HIV-1 Tat Recruits HDM2 E3 Ligase To Target IRF-1 for Ubiquitination and Proteasomal Degradation

Abstract

In addition to its ability to regulate HIV-1 promoter activation, the viral transactivator Tat also functions as a determinant of pathogenesis and disease progression by directly and indirectly modulating the host anti-HIV response, largely through the capacity of Tat to interact with and modulate the activities of multiple host proteins. We previously demonstrated that Tat modulated both viral and host transcriptional machinery by interacting with the cellular transcription factor interferon regulatory factor 1 (IRF-1). In the present study, we investigated the mechanistic basis and functional significance of Tat-IRF-1 interaction and demonstrate that Tat dramatically decreased IRF-1 protein stability. To accomplish this, Tat exploited the cellular HDM2 (human double minute 2 protein) ubiquitin ligase to accelerate IRF-1 proteasome-mediated degradation, resulting in a quenching of IRF-1 transcriptional activity during HIV-1 infection. These data identify IRF-1 as a new target of Tat-induced modulation of the cellular protein machinery and reveal a new strategy developed by HIV-1 to evade host immune responses.

Importance: Current therapies have dramatically reduced morbidity and mortality associated with HIV infection and have converted infection from a fatal pathology to a chronic disease that is manageable via antiretroviral therapy. Nevertheless, HIV-1 infection remains a challenge, and the identification of useful cellular targets for therapeutic intervention remains a major goal. The cellular transcription factor IRF-1 impacts various physiological functions, including the immune response to viral infection. In this study, we have identified a unique mechanism by which HIV-1 evades IRF-1-mediated host immune responses and show that the viral protein Tat accelerates IRF-1 proteasome-mediated degradation and inactivates IRF-1 function. Restoration of IRF-1 functionality may thus be regarded as a potential strategy to reinstate both a direct antiviral response and a more broadly acting immune regulatory circuit.

FIG 1

Tat affects IRF-1 stability. (A) HEK293 cells were transfected with a 3,500-bp fragment of the IRF-1 promoter linked to the luciferase reporter gene alone or in combination with increasing amounts of Tat-expressing vector. After 24 h, cells were treated for 4 h with 10 ng/ml of TNF-α (+), where indicated, and then processed for luciferase activity. Data shown are the means plus standard errors of the means (SEM) (error bars) from three separate experiments calculated after normalization with the Renilla activity. The values for untreated cells were set at 1. (B) HEK293 cells were transfected with increasing amounts of Tat-expressing vector. At 24 h after transfection, cells were harvested, and IRF-1 RNA levels were assessed using real-time RT-PCR. The levels were normalized to GAPDH mRNA abundance. The means plus SEM of three independent experiments are shown as relative expression units. The values for untreated cells were set at 1. (C) HEK293 cells were transfected with IRF-1 expression vector in the presence (+) or absence (−) of Flag-Tat expression vector. At 24 h posttransfection, the cells were treated with CHX for the indicated time. IRF-1 and Tat proteins were detected with anti-IRF-1 (α-IRF-1) and anti-Flag (α-Flag) antibodies, respectively. Data plotted in the graph represent the means ± SEM from three different assays of IRF-1 protein bands quantified from Western blots and normalized to actin protein levels as the loading control and presented as percentage values relative to those without CHX treatment set at 100%. (D) HEK293 cells were cotransfected with expression vectors for His6-Ub and IRF-1 in the presence or absence of Flag-Tat-expressing vector. His-Ub-conjugated proteins were captured by nickel-agarose beads, eluted, and analyzed by Western blotting with anti-IRF-1 antibody. Western blotting of cell lysates shows the expression of ectopically expressed proteins. (E) HEK293 cells were cotransfected with expression vectors for IRF-1 and Flag-Tat and then treated with MG132 for 2 h where indicated. IRF-1 and Tat expression was detected by Western blotting. Data plotted in the graph represent the means plus SEM from three different assays of IRF-1 protein bands quantified from Western blots and normalized to actin protein levels as the loading control. Results are presented as percentage values relative to basal IRF-1 expression set at 100. Blots are representative of at least three independent experiments with similar results.

