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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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Structural analysis of BBD-BH3 interactions.(A) Substitutions were generated in Bim BH3 at position φ1+1 (boxed), containing a conserved alanine in all identified BBD-interacting BH3 domains, to match collinear residues in BH3 domains refractory to interaction with BBD. Mutual substitution of φ1+1 to φ1+3 residues in the BH3 domains of Bim and closely related Bax were also generated. The altered and native Bim and Bax BH3 sequences were cloned into a bacterial expression vector for generation of recombinant GFP-BH3 fusion proteins. Grey-shaded bars in the sequence alignment correspond to coaligned hydrophobic residues; black shading indicates conserved colinear amino acids. (B) In vitro co-precipitation assays utilizing recombinant GFP-BH3 and GST-BBD fusion proteins were utilized to analyze BH3∶BBD interactions. Lane labels correspond to wild-type (wt) and mutated Bim (1–7) and Bax (mt) BH3 sequences indicated in panel A; GFP alone (v, vector) was used as a negative control. A BH3-related peptide sequence isolated from a phage-display library using GST-BBD as bait (see Materials and Methods) was also included in the binding assay (φ-seq*).
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ppat-1002748-g009: Structural analysis of BBD-BH3 interactions.(A) Substitutions were generated in Bim BH3 at position φ1+1 (boxed), containing a conserved alanine in all identified BBD-interacting BH3 domains, to match collinear residues in BH3 domains refractory to interaction with BBD. Mutual substitution of φ1+1 to φ1+3 residues in the BH3 domains of Bim and closely related Bax were also generated. The altered and native Bim and Bax BH3 sequences were cloned into a bacterial expression vector for generation of recombinant GFP-BH3 fusion proteins. Grey-shaded bars in the sequence alignment correspond to coaligned hydrophobic residues; black shading indicates conserved colinear amino acids. (B) In vitro co-precipitation assays utilizing recombinant GFP-BH3 and GST-BBD fusion proteins were utilized to analyze BH3∶BBD interactions. Lane labels correspond to wild-type (wt) and mutated Bim (1–7) and Bax (mt) BH3 sequences indicated in panel A; GFP alone (v, vector) was used as a negative control. A BH3-related peptide sequence isolated from a phage-display library using GST-BBD as bait (see Materials and Methods) was also included in the binding assay (φ-seq*).

Mentions: Comparisons of the BH3 domains of vIRF-1/BBD-interacting BOPs identified a single unique and conserved residue among the BBD-binding BH3 sequences, namely an alanine at position φ1+1 (Fig. 9A, left). Mutagenesis of this position within the context of Bim BH3 was undertaken to determine its significance with respect to BBD binding; it was changed to each of the collinear residues of the non-binding BH3 domains. Additionally, residues φ1+1 to φ1+3 (SEC) of BBD-refractory Bax BH3 were changed to the equivalents (AQE) in closely related Bim BH3 and the reciprocal changes were made in Bim BH3 to determine if these “diverged” residues in combination could, respectively, confer and abrogate BBD binding. The various changes made are shown in Fig. 9A (right). As before, these sequences were expressed as GFP fusions for use in GST-BBD-based coprecipitation assays. Other than wild-type Bim BH3, glutathione bead-precipitated GST-BBD was able to efficiently co-precipitate only cysteine- and serine-substituted alanine φ1+1, with weak binding apparent for the leucine-substituted BH3 (Fig. 9B). The AQE substitution of Bax SEC residues was able to confer at least some BBD-binding capacity to Bax, demonstrating the contribution of these residues, either directly or via structural influence, to binding; the converse substitution in Bim abrogated binding. Interestingly, a sequence isolated from a phage-display dodecamer-peptide library using GST-BBD as bait had some resemblance to BH3 sequences in respect of conserved basic residues and, importantly, possessed an alanine residue at the equivalent of position φ1+1. This sequence also showed some binding in the in vitro coprecipitation assay. Taken together, these data indicate the likely central importance of alanine at position φ1+1 for BBD interaction, although the residue's contribution is likely indirect and evidently context dependent, as particular collinear small side-chain residues (serine and cysteine) from non-binding BH3 domains can substitute for alanine in Bim BH3 and the AQE motif from Bim can confer only weak binding to the closely related BH3 domain of Bax.


