HIV blocks Type I IFN signaling through disruption of STAT1 phosphorylation

Cell culture preparation

Human embryonic kidney 293T cells (HEK 293T) and CEM cells (T lymphoblast) were obtained from the American Type Culture Collection (ATCC). HEK 293T cells were cultured and maintained in DMEM with 5% FBS and 1% penicillin/streptomycin. Phoenix-Ampho cells (purchased from ATCC) are a derivative of HEK 293T cells engineered to package retroviruses for transduction and were cultured similarly to the base HEK 293T cells.^23,24 CEM cells were cultured and maintained in Roswell Park Memorial Institute media with 5% FBS and 1% penicillin/streptomycin. Cells were incubated at 37°C and 5% CO₂ in a humid environment. All growth media and supplements were purchased from Gibco.

HIV plasmids

HIV plasmids Vpr, Vif, Nef were generously donated by WC Greene (University of California San Francisco) and Vpu was donated by K Strebel (National Institutes of Health, National Institute of Allergy and Infectious Diseases). FLAG-STAT1 and GFP-IRF9 were purchased from Addgene. FLAG-Tyk2 and HA-Jak1 were purchased from Origene. Gag components including matrix, capsid, nucleocapsid, p6 and retropepsin were also purchased from Origene. Vpu, Nef and Vif plasmids with a GFP tag were produced by PCR and introduced into the pBMN plasmid.

Luciferase reporter assay

Luciferase reporter assay was performed to measure the ISRE-containing promoter activity. HEK 293T cells were transfected in 24-well dishes as sets of triplicates. In brief, triplicates were transfected with X-tremeGENE™ 9 (Roche) at a ratio of 2:3 (transfection reagent: DNA). Each triplicate was transfected with 1250 ng of total plasmids with a mix of 200 ng of pGL3-ISRE luciferase reporter, 50 ng of pSV40-Renilla luciferase reporter and 1000 ng of expression plasmid. Transfection complex was incubated in 75 µl of DMEM with X-tremeGENE™ 9 per triplicate for 20 min. HEK 293T cells were plated at 1 × 10⁵ cells total per well. To each well 20 µl of transfection complex was added. Transfections were incubated for 24 h. The next day, transfections were stimulated with 1000 U of IFN-α for 6 h. Then, cells were lysed with passive lysis buffer from the Dual-Luciferase Reporter Assay kit (Promega). Luciferase readings were obtained by the GloMax®-Multi Detection System and analyzed using Microsoft Excel.

Vpu and Nef co-transfection

Co-transfection was performed to measure the expressions of IFN-α pathway components. HEK 293T cells were co-transfected with 1000 ng of control plasmid or an expression plasmid for Vpu or Nef plasmid and 1000 ng of one of the following plasmids: FLAG-Tyk2, HA-Jak1, FLAG-STAT1 or GFP-IRF9 in each well of a six-well plate. Transfection reagent, X-tremeGENE™ HP (Roche) was used at a ratio of 1:1 (extreme gene HP: DNA plasmids). Each transfection complex was incubated in 100 µl of DMEM/X-tremeGENE™ for 20 min. After incubation, 100 µl of transfection complex was added to each well, which had been plated with HEK 293T cells at a concentration of 4 × 10⁵ cells/ml (8 × 10⁵ cells total per well). Transfections were incubated for 48 h. Whole-cell lysates were obtained by incubating cells in 1X SDS loading buffer (1X Laemmli buffer (Bio-Rad) supplemented with 10% DTT). Samples were analyzed by Western blot analysis.

Stable CEM cell line establishment

Stable cell lines were established to measure the endogenous level of IFNAR-signaling components and their phosphorylated states. Phoenix-Ampho was used to package retroviruses with control, Vpu, Nef or Vif (as a control) that could be used to transduce CEM cells. Phoenix-Ampho cells were transfected with 2000 ng of plasmids using X-tremeGENE™ 9 (Roche) (2:3 ratio). Transfected cells were allowed to package viruses for 48 h. Retrovirus was harvested, and the supernatants were filtered through 0.22 µm syringes that had 0.4 µg of polybrene added to them. Retroviruses were added to CEM cells that were plated at 3 × 10⁵ cells per well. CEM cells were spinoculated at 800 g for 2 h in a room temperature (25°C) centrifuge to allow viral attachment. The media uses for packaging was removed and 2 ml of CEM media was added to each well. The cells were incubated for 2 d to allow for productive infection then neomycin was added to allow for selection for about 2 wk. Successfully transduced cells should be able to express EGFP as the control plasmid was GFP expressing and all of the HIV gene Open Reading Frames (ORFs) were fused in frame with Enhanced Green Fluorescent Protein (EGFP). The expression of transduced genes was monitored by GFP expression in the cell population by using flow cytometry. For cells that were selected for vesicular stomatitis virus (VSV)-GFP infections, the transduced ORFs did not contain GFP and thus were monitored by cell recovery after selection. ²⁵

IFN-α stimulation of transduced CEM cells experiment

Stably transduced CEM cells were plated at 3 × 10⁵ cells per well in a 24-well plate and stimulated with IFN-α for 4 h. Whole-cell lysates were obtained by incubating cells in 1X SDS-loading buffer (1X Laemmli buffer (Bio-Rad) supplemented with 10% DTT). Samples were analyzed by Western blot analysis.