FIG 2

HDM2 E3 ligase mediates Tat-induced IRF-1 turnover. (A) HEK293 cells were transfected with expression vectors encoding IRF-1 alone or in combination with increasing amounts of GFP-tagged HDM2 (GFP-HDM2), as indicated. The cells were treated with CHX for the indicated time, and cell lysates were then subjected to immunoblotting. Data plotted in the graph represent the means ± SEM from three different assays of IRF-1 protein bands quantified from Western blots and normalized to actin protein levels as the loading control and presented as percentage values relative to those without CHX treatment set at 100. (B) HEK293 cells were transfected with expression vectors for IRF-1, Flag-Tat, and HDM2-GFP expression vector as indicated. The cells were then treated with CHX, and IRF-1 expression was assessed by immunoblotting. Data plotted in the graph are calculated and presented as in panel A. (C) HEK293 cells were transfected with expression vectors encoding IRF-1, GFP-HDM2, and wild-type Flag-Tat (Flag-Tat^wt) or Flag-Tat^C22G. The cells were then treated with CHX, and IRF-1 expression was assessed as described above for panel A. (D) HEK293 cells were transfected with expression vectors for Flag-IRF-1 and Tat and/or GFP-HDM2, as indicated. Whole-cell extracts were incubated with anti-Flag (α-Flag)-conjugated resin or control IgG, and immunoprecipitated (IP) complexes were separated by SDS-PAGE and subsequently probed with anti-Tat, anti-GFP, or anti-IRF-1 antibodies, respectively. The levels of ectopically expressed proteins are shown in the INPUT blots.

FIG 3

Tat increases HDM2-mediated K48 polyubiquitination of IRF-1. (A) IRF-1 ubiquitination in the presence of Flag-Tat, GFP-HDM2, or both was monitored as described in the legend to Fig. 1D. Western blots show the expression, in whole-cell extracts (WCE), of ectopically expressed proteins. (B) HEK293 cells were cotransfected with Flag-tagged IRF1, GFP-HDM2, and Tat alone or in combination, as indicated. Two-hour treatment with MG132 (lane 2) or IL-1β (lane 7) was used for positive internal controls. Immunoprecipitation (IP) with Flag-conjugated resin was performed, and the IRF1 ubiquitination forms were detected by Western blotting or immunoblotting (IB) with anti-K48-linked ubiquitin (α-K48-Ub) or anti-K63-linked ubiquitin (α-K63-Ub) or control IgG. Western blots show the expression, in whole-cell extracts, of ectopically expressed proteins.

FIG 4

Tat-mediated IRF-1 degradation requires the IRF-1 C-terminal domain. (A) HEK293 cells were cotransfected with expression vectors for an IRF-1 mutant with the 34-aa COOH terminus deleted (IRF1²⁹¹), Flag-Tat, and GFP-HDM2, alone or in combination. One day after transfection, the cells were treated with CHX for the indicated time points, and expression of IRF1²⁹¹, Tat, and HDM2 was detected by Western blotting using specific antibodies, as indicated. (B) IRF1²⁹¹ ubiquitination in the presence of Flag-Tat, GFP-HDM2, or both, was monitored as described in the legend to Fig. 3A. Western blots show the expression of ectopically expressed proteins in whole-cell extracts. (C) HEK293 cells were cotransfected with the indicated expression vectors, immunoprecipitated with anti-IRF-1 antibodies, and Tat and HDM2 were detected by Western blotting using the indicated antibodies. INPUT shows the level of ectopically expressed proteins.

FIG 5

Inhibition of IRF-1-dependent transcription by HDM2 is accelerated in the presence of Tat. Transcription of the IRF-1-responding constructs pTA-ISRE-Luc (Luc stands for luciferase) (A) and the human p21 gene promoter linked to the luciferase reporter gene (B) was measured by dual-luciferase assay in whole-cell extracts from HEK293 cells 24 h after transfection with expression vectors for IRF-1, Tat, and HDM2 as indicated. Means plus standard deviations (SD) from three separate experiments were calculated after normalization with the Renilla activity are shown. Values that are significantly different are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