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)

Structural analysis of BBD-BH3 interactions.(A) Substitutions were generated in Bim BH3 at position φ1+1 (boxed), containing a conserved alanine in all identified BBD-interacting BH3 domains, to match collinear residues in BH3 domains refractory to interaction with BBD. Mutual substitution of φ1+1 to φ1+3 residues in the BH3 domains of Bim and closely related Bax were also generated. The altered and native Bim and Bax BH3 sequences were cloned into a bacterial expression vector for generation of recombinant GFP-BH3 fusion proteins. Grey-shaded bars in the sequence alignment correspond to coaligned hydrophobic residues; black shading indicates conserved colinear amino acids. (B) In vitro co-precipitation assays utilizing recombinant GFP-BH3 and GST-BBD fusion proteins were utilized to analyze BH3∶BBD interactions. Lane labels correspond to wild-type (wt) and mutated Bim (1–7) and Bax (mt) BH3 sequences indicated in panel A; GFP alone (v, vector) was used as a negative control. A BH3-related peptide sequence isolated from a phage-display library using GST-BBD as bait (see Materials and Methods) was also included in the binding assay (φ-seq*).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369933&req=5

ppat-1002748-g009: Structural analysis of BBD-BH3 interactions.(A) Substitutions were generated in Bim BH3 at position φ1+1 (boxed), containing a conserved alanine in all identified BBD-interacting BH3 domains, to match collinear residues in BH3 domains refractory to interaction with BBD. Mutual substitution of φ1+1 to φ1+3 residues in the BH3 domains of Bim and closely related Bax were also generated. The altered and native Bim and Bax BH3 sequences were cloned into a bacterial expression vector for generation of recombinant GFP-BH3 fusion proteins. Grey-shaded bars in the sequence alignment correspond to coaligned hydrophobic residues; black shading indicates conserved colinear amino acids. (B) In vitro co-precipitation assays utilizing recombinant GFP-BH3 and GST-BBD fusion proteins were utilized to analyze BH3∶BBD interactions. Lane labels correspond to wild-type (wt) and mutated Bim (1–7) and Bax (mt) BH3 sequences indicated in panel A; GFP alone (v, vector) was used as a negative control. A BH3-related peptide sequence isolated from a phage-display library using GST-BBD as bait (see Materials and Methods) was also included in the binding assay (φ-seq*).
Mentions: Comparisons of the BH3 domains of vIRF-1/BBD-interacting BOPs identified a single unique and conserved residue among the BBD-binding BH3 sequences, namely an alanine at position φ1+1 (Fig. 9A, left). Mutagenesis of this position within the context of Bim BH3 was undertaken to determine its significance with respect to BBD binding; it was changed to each of the collinear residues of the non-binding BH3 domains. Additionally, residues φ1+1 to φ1+3 (SEC) of BBD-refractory Bax BH3 were changed to the equivalents (AQE) in closely related Bim BH3 and the reciprocal changes were made in Bim BH3 to determine if these “diverged” residues in combination could, respectively, confer and abrogate BBD binding. The various changes made are shown in Fig. 9A (right). As before, these sequences were expressed as GFP fusions for use in GST-BBD-based coprecipitation assays. Other than wild-type Bim BH3, glutathione bead-precipitated GST-BBD was able to efficiently co-precipitate only cysteine- and serine-substituted alanine φ1+1, with weak binding apparent for the leucine-substituted BH3 (Fig. 9B). The AQE substitution of Bax SEC residues was able to confer at least some BBD-binding capacity to Bax, demonstrating the contribution of these residues, either directly or via structural influence, to binding; the converse substitution in Bim abrogated binding. Interestingly, a sequence isolated from a phage-display dodecamer-peptide library using GST-BBD as bait had some resemblance to BH3 sequences in respect of conserved basic residues and, importantly, possessed an alanine residue at the equivalent of position φ1+1. This sequence also showed some binding in the in vitro coprecipitation assay. Taken together, these data indicate the likely central importance of alanine at position φ1+1 for BBD interaction, although the residue's contribution is likely indirect and evidently context dependent, as particular collinear small side-chain residues (serine and cysteine) from non-binding BH3 domains can substitute for alanine in Bim BH3 and the AQE motif from Bim can confer only weak binding to the closely related BH3 domain of Bax.

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