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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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Biological significance of Bid in HHV-8 replication.(A) TIME cells latently infected with BCBL-1 PEL culture-derived virus were transduced with non-silencing (NS), Bid-specific (sh1, sh2), or Bim-specific shRNAs using lentiviral vectors (see Materials and Methods). After 48 h, these TIME cultures were treated with TPA to induce lytic reactivation, and media and cells were harvested at 0, 3 and 6 days post-induction for determinations of encapsidated genome copy numbers and Bid and Bim expression, respectively. Quantitative PCR was used to determine genome copies following pre-treatment with DNase I to remove any unencapsidated, contaminating viral DNA released from disrupted cells (see Materials and Methods). Immunoblotting confirmed both induction of Bid and Bim in induced cultures (detected even though a minority of cells support lytic replication [23], [8]) and their specific suppression by the respective transduced shRNAs. (B) Similar experiments were undertaken in control (NS shRNA-transduced) and Bid-depleted (Bid sh1-transduced) TIME-TRE/RTA cells using Dox (1 µg/ml) to induce lytic reactivation. Higher reactivation frequencies possible in these cells enabled relative virus titers from these cultures to be measured reliably by using an infectious assay, in which LANA+ cells were detected by immunofluorescence assay. Naïve TIME cultures were inoculated with ultracentrifuge-concentrated virus from Dox-treated HHV-8+ TIME-TRE/RTA cell culture media, cumulatively collected over 5 days following Dox addition. Numbers of infected, LANA+ TIME cells were counted from multiple random fields to derive average values. Results from two independent experiments are shown, with titers expressed relative to those obtained from NS shRNA-transduced cells (set at 1); the error bar represents deviation from the average NS/Bid titer ratios. Cells were harvested at day 5 for mRNA preparation and RT-PCR confirmation of Bid depletion (right).
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ppat-1002748-g010: Biological significance of Bid in HHV-8 replication.(A) TIME cells latently infected with BCBL-1 PEL culture-derived virus were transduced with non-silencing (NS), Bid-specific (sh1, sh2), or Bim-specific shRNAs using lentiviral vectors (see Materials and Methods). After 48 h, these TIME cultures were treated with TPA to induce lytic reactivation, and media and cells were harvested at 0, 3 and 6 days post-induction for determinations of encapsidated genome copy numbers and Bid and Bim expression, respectively. Quantitative PCR was used to determine genome copies following pre-treatment with DNase I to remove any unencapsidated, contaminating viral DNA released from disrupted cells (see Materials and Methods). Immunoblotting confirmed both induction of Bid and Bim in induced cultures (detected even though a minority of cells support lytic replication [23], [8]) and their specific suppression by the respective transduced shRNAs. (B) Similar experiments were undertaken in control (NS shRNA-transduced) and Bid-depleted (Bid sh1-transduced) TIME-TRE/RTA cells using Dox (1 µg/ml) to induce lytic reactivation. Higher reactivation frequencies possible in these cells enabled relative virus titers from these cultures to be measured reliably by using an infectious assay, in which LANA+ cells were detected by immunofluorescence assay. Naïve TIME cultures were inoculated with ultracentrifuge-concentrated virus from Dox-treated HHV-8+ TIME-TRE/RTA cell culture media, cumulatively collected over 5 days following Dox addition. Numbers of infected, LANA+ TIME cells were counted from multiple random fields to derive average values. Results from two independent experiments are shown, with titers expressed relative to those obtained from NS shRNA-transduced cells (set at 1); the error bar represents deviation from the average NS/Bid titer ratios. Cells were harvested at day 5 for mRNA preparation and RT-PCR confirmation of Bid depletion (right).

Mentions: Previous studies from this laboratory identified Bim as a potent inhibitor of virus productive replication and the importance of vIRF-1 BBD-mediated interactions in countering lytic cycle-induced apoptosis and promoting HHV-8 production in TIME cells [23], [8]. In view of the present findings that vIRF-1 inhibits Bid activity in a BBD-dependent fashion (Fig. 7) and that Bid is induced in lytically reactivated TIME cells (Fig. 4A), we wanted to test the significance of Bid in HHV-8 replication. To do this, we utilized lentiviral vector-delivered shRNAs directed to Bid mRNA sequences to deplete Bid in HHV-8+ (latently infected) TIME cells and compared levels of cell-released encapsidated viral genomes produced following TPA induction to those obtained from non-silencing (NS) shRNA-transduced control cultures. Bim shRNA-transduced TIME cells were also included to provide a positive control for the experiment. The data from this experiment (Fig. 10A) revealed that each of the two Bid shRNAs led to small but significant increases in virus production from TPA-treated TIME cells; as before, Bim depletion led to substantial amplification of virus titers. Similar experiments were undertaken in TIME-TRE/RTA cells to confirm the effect on replication of Bid depletion; here, lytic replication was induced by addition of doxycycline to the cultures. Again, Bid depletion led to increased virus production (Fig. 10B), measured in this experiment by titration of released infectious virus in culture media via inoculation of naïve TIME cells and detection of virus infection by immunofluorescence assay for latency-associated nuclear antigen (LANA). These data demonstrate that Bid, in addition to Bim, is a contributor to negative regulation of HHV-8 infection and suggest that its control by vIRF-1 is likely to be important for optimal virus productive replication. It is important to note, however, that the positive effects on virus replication of Bid and Bim depletion by shRNA transduction demonstrate also that vIRF-1 is not completely effective at suppressing the activities of these BOPs. This situation is not unexpected and is probably universal amongst such viral regulators of cellular activities.


