K digestion, demonstrating peripheral association of vIRF-1 with mitochondria in HHV-8 infected cells (Fig. 6C). vIRF-1 has been reported previously to be expressed during latency in PEL cells but to be induced during lytic reactivation [51], consistent with our detected patterns of vIRF-1 expression in BCBL1-TRE/RTA cells. Mitochondrial localization of vIRF-1 was verified by IFA in TIME-TRE/vIRF-1 cells [23] in which vIRF-1 expression is inducible upon addition of doxycycline to culture medium. These results demonstrated localization of detectable levels of vIRF-1 to a subset of loci staining positively for mitochondrial marker TOM20 (Fig. 6D). In both Dox-inducible BCBL1-TRE/RTA PEL cells and HHV-8-infected TIME-TRE/RTA endothelial cells (see Materials and Methods and Fig.S1), vIRF-1 was found by immunoblotting of mitochondrial fractions to localize in part to mitochondria during productive replication (Fig. 6E). The proportion of vIRF-1 localizing to mitochondria in the BCBL-1 cells was comparable with that measured in transfected cells (Fig. 6A); the level in the TIME cells (+Dox) was >4 times higher at 9%. For the PEL cells, which express vIRF-1 also during latency but at reduced levels, the proportion of mitochondrial-localized vIRF-1 was increased from 0.9% to 2% in Dox-treated, lytically-induced cultures, possibly reflecting biological significance of vIRF-1 activity at this site during productive replication. It should be noted that as only a subset of these cells support lytic reactivation, the proportion of vIRF-1 localized to mitochondria in lytically infected cells is likely to be substantially greater than the 2% level observed for the culture as a whole. Similar fractionation experiments in HHV-8+ TIME-TRE/RTA cells treated with Dox verified Bid, as well as vIRF-1, localization to mitochondria during lytic reactivation (Fig. 6F). Confocal immunofluorescence microscopy detected at least some colocalization of vIRF-1 and Bid in these cells (Fig.S2). Combined, our data provide evidence of mitochondrial association of vIRF-1, in both transduced and infected cells, and indicate that vIRF-1 may target BOPs for inhibition at this site.

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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Related in: MedlinePlus