Bim nuclear translocation and inactivation by viral interferon regulatory factor

Abstract

Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of the host cell. Human herpesvirus 8 (HHV-8) uses several strategies to block the host's innate antiviral defenses via interference with interferon and apoptotic signaling. Contributors include the four viral interferon regulatory factors (vIRFs 1-4), which function in dominant negative fashion to block cellular IRF activities in addition to targeting IRF signaling-induced proteins such as p53 and inhibiting other inducers of apoptosis such as TGFbeta receptor-activated Smad transcription factors. Here we identify direct targeting by vIRF-1 of BH3-only pro-apoptotic Bcl-2 family member Bim, a key negative regulator of HHV-8 replication, to effect its inactivation via nuclear translocation. vIRF-1-mediated relocalization of Bim was identified in transfected cells, by both immunofluorescence assay and western analysis of fractionated cell extracts. Also, co-localization of vIRF-1 and Bim was detected in nuclei of lytically infected endothelial cells. In vitro co-precipitation assays using purified vIRF-1 and Bim revealed direct interaction between the proteins, and Bim-binding residues of vIRF-1 were mapped by deletion and point mutagenesis. Generation and experimental utilization of Bim-refractory vIRF-1 variants revealed the importance of vIRF-1:Bim interaction, specifically, in pro-replication and anti-apoptotic activity of vIRF-1. Furthermore, blocking of the interaction with cell-permeable peptide corresponding to the Bim-binding region of vIRF-1 confirmed the relevance of vIRF-1:Bim association to vIRF-1 pro-replication activity. To our knowledge, this is the first report of an IRF protein that interacts with a Bcl-2 family member and of nuclear sequestration of Bim or any other member of the family as a means of inactivation. The data presented reveal a novel mechanism utilized by a virus to control replication-induced apoptosis and suggest that inhibitory targeting of vIRF-1:Bim interaction may provide an effective antiviral strategy.

Figure 1. Nuclear localization of Bim during HHV-8 productive replication.

(A) Telomerase-immortalized endothelial (TIME) cells latently infected with HHV-8 were treated with TPA to induce lytic replication. Cells supporting lytic reactivation were identified by immunofluorescence staining for K8.1 late lytic membrane protein and Bim was detected by co-staining with appropriate immunological reagents (see Materials and Methods), six days after lytic induction. The left panel shows merged immunofluorescence staining for K8.1 (green) and Bim (red) together with Hoechst nuclear staining (blue). The right panels show individual staining for K8.1 and Bim, emphasizing the correlation of Bim nuclear staining with lytic antigen (K8.1) expression. (B) Analogous confocal immunofluorescence analyses verified Bim nuclear localization specifically in lytically reactivated cells, expressing ORF59-encoded and vIRF-1 early nuclear antigens in addition to K8.1 late membrane protein, and revealed colocalization of vIRF-1 and Bim staining patterns.

Figure 2. vIRF-1 induced Bim nuclear localization and vIRF-1:Bim interaction.

(A) vIRF-1 and selected other HHV-8 nuclear proteins, or GFP (negative control), were tested for their abilities to induce nuclear translocation of Bim_EL-Flag in transfected HEK293T cells by western analysis of cytoplasmic and nuclear fractions of transfected cells. vIRF-1, specifically, induced nuclear localization of Bim. (B) IFA-determined nuclear translocation of Flag-Bim_EL (but not Flag-Puma, control BH3-only protein) induced by vIRF-1 in expression vector-transfected HEK293T cells. Nuclear localization of Bim in vIRF-1 co-transfected cells correlated with strong nuclear vIRF-1 staining. (C) Physical association of vIRF-1 and Bim was detected by co-precipitation assay employing GST-fused vIRF-1 mixed with extracts of Flag-Bim_EL vector-transfected HEK293T or untransfected BCBL-1 cells , the latter expressing high levels of endogenous Bim. Western analysis of glutathione bead-precipitated material identified Bim precipitated with GST-vIRF-1 protein, but not with GST alone (top panels). Lower panels show precipitated GST protein; bracketed bands (*) correspond to GST-vIRF-1 protein degradation products. (D) Association of Bim and vIRF-1 in intact cells as evidenced by vIRF-1 co-precipitation with Flag antibody-immunoprecipitated (IP) Flag-Bim_EL from cell lysate of appropriately transfected cells. Cntl IgG, control (non-specific) IgG. (E) In vitro binding assay using bacterially expressed and purified recombinant GST-vIRF-1 and chitin-binding domain (CBD)-fused Bim_S, Bim_L and Bim_EL isoforms, precipitable with chitin beads. All Bim-CBD constructions, but not CBD alone, were able to co-precipitate GST-vIRF-1, demonstrating direct binding of each Bim isoform to vIRF-1.

Figure 3. Mapping Bim interaction site of vIRF-1.

(A) Recombinant GST-vIRF-1 (progressively deleted) and Bim_EL-CBD proteins were used in co-precipitation-based binding experiments to determine the “Bim-binding domain” (BBD) of vIRF-1. The C-terminal hydrophobic region of Bim_EL (last 18 amino acids) was deleted to enhance solubility of the protein in bacteria. The binding assay employed was analogous to that of Fig. 2E. (B) Mutations introduced into the deletion-mapped BBD (residues 170–187) identified residues 174–181 as necessary for interaction with Bim.

