Evidence for BAG3 modulation of HIV-1 gene transcription

Abstract

A family of co-chaperone proteins that share the Bcl-2-associated athanogene (BAG) domain are involved in a number of cellular processes, including proliferation and apoptosis. Among these proteins, BAG3 has received increased attention due to its high levels in several disease models and ability to associate with Hsp70 and a number of other molecular partners. BAG3 expression is stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, and certain drugs. Here, we demonstrate that BAG3 expression is elevated upon HIV-1 infection of human lymphocytes and fetal microglial cells. Furthermore, BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies, suggesting that induction of BAG3 is part of the host cell response to viral infection. To assess the impact of BAG3 upregulation on HIV-1 gene expression, we performed transcription assays and demonstrated that BAG3 can suppress transcription of the HIV-1 long terminal repeat (LTR) in microglial cells. This activity was mapped to the kappaB motif of the HIV-1 LTR. Results from in vitro and in vivo binding assays revealed that BAG3 suppresses interaction of the p65 subunit of NF-kappaB with the kappaB DNA motif of the LTR. Results from binding and transcriptional assay identified the C-terminus of BAG3 as a potential domain involved in the observed inhibitory effect of BAG3 on p65 activity. These observations reveal a previously unrecognized cell response, that is, an increase in BAG3, elicited by HIV-1 infection, and may provide a new avenue for the suppression of HIV-1 gene expression.

Fig. 1

Detection of BAG3 in HIV-1-associated encephalopathy during the course of HIV-1 infection. A: Astrocytes in the white matter of normal brain (original magnification 400×) and in an area of gliosis of a case of HIV-encephalopathy (original magnification 200×) were analyzed by immunohistochemistry (IHC) with an anti-BAG3 polyclonal antibody. B: SupT1 cells in the log phase of growth were infected with JR-FL of HIV and harvested on days 5, 10, and 15. Five micrograms of total cell lysates were examined for the amount of BAG3, and α-tubulin was used as a control to monitor equal loading conditions.

Fig. 2

Effect of BAG3 overexpression on HIV-1 LTR is dependent on κB DNA-binding motifs. A: Schematic illustration of HIV-1 LTR representing two copies of the κB domain, three GC-rich motifs, TATA box, and the TAR region, all spanning within −120 to +66. An arrow points to the transcription start site (+1), and the direction of LTR transcription. B: Human microglial cells were transfected with plasmids containing the sequences −120 to +66 and −80/+66 of the HIV-1 LTR promoter. CMV-p65- and CMV-BAG3-expressing plasmids were used in co-transfection and luciferase activity was determined as described in the Materials and Methods Section. C: Human microglial cells were co-transfected with a luciferase reporter plasmid containing the sequence from nucleotides −120 to +66 of LTR and various combinations of CMV-p65-, CMV-Tat-, and CMV-BAG3-expressing plasmids. Luciferase activity was measured after 48 h in duplicates. Bar graphs depict the means±standard deviations of fold increases with respect to promoter basal activity. Similar results were obtained in other two independent experiments.

Fig. 3

Suppression of BAG3 by siRNA restores the ability of p65 to enhance LTR activity. A: U87MG cells were transfected with a plasmid overexpressing BAG3 and with a specific siRNA targeting BAG3 mRNA or a Scrambled siRNA, together with a CMV-p65 plasmid. Luciferase activity was determined as described in Materials and Methods section. B: BAG3 and NFκB p65 protein levels were assessed by Western blot and Grb2 was used as a control to monitor equal loading conditions.

Fig. 4

Effect of BAG3 deleted mutants on HIV-1 LTR activation mediated by p65. A: Structural organization of full-length BAG3 and the effect of BAG3 and its mutants on p65 activation of the LTR. U87MG cells were transfected as described in Experimental Procedures with a plasmid containing the sequence from nucleotides −120 to +66 of LTR. Cells were transfected with an NF-κB p65-expressing plasmid and BAG3 deletion mutant plasmids. Luciferase activity was determined; the percentages of inhibition of p65 activity on HIV-1 LTR promoter by BAG3 FL protein and by the deletion mutants were calculated. B: Interaction of BAG3 and its mutants with p65. U87MG cells were transfected with the BAG3 FL plasmid and the deletion mutants. After 48 h cells were harvested and whole cell lysate were used in a co-immunoprecipitation assay using a Myc-Tag antibody that detects full-length BAG3 (BAG3 FL) and its mutants BAG3 1-502 and BAG3 1-418. C: IHC with a fluorescein-tagged secondary antibody demonstrated the presence of BAG3 in the cytoplasm of astrocytic cells. The NF-κB p65 subunit was also found in the cytoplasm of neoplastic astrocytes. Double labeling demonstrated the co-localization of both proteins in the cytoplasmic compartment of neoplastic cells (all parts: original magnification 1,000×).

Fig. 5

Suppression of NF-κB p65 binding to the κB motif of the HIV-1 promoter by BAG3. A: Nuclear extracts from cells expressing BAG3, BAG3 (1-502) and p65 were incubated with a [³²P]-labeled probe containing the nucleotide sequence of the κB-binding site on LTR. The nucleoprotein complexes were analyzed by native PAGE. Arrows depict the formation of the p65:κB nucleoprotein complex. B: Chromatin immunoprecipitation assay was performed using a p65-specific antibody or control sera. Each sample was used in PCR using specific primers that flank the κB motif of the LTR. The control sera and input were used as negative and positive controls. C: A fraction from the eluted complexes was analyzed by Western blot to verify the presence of p65 in the samples.