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)

Biological significance of Bid in HHV-8 replication.(A) TIME cells latently infected with BCBL-1 PEL culture-derived virus were transduced with non-silencing (NS), Bid-specific (sh1, sh2), or Bim-specific shRNAs using lentiviral vectors (see Materials and Methods). After 48 h, these TIME cultures were treated with TPA to induce lytic reactivation, and media and cells were harvested at 0, 3 and 6 days post-induction for determinations of encapsidated genome copy numbers and Bid and Bim expression, respectively. Quantitative PCR was used to determine genome copies following pre-treatment with DNase I to remove any unencapsidated, contaminating viral DNA released from disrupted cells (see Materials and Methods). Immunoblotting confirmed both induction of Bid and Bim in induced cultures (detected even though a minority of cells support lytic replication [23], [8]) and their specific suppression by the respective transduced shRNAs. (B) Similar experiments were undertaken in control (NS shRNA-transduced) and Bid-depleted (Bid sh1-transduced) TIME-TRE/RTA cells using Dox (1 µg/ml) to induce lytic reactivation. Higher reactivation frequencies possible in these cells enabled relative virus titers from these cultures to be measured reliably by using an infectious assay, in which LANA+ cells were detected by immunofluorescence assay. Naïve TIME cultures were inoculated with ultracentrifuge-concentrated virus from Dox-treated HHV-8+ TIME-TRE/RTA cell culture media, cumulatively collected over 5 days following Dox addition. Numbers of infected, LANA+ TIME cells were counted from multiple random fields to derive average values. Results from two independent experiments are shown, with titers expressed relative to those obtained from NS shRNA-transduced cells (set at 1); the error bar represents deviation from the average NS/Bid titer ratios. Cells were harvested at day 5 for mRNA preparation and RT-PCR confirmation of Bid depletion (right).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002748-g010: Biological significance of Bid in HHV-8 replication.(A) TIME cells latently infected with BCBL-1 PEL culture-derived virus were transduced with non-silencing (NS), Bid-specific (sh1, sh2), or Bim-specific shRNAs using lentiviral vectors (see Materials and Methods). After 48 h, these TIME cultures were treated with TPA to induce lytic reactivation, and media and cells were harvested at 0, 3 and 6 days post-induction for determinations of encapsidated genome copy numbers and Bid and Bim expression, respectively. Quantitative PCR was used to determine genome copies following pre-treatment with DNase I to remove any unencapsidated, contaminating viral DNA released from disrupted cells (see Materials and Methods). Immunoblotting confirmed both induction of Bid and Bim in induced cultures (detected even though a minority of cells support lytic replication [23], [8]) and their specific suppression by the respective transduced shRNAs. (B) Similar experiments were undertaken in control (NS shRNA-transduced) and Bid-depleted (Bid sh1-transduced) TIME-TRE/RTA cells using Dox (1 µg/ml) to induce lytic reactivation. Higher reactivation frequencies possible in these cells enabled relative virus titers from these cultures to be measured reliably by using an infectious assay, in which LANA+ cells were detected by immunofluorescence assay. Naïve TIME cultures were inoculated with ultracentrifuge-concentrated virus from Dox-treated HHV-8+ TIME-TRE/RTA cell culture media, cumulatively collected over 5 days following Dox addition. Numbers of infected, LANA+ TIME cells were counted from multiple random fields to derive average values. Results from two independent experiments are shown, with titers expressed relative to those obtained from NS shRNA-transduced cells (set at 1); the error bar represents deviation from the average NS/Bid titer ratios. Cells were harvested at day 5 for mRNA preparation and RT-PCR confirmation of Bid depletion (right).
Mentions: Previous studies from this laboratory identified Bim as a potent inhibitor of virus productive replication and the importance of vIRF-1 BBD-mediated interactions in countering lytic cycle-induced apoptosis and promoting HHV-8 production in TIME cells [23], [8]. In view of the present findings that vIRF-1 inhibits Bid activity in a BBD-dependent fashion (Fig. 7) and that Bid is induced in lytically reactivated TIME cells (Fig. 4A), we wanted to test the significance of Bid in HHV-8 replication. To do this, we utilized lentiviral vector-delivered shRNAs directed to Bid mRNA sequences to deplete Bid in HHV-8+ (latently infected) TIME cells and compared levels of cell-released encapsidated viral genomes produced following TPA induction to those obtained from non-silencing (NS) shRNA-transduced control cultures. Bim shRNA-transduced TIME cells were also included to provide a positive control for the experiment. The data from this experiment (Fig. 10A) revealed that each of the two Bid shRNAs led to small but significant increases in virus production from TPA-treated TIME cells; as before, Bim depletion led to substantial amplification of virus titers. Similar experiments were undertaken in TIME-TRE/RTA cells to confirm the effect on replication of Bid depletion; here, lytic replication was induced by addition of doxycycline to the cultures. Again, Bid depletion led to increased virus production (Fig. 10B), measured in this experiment by titration of released infectious virus in culture media via inoculation of naïve TIME cells and detection of virus infection by immunofluorescence assay for latency-associated nuclear antigen (LANA). These data demonstrate that Bid, in addition to Bim, is a contributor to negative regulation of HHV-8 infection and suggest that its control by vIRF-1 is likely to be important for optimal virus productive replication. It is important to note, however, that the positive effects on virus replication of Bid and Bim depletion by shRNA transduction demonstrate also that vIRF-1 is not completely effective at suppressing the activities of these BOPs. This situation is not unexpected and is probably universal amongst such viral regulators of cellular activities.

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