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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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Nuclear localization-independent inhibition of Bim and Bid by vIRF-1.(A) Amino acid sequence of vIRF-1, showing putative nuclear localization signals (NLS, boxed), targeted for mutagenesis (basic-to-alanine residues). (B) Residues 159–163 (shaded box, panel A) were found to be required for nuclear localization of vIRF-1 in transfected HEK293T cells, as determined by immunofluorescence assay. Mutation of the other putative NLS sequences did not significantly affect vIRF-1 localization (data not shown). (C) Functional confirmation of nuclear exclusion of vIRF-1(RGRRR163AGAAA) (NLSX) using a p53-reporter assay, showing inhibition of nuclear-localized p53 activity by wild-type vIRF-1 (and also vIRF-1ΔBBD) but not by vIRF-1.NLSX. (D) Equivalent suppression of Bim and Bid apoptotic activity by wild-type and NLS-mutated vIRF-1, as determined by GFP-based cell viability assay applied to appropriately transfected HEK293T cells. For panels C and D, error bars represent standard deviations from the average values obtained from triplicate samples.
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ppat-1002748-g005: Nuclear localization-independent inhibition of Bim and Bid by vIRF-1.(A) Amino acid sequence of vIRF-1, showing putative nuclear localization signals (NLS, boxed), targeted for mutagenesis (basic-to-alanine residues). (B) Residues 159–163 (shaded box, panel A) were found to be required for nuclear localization of vIRF-1 in transfected HEK293T cells, as determined by immunofluorescence assay. Mutation of the other putative NLS sequences did not significantly affect vIRF-1 localization (data not shown). (C) Functional confirmation of nuclear exclusion of vIRF-1(RGRRR163AGAAA) (NLSX) using a p53-reporter assay, showing inhibition of nuclear-localized p53 activity by wild-type vIRF-1 (and also vIRF-1ΔBBD) but not by vIRF-1.NLSX. (D) Equivalent suppression of Bim and Bid apoptotic activity by wild-type and NLS-mutated vIRF-1, as determined by GFP-based cell viability assay applied to appropriately transfected HEK293T cells. For panels C and D, error bars represent standard deviations from the average values obtained from triplicate samples.

Mentions: (A) TIME-TRE/RTA cells were infected with HHV-8 and latency was allowed to establish. Cells were then reactivated by addition of 1 µg/ml doxycycline (Dox) to culture media and after 48 h cells were fixed and dually immunostained for detection of Bid and lytic antigen (vIRF-1). HHV-8+ TIME-TRE/RTA cells were also stained for detection of Bid or Bim in the absence and presence of Dox. Both BOPs were induced by Dox treatment (right panels), with general coincidence of Bid and lytic antigen immunofluorescence (left panels). Strong nuclear staining was evident only for Bim, with Bid localization remaining predominantly cytoplasmic. (B) HEK293T cells were transfected with expression vectors for Flag-tagged BimL or tBid and either empty vector (−vIRF-1) or vIRF-1 expression plasmid (+vIRF-1). Cells were immunofluorescence-stained to detect Flag (green) and vIRF-1 (red) and counterstained with DAPI to visualize nuclei (blue). Representative examples are shown. Nuclear localization of Bim but not Bid was induced by vIRF-1. (C) Nuclear and cytoplasmic extracts of similarly transfected cells were prepared and immunoblotted to provide independent analysis of potential vIRF-1 influence on BidL and tBid nuclear-cytoplasmic distribution. Extracts were prepared and fractionated as described in Materials and Methods and quality-checked by probing with cytoplasmic-localized lactate dehydrogenase (LDH) and nuclear-localized histone deacetylase 1 (HDAC1). Bim but not Bid relocalization in the presence of vIRF-1 co-expression was detected. Included in this experiment was a nuclear localization-defective variant of vIRF-1, vIRF-1.NLSX (see Fig. 5 and associated legend and text), which was unable to induce Bim nuclear localization (v'1.NLSX lane, α-Flag). (arrowhead, vIRF-1; asterisk, non-vIRF-1 α-Flag immunoreactive band).


