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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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Bid localization in lytically reactivated and vIRF-1 transfected cells.(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).
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ppat-1002748-g004: Bid localization in lytically reactivated and vIRF-1 transfected cells.(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).

Mentions: In HHV-8 lytically reactivated endothelial cells, Bim is found predominantly in the nucleus, and nuclear location of Bim can be induced by vIRF-1 in transfected HEK293T cells [23]. As nuclear-localized Bim is inactive in respect of apoptotic induction, its nuclear sequestration represents a mechanism of BOP inactivation. To determine the nuclear-cytoplasmic distribution of Bid during HHV-8 lytic reactivation, dual-label immunofluorescence assays (IFA) were undertaken to identify Bid induction and distribution in reactivated cells expressing lytic antigen (vIRF-1). Like Bim, Bid was induced during lytic reactivation in telomerase-immortalized endothelial (TIME) cells [46], here engineered to express HHV-8 immediate-early protein RTA in response to doxycycline (see Materials and Methods and Fig.S1) (Fig. 4A). However, little or no nuclear localization of Bid was apparent, in sharp contrast to the predominant nuclear localization of Bim in lytically reactivated cultures [23] (Fig. 4A). In cells transfected with BidL or tBid expression vectors together with an empty or vIRF-1 expression plasmid, the nuclear-cytoplasmic distribution of each Bid protein was refractory to vIRF-1 influence (Fig. 4B). It is notable that some nuclear localization of BidL was apparent, consistent with previous reports of nuclear localization and associated activities of Bid [47]–[49], but no nuclear staining was evident for tBid. In contrast to Bid, and consistent with previous findings [23], Bim distribution was altered in the presence of vIRF-1, with strong nuclear staining apparent exclusively with vIRF-1 co-expression (Fig. 4B). That vIRF-1 indeed did not influence nuclear-cytoplasmic distribution of Bid was verified by using immunoblotting of cytoplasmic and nuclear fractions of transfected cells. Again, while nuclear localization of a proportion of BidL was detected, this was not detectably influenced by vIRF-1 co-expression, and tBid localization was restricted to the cytoplasm in the absence and presence of vIRF-1 (Fig. 4C). Furthermore, a nuclear localization-defective vIRF-1 variant (see below) also did not influence the nuclear-cytoplasmic distribution of BidL, although induction of Bim nuclear localization was abolished (Fig. 4C).


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)

Bid localization in lytically reactivated and vIRF-1 transfected cells.(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).
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ppat-1002748-g004: Bid localization in lytically reactivated and vIRF-1 transfected cells.(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).
Mentions: In HHV-8 lytically reactivated endothelial cells, Bim is found predominantly in the nucleus, and nuclear location of Bim can be induced by vIRF-1 in transfected HEK293T cells [23]. As nuclear-localized Bim is inactive in respect of apoptotic induction, its nuclear sequestration represents a mechanism of BOP inactivation. To determine the nuclear-cytoplasmic distribution of Bid during HHV-8 lytic reactivation, dual-label immunofluorescence assays (IFA) were undertaken to identify Bid induction and distribution in reactivated cells expressing lytic antigen (vIRF-1). Like Bim, Bid was induced during lytic reactivation in telomerase-immortalized endothelial (TIME) cells [46], here engineered to express HHV-8 immediate-early protein RTA in response to doxycycline (see Materials and Methods and Fig.S1) (Fig. 4A). However, little or no nuclear localization of Bid was apparent, in sharp contrast to the predominant nuclear localization of Bim in lytically reactivated cultures [23] (Fig. 4A). In cells transfected with BidL or tBid expression vectors together with an empty or vIRF-1 expression plasmid, the nuclear-cytoplasmic distribution of each Bid protein was refractory to vIRF-1 influence (Fig. 4B). It is notable that some nuclear localization of BidL was apparent, consistent with previous reports of nuclear localization and associated activities of Bid [47]–[49], but no nuclear staining was evident for tBid. In contrast to Bid, and consistent with previous findings [23], Bim distribution was altered in the presence of vIRF-1, with strong nuclear staining apparent exclusively with vIRF-1 co-expression (Fig. 4B). That vIRF-1 indeed did not influence nuclear-cytoplasmic distribution of Bid was verified by using immunoblotting of cytoplasmic and nuclear fractions of transfected cells. Again, while nuclear localization of a proportion of BidL was detected, this was not detectably influenced by vIRF-1 co-expression, and tBid localization was restricted to the cytoplasm in the absence and presence of vIRF-1 (Fig. 4C). Furthermore, a nuclear localization-defective vIRF-1 variant (see below) also did not influence the nuclear-cytoplasmic distribution of BidL, although induction of Bim nuclear localization was abolished (Fig. 4C).

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