Western blot

Whole-cell lysates were passed through a 21-Gauge needle three times to shear cellular DNA and membranes, then boiled for 5 min to denature protein. Samples were clarified by spinning at 14,200 g for 10 min in a microcentrifuge. Then 30 µl of each sample were run in a pre-made TGX gel (Bio-Rad) for 30 min at 200 mV in 1X Tris-SDS running buffer (Bio-Rad). Proteins were transferred using the Trans-Blot Turbo Transfer system (Bio-Rad). Each membrane was blocked in 5% milk TBST for 1 h at room temperature on a shaker. Specific primary Abs were added and incubated overnight in 5% milk TBST at 4°C with agitation. The Abs used were anti-FLAG M2 Ab (Sigma) (1:1000), anti-GFP (FL) Ab (Santa Cruz Biotech) (1:500), (1:500), anti-HA Ab (Sigma) (1:10,000), anti-STAT1 Ab (Cell Signaling) (1:3000), anti-pSTAT1 Ab (Cell Signaling) (1:1000), anti-Tyk2 (Cell Signaling) (1:1000), anti-actin (Cell Signaling) (1:10,000) and anti-tubulin (Cell Signaling) (1:10,000). For pSTAT1 Ab, 5% BSA TBST was used instead of 5% milk TBST for membrane blocking and primary Ab incubation. Membranes were washed the next day three times (5 min each time) with 5% milk TBST. Membranes were incubated in secondary Ab, either goat anti-mouse (Santa Cruz Biotech) (1:3000) or goat anti-rabbit (Santa Cruz Biotech) (1:3000) for 1 h. Then, membranes were washed three times with 5% milk TBST (15 min each time) and three times with TBST (5 min each time) at room temperature. All the washes were done on a Belly Dancer shaker. Membranes were incubated in Luminol reagent (Santa Cruz Biotechnology) and proteins were detected using digital light detection using a Gel Doc XR+ System and analyzed by Image Lab (BioRad).

Note on values graphed in figures

In an effort to show genuine quantitative data, we do not show fold induction for any luciferase experiment but instead show the actual values obtained in an experiment normalized to the internal standard. We feel this gives a more genuine representation of data and that the data are not masked through the use of fold-induction values, especially in reporter assays. Consequently, the values in different experiments may vary but the interpretation remains the same and is more consistent with the biology of each individual experiment. Relative levels are added above the real data graphed.

Vpu and Nef reduce IFN-α activation of ISRE promoter activity

To begin investigating if HIV modulates IFN-α signaling, we first sought to identify which of the HIV proteins are involved in controlling IFN-α signaling. IFN-α, induced during viral infection, activates the transcription factor ISGF3, which binds to the promoter element ISRE to start transcribing ISGs. ²⁶ Therefore, we measured the activity of the ISRE promoter in the presence of HIV proteins using luciferase assays. We screened all the possible HIV protein products by co-transfecting HEK 293T cells with plasmids individually expressing each of the HIV proteins and the ISRE luciferase plasmid. We also included a positive control, protein inhibitor of activated STAT, which is known to inhibit JAK/STAT signaling. ²⁷ The cells were transfected with the corresponding plasmids for 24 h and then stimulated for 6 h with 1000 U of IFN-α. Cell lysates were obtained and the luminescence levels were measured (Figure 1). Data obtained were normalized to the levels of Renilla, which was used as an internal control.

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Figure 1.

HIV Open Reading Frames (ORFs) block IFN-α-induced IFN-stimulated response element (ISRE) activation. HEK 293T cells were transfected with plasmids expressing individual HIV proteins for 24 h and then stimulated with 1000 U of IFN-α for 6 h. ISRE reporter activity was measured and graphed in relative luciferase units normalized to the internal control Renilla. Graphed data represent the average of a triplicate done on the same day and each experiment was repeated three times. The graph displays data from transfection with (a) Vpu and Nef, (b) gag, gp120, Vpr, Vif, (c) nucleocapsid (Nc), matrix (Ma), protease (Pro), p6 and capsid (Ca). Fold changes after IFN-α stimulation are noted above the respective bars.