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)

Nuclear localization-independent inhibition of Bim and Bid by vIRF-1.(A) Amino acid sequence of vIRF-1, showing putative nuclear localization signals (NLS, boxed), targeted for mutagenesis (basic-to-alanine residues). (B) Residues 159–163 (shaded box, panel A) were found to be required for nuclear localization of vIRF-1 in transfected HEK293T cells, as determined by immunofluorescence assay. Mutation of the other putative NLS sequences did not significantly affect vIRF-1 localization (data not shown). (C) Functional confirmation of nuclear exclusion of vIRF-1(RGRRR163AGAAA) (NLSX) using a p53-reporter assay, showing inhibition of nuclear-localized p53 activity by wild-type vIRF-1 (and also vIRF-1ΔBBD) but not by vIRF-1.NLSX. (D) Equivalent suppression of Bim and Bid apoptotic activity by wild-type and NLS-mutated vIRF-1, as determined by GFP-based cell viability assay applied to appropriately transfected HEK293T cells. For panels C and D, error bars represent standard deviations from the average values obtained from triplicate samples.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002748-g005: Nuclear localization-independent inhibition of Bim and Bid by vIRF-1.(A) Amino acid sequence of vIRF-1, showing putative nuclear localization signals (NLS, boxed), targeted for mutagenesis (basic-to-alanine residues). (B) Residues 159–163 (shaded box, panel A) were found to be required for nuclear localization of vIRF-1 in transfected HEK293T cells, as determined by immunofluorescence assay. Mutation of the other putative NLS sequences did not significantly affect vIRF-1 localization (data not shown). (C) Functional confirmation of nuclear exclusion of vIRF-1(RGRRR163AGAAA) (NLSX) using a p53-reporter assay, showing inhibition of nuclear-localized p53 activity by wild-type vIRF-1 (and also vIRF-1ΔBBD) but not by vIRF-1.NLSX. (D) Equivalent suppression of Bim and Bid apoptotic activity by wild-type and NLS-mutated vIRF-1, as determined by GFP-based cell viability assay applied to appropriately transfected HEK293T cells. For panels C and D, error bars represent standard deviations from the average values obtained from triplicate samples.
Mentions: (A) TIME-TRE/RTA cells were infected with HHV-8 and latency was allowed to establish. Cells were then reactivated by addition of 1 µg/ml doxycycline (Dox) to culture media and after 48 h cells were fixed and dually immunostained for detection of Bid and lytic antigen (vIRF-1). HHV-8+ TIME-TRE/RTA cells were also stained for detection of Bid or Bim in the absence and presence of Dox. Both BOPs were induced by Dox treatment (right panels), with general coincidence of Bid and lytic antigen immunofluorescence (left panels). Strong nuclear staining was evident only for Bim, with Bid localization remaining predominantly cytoplasmic. (B) HEK293T cells were transfected with expression vectors for Flag-tagged BimL or tBid and either empty vector (−vIRF-1) or vIRF-1 expression plasmid (+vIRF-1). Cells were immunofluorescence-stained to detect Flag (green) and vIRF-1 (red) and counterstained with DAPI to visualize nuclei (blue). Representative examples are shown. Nuclear localization of Bim but not Bid was induced by vIRF-1. (C) Nuclear and cytoplasmic extracts of similarly transfected cells were prepared and immunoblotted to provide independent analysis of potential vIRF-1 influence on BidL and tBid nuclear-cytoplasmic distribution. Extracts were prepared and fractionated as described in Materials and Methods and quality-checked by probing with cytoplasmic-localized lactate dehydrogenase (LDH) and nuclear-localized histone deacetylase 1 (HDAC1). Bim but not Bid relocalization in the presence of vIRF-1 co-expression was detected. Included in this experiment was a nuclear localization-defective variant of vIRF-1, vIRF-1.NLSX (see Fig. 5 and associated legend and text), which was unable to induce Bim nuclear localization (v'1.NLSX lane, α-Flag). (arrowhead, vIRF-1; asterisk, non-vIRF-1 α-Flag immunoreactive band).

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