IRF-1 degradation is increased in cells expressing Tat protein. (A) HEK293 cells were transfected with the indicated doses of expression vector encoding Flag-Tat, and one day after transfection, the cells were treated with 10 ng/ml of TNF-α for 4 h. Cell lysates were then subjected to immunoblotting using anti-IRF-1 and anti-Flag antibodies. Data plotted in the graph represent the means plus SEM from three different assays of IRF-1 and Tat protein bands quantified from Western blots and normalized to actin protein levels as the loading control. Results are presented as percentage values relative to IRF-1 expression in control TNF-treated cells and ectopically expressed Tat (2 µg) set at 100. (B) Jurkat A2 cells were induced by TNF-α for 48 h to express Tat, and the levels of IRF-1 and Tat proteins were determined by Western blotting analysis using specific anti-IRF-1 and anti-Tat antibodies, respectively. Means plus SD from three separate experiments calculated after normalization with actin and with the control set at 100% are shown (***, P < 0.001). (C) Endogenous IRF-1 protein was immunoprecipitated using anti-IRF-1 antibody from A72 and A2 Jurkat cell lines treated with TNF-α (+) or not treated with TNF-α (−). IRF-1 ubiquitination was detected upon blotting with anti-K48 ubiquitin antibody (α-K48). The levels of ectopically expressed proteins are shown in the INPUT blots.

FIG 7

IRF-1 is downregulated and K48 polyubiquitinated in HIV-1-infected Jurkat T cells and during HIV-1 de novo infection of human primary CD4⁺ T cells when Tat is maximally expressed. (A, top) Tat/Rev RNA levels were measured by real-time RT-PCR as described in Materials and Methods. Means plus SD from three separate experiments calculated after normalization with GAPDH are shown (*, P < 0.05; ***, P < 0.001). (Bottom) WCE were prepared at the indicated time points from infected and uninfected Jurkat cells and then probed with anti-IRF-1, anti-HDM2, and anti-actin antibodies, respectively. Representative Western blots are shown. Data plotted in the graphs represent the means ± SEM from three different assays of IRF-1 and HDM2 protein bands quantified from Western blots and normalized to actin protein levels as the loading control (CTR). Results are presented as percentage values relative to basal IRF-1 and HDM2 expression set at 100. (B) WCE were prepared at the indicated time points from control and HIV-1-infected cells and immunoprecipitated with anti-IRF-1 antibody. IRF-1 ubiquitinated forms were detected using anti-K48 ubiquitin antibody (α-K48). The Western blot is representative of at least three independent experiments with similar results. (Bottom) The levels of IRF-1 in control cells, HIV-1-infected cells, and HIV-1-infected cells in the presence of MG132 are shown. Data plotted in the graphs are the means plus SD of IRF-1-specific bands quantified from Western blots normalized to actin from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) WCE as in panel A were probed with anti-p21 and anti-CDK2 antibodies, respectively. (Top) Quantification of p21 and CDK2 calculated as in panel A. (Bottom) Representative Western blots. (D) Purified human primary CD4⁺ T cells were infected with HIV-1 as described in Materials and Methods, and WCE were subjected to Western blot analysis with specific anti-IRF-1 antibody. Data plotted in the graph represent the means plus SD from three independent experiments (*, P < 0.05; **, P < 0.01). The time (in hours postinfection [hr p.i.]) is shown below the blot. (E) Total RNA was extracted at the indicated time points from cells as described above for panel D and analyzed by real-time RT-PCR for the doubly spliced (Tat/Rev) transcript as described in Materials and Methods (**, P < 0.01; ***, P < 0.001).

FIG 8

Schematic representation of the dual effect of Tat on IRF-1 activity in the course of HIV-1 infection. In early phases of HIV-1 replication, IRF-1 is transcriptionally stimulated by viral infection, and it is recruited by small amounts of Tat on the viral promoter to drive, with NF-κB, initial transcription of the integrated provirus. Later, when discrete amounts of Tat are produced and IRF1 activity on LTR is dispensable for the virus to replicate, Tat nullifies the function of IRF-1, accelerating its proteasome-mediated degradation upon recruitment of the HDM2 E3 ligase, thus quenching its activity on target gene promoters.