Upon transfection of HIV plasmids, compared to the control transfection, we found that IFN-α-induced ISRE-luciferase signal was lower in the Vpu and Nef transfected cells (Figure 1a). ISRE promoter activity was significantly reduced in the presence of Vpu and Nef compared to transfection with the other HIV proteins including gp120, Vpr, Vif, protease (retropepsin) and the Gag components: nucleocapsid, capsid, matrix, p6 (Figure 1b and 1c).

These experiments were repeated in a dose-dependent manner with increasing amounts of Vpu and Nef. As seen in Figure 2a for Vpu and 2b for Nef, the levels of the ISRE activity decreased as Vpu and Nef increased. Therefore, the ISRE promoter activity was inhibited specifically by Vpu and Nef but not by other HIV components.

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Figure 2.

Vpu and Nef specifically block IFN-stimulated response element (ISRE) activation. (a and b) HEK 293T cells were transfected with plasmids expressing individual HIV proteins for 24 h and then stimulated with 1000 U of IFN-α for 6 h. ISRE reporter activity was measured and graphed in relative luciferase units normalized to the internal control Renilla. Graphed data represent the average of a triplicate done on the same day and each experiment was repeated three times. Transfections for the luciferase assay were repeated in a dose-dependent manner with 0.5 µg, 1.0 µg and 1.5 µg of Vpu in (a) or Nef in (b). Graphed data represent the average of a triplicate done on the same day and each experiment was repeated three times.

Jak1 and Tyk2 protein expressions are unaffected by Vpu and Nef

Because ISRE promoter activity was inhibited by Vpu and Nef, we hypothesized that Vpu and Nef must interact with a cellular component somewhere in the IFN-α signaling pathway. To determine at which step of the signaling pathway Vpu and Nef have an effect, we first looked at the stability of components of the JAK/STAT pathway and assessed the level of expression of the two kinases, Jak1 and Tyk2 in the presence of Vpu and Nef. Jak1 and Tyk2 kinases are activated following the binding of IFN-α to the IFNAR receptor. ²⁸ Jak1 and Tyk2 activate intermediate proteins, which include STAT1 and STAT2 downstream.

HEK 293T cells were co-transfected with HA-tagged Jak1 or FLAG-tagged Tyk2 plasmids along with Vpu or Nef plasmids. The protein levels of Jak1 and Tyk2 were not significantly reduced by Vpu or Nef (Figure 3a and b). Therefore, Vpu and Nef do not affect the expression of Jak1 and Tyk2 even though ISRE activity was inhibited by Vpu and Nef.

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Figure 3.

Components of IFN-α/β receptor signaling are stable in the presence of Vpu or Nef. HEK 293T cells were co-transfected with HIV plasmids (Vpu or Nef) and (a) HA-tagged Jak1, (b) FLAG-tagged Tyk2, (c) FLAG-tagged STAT1 or (d) GFP-tagged IRF9. After 48 h of incubation, protein lysates were obtained by re-suspending in 1X SDS loading buffer for Western blot analysis using actin as a loading control. After experimental setup and establishment as well as Ab titrations, each Western blot was additionally repeated an additional three times as displayed. The relative protein expressions relative to actin levels for four replicates of the Western blot were graphed along with standard deviation.

STAT1 and IRF9 are unaffected by Vpu and Nef

Next, we looked at the two proteins downstream of the JAK/STAT pathway. STAT1 is phosphorylated by activated Jak1 kinase. ²⁹ When phosphorylated, STAT1 is able to dimerize with phosphorylated STAT2. This heterodimer can recruit IRF9 to form a transcription factor complex called ISGF3 that can then enter the nucleus to activate ISRE-containing promoters. Here, we examined the expression levels of STAT1 and IFR9 in the presence of Vpu and Nef.

HEK 293T cells were co-transfected similarly to the experiment above with FLAG-tagged STAT1 or GFP-tagged IRF9 plasmids along with Vpu or Nef plasmids. Whole-cell lysates were obtained following 48 h of incubation. Surprisingly, both STAT1 and IRF9 were unaffected by Vpu and Nef as seen in Figure 3c and d. Thus, although Vpu and Nef reduce IFN-α activation of ISRE, they do not affect the stability of the kinases Jak1 or Tyk2, or two key components of the ISFG3 activator, STAT1 or IRF9.

Phosphorylation of STAT1 is inhibited by Vpu and Nef

So far, expression of the proteins in the IFN-α pathway was shown to be unaffected by Vpu and Nef. Because ISRE activation is being blocked, we hypothesized that perhaps the phosphorylation of STAT1 is affected by Vpu or Nef. To understand the phosphorylation activity, we looked at the endogenous STAT1 and its phosphorylation state. We engineered CD4⁺ T cell lines that stably express HIV proteins by transducing CEM cells using retrovirus produced from Phoenix-Ampho cells. We used Vpu, Nef and a control GFP plasmid to obtain three different cell lines. Flow cytometry was performed to monitor uniform gene expression by looking at GFP-expression in the cell population (Figure 4f). Stably transduced CEM cells were plated and stimulated with IFN-α for 2 and 4 h. Protein levels for Tyk2, STAT1 or phosphorylated STAT1 were monitored by Western blot.