Figure 4. Sufficiency and requirement of vIRF-1 BBD for Bim interaction.

(A) Sufficiency of BBD for Bim binding intracellularly was determined by immunofluorescence assay (IFA) in cells transfected with plasmid vectors expressing nuclear localization signal (NLS)-fused BBD (also linked to GFP) and Flag-Bim_EL. NLS-GFP was used as a negative control. (B) Analogous IFA studies employing BBD core residue-deleted (Δ174-181) or -substituted (GK₁₇₉AA) versions of vIRF-1 verified the importance of BBD in the context of full-length vIRF-1 for interaction with and nuclear localization of Bim. (C) BBD-altered vIRF-1 proteins also were unable to bind Bim_EL in immunoprecipitation (IP) binding assays (analogous to those in represented in Fig. 2D). (D) Reporter assays were used to assess the specificity of effects of BBD alteration, as BBD lies within (large) regions of vIRF-1 previously shown to interact in inhibitory fashion with p53, Smads 3 and 4, and IRF-1 , , . Luciferase reporters responsive to these proteins were inhibited equivalently by wild-type and BBD-mutated vIRF-1 proteins, in the presence of p53, Smad3 or IRF-1. Results are from three transfections; error bars represent standard deviations from means values.

Figure 5. Functional significance of vIRF-1:Bim binding and Bim nuclear translocation.

(A) vIRF-1 inhibition of Bim-induced cell death and apoptosis was identified using GFP-based cell viability and TUNEL assays, respectively. The left panels show examples of fluorescence microscopy results from which quantified data (right panels, charts) were derived. (B) Using the GFP-based assay and quantifying GFP fluorescence by fluorometry, Bim-inhibitory activity of wild-type vIRF-1 was compared against activities of BBD-altered vIRF-1(GK₁₇₉AA) and vIRF-1Δ174-181. The latter were unable to inhibit Bim_EL activity. (C) Using the same assay, the functional consequence of nuclear translocation of Bim was tested using an NLS-fused version of Bim_EL (Flag-tagged, “fBim”). NLS-fBim_EL, localizing predominantly in the nuclei of transfected cells as expected (IFA, bottom panels), was significantly inhibited relative to native fBim_EL in respect of pro-death activity (chart, middle panel). Results are expressed in relation to GFP fluorescence values (100%) derived from control cultures transfected with empty vector instead of Bim vectors. Western blotting (right panels) verified equivalent expression of native and NLS-fused versions of Flag-tagged Bim_EL; fBim- and NLS-fBim-containing extracts were processed identically and in parallel, and therefore band intensities are directly comparable. Data from apoptosis assays were derived from triplicate (panels A and B) or duplicate (panel C) transfections; error bars show standard deviations from mean values. GFP fluorescence measurements were undertaken 24 h posttransfection (panels B and C).

Figure 6. vIRF-1 function and significance of vIRF-1:Bim interaction in the context of virus infection.

(A) Two lentiviral-cloned shRNAs effective for vIRF-1 depletion (top panels) were transduced into HHV-8 (latently) infected TIME cells and lytic reactivation then induced with TPA. After six days, encapsidated (DNaseI-resistant) viral genomes released into culture media were quantified by qPCR . vIRF-1 shRNA-specific reductions of viral titers were observed. NS, non-silencing shRNA control. Data were obtained from triplicate qPCR reactions; error bars show standard deviations from mean values. (B) Effects of wild-type and BBD-altered [GK₁₉₇AA (“AA”) and Δ171-184 (“Δ”)] vIRF-1 proteins on virus replication were tested using TIME cell lines expressing each of the proteins in a doxycycline (Dox)-inducible fashion. EV, empty vector control. Data were derived from triplicate PCR reactions; error bars show deviations from mean values. Western analysis of nuclear extracts revealed induced nuclear localization of endogenous Bim only in wild-type vIRF-1 expressing cells. (C) In analogous experiments, TUNEL analysis of apoptosis revealed effective protection from lytic cycle-induced apoptosis (+TPA) by overexpression of wild-type vIRF-1 (+Dox), but not by Dox-induced vIRF-1 variants GK₁₇₉AA or Δ174-181. Data were derived from multiple random fields for each condition; error bars represent standard deviations from mean values. (D) Similar analysis of apoptosis in vIRF-1-depleted and control non-silencing (NS) shRNA-transduced TIME cultures revealed significantly increased rates of HHV-8 replication-induced apoptosis in vIRF-1 shRNA (sh1, sh2) expressing cultures, demonstrating anti-apoptotic function of endogenously expressed vIRF-1. Data were derived multiple random fields from duplicate cultures; error bars reflect deviations from mean values obtained from each.

Figure 7. BBD peptide-mediated disruption of vIRF-1:Bim interaction.

The biological significance of Bim binding by vIRF-1 and feasibility of targeted disruption of vIRF-1:Bim interaction were tested using cell-permeable (Tat-fused) BBD peptides, wild-type and AA-mutated. These were applied (at 10 µM final concentration) to HHV-8 infected TIME cultures prior to TPA induction. Inhibition of virus production specifically by wild-type BBD (top) reflected the relative abilities of the wild-type and mutated peptides to interfere with vIRF-1:Bim interaction in vitro (lower panels). Replication data were derived from triplicate PCR reactions; error bars indicate deviations from mean values.