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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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Additional targets of vIRF-1 BBD.(A) Recombinant proteins comprising GFP-fused BH3 domains of various BOPs and multi-BH-domain pro- and anti-apoptotic Bcl-2 family members were used together with GST-BBD (or GST, negative control) in co-precipitation assays, as illustrated. In addition to Bid and Bim, Bik, Bmf, Hrk and Noxa were co-precipitated with glutathione bead-sedimented GST-BBD (but not by GST alone). None of the non-BOP BH3 domains tested were co-precipitated. (B) Largely reflecting the in vitro binding data, Flag-tagged full-length BOPs containing the BBD-binding BH3 domains were, with the exception of Bik, able to co-precipitate vIRF-1 from lysates of HEK293T cells co-transfected with the appropriate expression vector pairs. Puma was included as an additional potential BOP target of vIRF-1. Bcl-2 served as a negative control. Arrows indicate bands corresponding to the full-length proteins of the expected sizes.
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ppat-1002748-g008: Additional targets of vIRF-1 BBD.(A) Recombinant proteins comprising GFP-fused BH3 domains of various BOPs and multi-BH-domain pro- and anti-apoptotic Bcl-2 family members were used together with GST-BBD (or GST, negative control) in co-precipitation assays, as illustrated. In addition to Bid and Bim, Bik, Bmf, Hrk and Noxa were co-precipitated with glutathione bead-sedimented GST-BBD (but not by GST alone). None of the non-BOP BH3 domains tested were co-precipitated. (B) Largely reflecting the in vitro binding data, Flag-tagged full-length BOPs containing the BBD-binding BH3 domains were, with the exception of Bik, able to co-precipitate vIRF-1 from lysates of HEK293T cells co-transfected with the appropriate expression vector pairs. Puma was included as an additional potential BOP target of vIRF-1. Bcl-2 served as a negative control. Arrows indicate bands corresponding to the full-length proteins of the expected sizes.

Mentions: We next investigated whether BBD could target additional BH3 domains, in particular those of other BOPs. The respective BH3 coding sequences were cloned in-frame with the GFP open reading frame in plasmid vector pTYB4 and expressed in and purified from bacteria; BBD was expressed and isolated similarly, as a GST fusion protein (Materials and Methods). BH3 domains tested comprised those of BOPs Bad, Bmf, Bnip3L, Hrk and Noxa, along with Bim, Bid and “BH3-only” beclin [52], and BH3s from multi-BH domain proteins Bcl-2 and Mcl-1 (anti-apoptotic) and Bak, Bax and Bok (pro-apoptotic). Results from these co-precipitation assays identified Bik, Bmf, Hrk and Noxa BH3 domains as additional targets of BBD interaction (Fig. 8A). Interactions between the corresponding full-length proteins and vIRF-1 were tested by co-immunoprecipitation of vIRF-1 with Flag-tagged BOPs from transfected cell lysates; all but Bik were able to co-precipitate vIRF-1 in this experiment (Fig. 8B). Bcl-2 was essentially negative, with barely detectable levels of vIRF-1 in the co-precipitate, as was BOP Puma [not included in the BBD∶BH3 experiment (Fig. 8A)]. Therefore, of the Bcl-2 protein family members tested, BOPs Bim, Bid, Bmf, Hrk and Noxa are demonstrably targeted by vIRF-1, via BBD∶BH3 association, and Bik BH3 can also bind BBD.


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)

Additional targets of vIRF-1 BBD.(A) Recombinant proteins comprising GFP-fused BH3 domains of various BOPs and multi-BH-domain pro- and anti-apoptotic Bcl-2 family members were used together with GST-BBD (or GST, negative control) in co-precipitation assays, as illustrated. In addition to Bid and Bim, Bik, Bmf, Hrk and Noxa were co-precipitated with glutathione bead-sedimented GST-BBD (but not by GST alone). None of the non-BOP BH3 domains tested were co-precipitated. (B) Largely reflecting the in vitro binding data, Flag-tagged full-length BOPs containing the BBD-binding BH3 domains were, with the exception of Bik, able to co-precipitate vIRF-1 from lysates of HEK293T cells co-transfected with the appropriate expression vector pairs. Puma was included as an additional potential BOP target of vIRF-1. Bcl-2 served as a negative control. Arrows indicate bands corresponding to the full-length proteins of the expected sizes.
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Related In: Results  -  Collection

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ppat-1002748-g008: Additional targets of vIRF-1 BBD.(A) Recombinant proteins comprising GFP-fused BH3 domains of various BOPs and multi-BH-domain pro- and anti-apoptotic Bcl-2 family members were used together with GST-BBD (or GST, negative control) in co-precipitation assays, as illustrated. In addition to Bid and Bim, Bik, Bmf, Hrk and Noxa were co-precipitated with glutathione bead-sedimented GST-BBD (but not by GST alone). None of the non-BOP BH3 domains tested were co-precipitated. (B) Largely reflecting the in vitro binding data, Flag-tagged full-length BOPs containing the BBD-binding BH3 domains were, with the exception of Bik, able to co-precipitate vIRF-1 from lysates of HEK293T cells co-transfected with the appropriate expression vector pairs. Puma was included as an additional potential BOP target of vIRF-1. Bcl-2 served as a negative control. Arrows indicate bands corresponding to the full-length proteins of the expected sizes.
Mentions: We next investigated whether BBD could target additional BH3 domains, in particular those of other BOPs. The respective BH3 coding sequences were cloned in-frame with the GFP open reading frame in plasmid vector pTYB4 and expressed in and purified from bacteria; BBD was expressed and isolated similarly, as a GST fusion protein (Materials and Methods). BH3 domains tested comprised those of BOPs Bad, Bmf, Bnip3L, Hrk and Noxa, along with Bim, Bid and “BH3-only” beclin [52], and BH3s from multi-BH domain proteins Bcl-2 and Mcl-1 (anti-apoptotic) and Bak, Bax and Bok (pro-apoptotic). Results from these co-precipitation assays identified Bik, Bmf, Hrk and Noxa BH3 domains as additional targets of BBD interaction (Fig. 8A). Interactions between the corresponding full-length proteins and vIRF-1 were tested by co-immunoprecipitation of vIRF-1 with Flag-tagged BOPs from transfected cell lysates; all but Bik were able to co-precipitate vIRF-1 in this experiment (Fig. 8B). Bcl-2 was essentially negative, with barely detectable levels of vIRF-1 in the co-precipitate, as was BOP Puma [not included in the BBD∶BH3 experiment (Fig. 8A)]. Therefore, of the Bcl-2 protein family members tested, BOPs Bim, Bid, Bmf, Hrk and Noxa are demonstrably targeted by vIRF-1, via BBD∶BH3 association, and Bik BH3 can also bind BBD.

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