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Figure 4.

Vpu and Nef block IFN-α stimulated STAT1 phosphorylation. Stable CEM cell lines expressing Vpu and Nef were established. Transduced cells were selected between 10–14 d and monitored by flow cytometry based on the presence of GFP. Cells were plated and stimulated with 1000 U of IFN-α for 2 or 4 h. Cell lysates were obtained in SDS loading buffer and analyzed by Western blotting for (a) phosphorylated STAT1 or (b) total STAT1 along with tubulin for each. After the experimental setup and establishment as well as Ab titrations, each Western blot was additionally repeated an additional three times as displayed. The relative protein expressions relative to actin levels for four replicates of the Western blot were graphed along with standard deviation. Relative fold of protein levels for phosphorylated STAT1 were graphed in (c) and for total STAT1 were graphed in (d). Levels of phosphorylated STAT1 compared to the corresponding STAT1 were graphed in (e). (f) Displays GFP levels of the transduced population to show that all cell lines were similar in terms of transduction and stable expression of genes. (g) Total Tyk2 levels at 0 and 4 h after IFN-a stimulation. The relative protein expressions relative to actin levels for four replicates of the Western blot were graphed along with standard deviation. Relative fold of protein levels for Tyk2 were graphed in (h).

As shown in Figure 4, there is a reduction of phosphorylated STAT1 after IFN-α stimulation in the Vpu- and Nef-expressing cells (Figure 4a). Nef seemed to have more impact in decreasing the phosphorylation of STAT1 than Vpu. Meanwhile, GFP did not have any effect on the phosphorylation state of STAT1. The data were consistent with the luciferase assay result where Nef also seemed to have stronger inhibition of ISRE promoter activation compared to Vpu. In another set of experiments when blotting for STAT1 and Tyk2, the expression levels of these proteins were not affected by Vpu or Nef as seen in the results (Figure 4b and 4g). Fold of protein levels was plotted for four replicates of each graph (Figure 4). These data were also consistent with the results in Figure 3, where the expression levels of Tyk2 and STAT1 were not affected in the transient transfection experiment.

Vpu increases VSV replication in the presence of exogenous IFN-α

To better understand the physiological impact of targeting STAT1 phosphorylation as a mechanism of diminishing ISRE and ISG activation, we utilized an IFN-induced antiviral efficacy assay. If Vpu disrupts the expression of ISGs in the stable cell lines, we expect to see a lack of viral protection in these cells. We designed an experiment to understand the infection rate of VSV-GFP in the presence of HIV proteins after those cells were stimulated with IFN-α. The stable cells were stimulated with IFN-α for 4 h followed by VSV-GFP infection for 24 h. The cells were collected and analyzed by flow cytometry looking at the level of GFP.

VSV-GFP expresses GFP if infection is allowed to proceed (Figure 5a vs ⁵ b). However, the addition of IFN-α leads to a decreased level of GFP (Figure 5b vs ⁵ c). As expected, comparing stable Vpu cells to stable Vif and CEM cells, GFP level was increased in Vpu stable cells relatively (Figure 5e vs ⁵ h). Therefore, the rate of VSV replication was higher when there was Vpu because the antiviral activities were probably reduced. Additionally, when treated with IFN-α to activate the antiviral state, the control CEM cells and those stably expressing Vif cells were rescued as shown by decreased VSV-GFP levels (Figure 5c and 5f). Meanwhile, in the presence of Vpu, the IFN-α-induced rescue effect did not take place. There was no difference with and without treatment of IFN-α as Vpu seems to disrupt the function of IFN-α interfering with VSV-GFP replication (Figure 5h vs ⁵ i). In short, Vpu decreases the ability of IFN-α to induce an efficacious antiviral state and allowed for the increased replication and spread of VSV. Representative fluorescent pictures of the cells analyzed in Figure 5a–5i are shown in Figure 5j.

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Figure 5.

Vpu blocks IFN-α stimulated establishment of an antiviral state. (a–i) Stable CEM cell lines expressing Vpu or Vif were established. Cells were plated and stimulated with 1000 U of IFN-α for 6 h and infected with a GFP expressing vesicular stomatitis virus. Cells were monitored by flow cytometry based on the presence of GFP. The percentage of GFP-positive cells is indicated at the top right corner. (j) Fluorescent pictures of the cells were taken, and representative pictures are displayed. The scale bar is at 200 µm.