Table 1.
Kinase and phosphatase inhibitors used in spread assay.
Fig 1.
Summary of multiplexed mass spectrometry proteomic and phosphoproteomic analyses.
(A) Schematic of experimental design for the phosphoproteomic and proteomic analyses. HIV expressing Jurkat cells grown in light media are mixed with target SupT1 cells grown in heavy media for 0, 5 or 60 minutes, with three or four replicates for each condition. Samples were harvested, peptides digested and the peptides in each sample were ligated to a tandem mass tag (TMT). IMAC chromatography was used to enrich phosphoproteins and liquid chromatography mass spec (LC-MS) was used to resolve the light and heavy samples, and a second MS was run to resolve the TMT. (B) HIV proteins with altered phosphorylations 5 and 60 min after coculture.
Table 2.
Phosphorylation changes after VS formation.
Fig 2.
Multiplex analysis used for MS analysis of HIV Env-mediated cell signaling.
(A) Schematic showing 3 different multiplexes used in TMT-mass spec analysis. Multiplex 1 contained full HIV expressing producer cells mixed with uninfected target cells for 0 min. (quadruplicate), 5 min. (triplicate) and 60 min (triplicate). Multiplex 2 had duplicates of WT full virus at 0 and 5 min and duplicates of HIV lacking Env (ΔEnv) at 0 and 5 min. Multiplex 3 consisted of WT full virus in duplicate at 0 and 60 min and duplicates of cells only expressing Env (Env only). (B) Tabular comparison of shared total protein and phosphopeptide changes identified with the control samples in multiplexes 2 and 3 are shown. (C) Venn diagrams indicating relative Env dependent changes at 5 and 60 minutes are shown below.
Fig 3.
Functional enrichment of protein subnetworks in HIV-1 producer cells after co-culture with uninfected cells.
Prize-Collecting Steiner Forest (PCSF) analysis was used to generate subnetworks from proteins with significant changes after 5 or 60 minutes of co-culture. The top 30 enriched Gene Ontology (GO) biological process terms from both 5 min and 60 min subnetworks were divided into those that primarily exhibited (A) early or (B) late enrichment after co-culture. Adjusted p-value indicates the Benjamini-Hochberg corrected p-value. (C) Heat map of the kinase activity score generated by PhosFate Profiler analysis [61], which infers kinase activity from changes in the phosphorylation states of known substrates. Selected cell cycle regulatory kinases and known HIV-1 effectors are shown. A positive score indicates increased phosphorylation while a negative score indicates decreased kinase-specific substrate phosphorylation in HIV-1 producer cells. (D) Selected regions of the protein-protein interaction subnetworks created using the PCSF algorithm from significantly differentiated proteins and phosphopeptides. The subnetworks depict all proteins in the same connected component as AURKB. The 5 min combined subnetwork shows edges with 75% or greater confidence, and the 60 min combined subnetwork shows edges with 90% or greater confidence. Vertex color of the elliptical vertices represents the magnitude of the log-transformed q-values, which were used as protein prizes. Steiner nodes, vertices that were not significantly changed between time points but were included as important connective proteins by the PCSF algorithm, are shown as rectangles. Proteins conserved between 5 and 60 min time points are outlined in red.
Fig 4.
Inhibition of cellular kinases alters cell to cell mediated viral spread.
(A) Schematic of TZM-bl assay. HIV expressing producer cells are pelleted and washed to remove extracellular viral like particles (VLP). The cells were resuspended in media containing kinase inhibitor and incubated for 2hr. A sample was removed and tested for effects of the chemical on producer cell viability. Treated cells were then mixed with TZM-bl reporter cells (HeLa cells that express the HIV CD4 receptor and co-receptors and have a TAT responsive promoter upstream of the firefly luciferase reporter), diluting the chemical 5-fold and incubated for 2 hours. The treated producer cells were then removed from the TZM-bl cells by washing and the cells were incubated for 48 hours to allow for infection and reporter gene expression. At 48 hours, cell viability and viral transfer were assayed. (B) The effects of kinase inhibitors targeting AURKB (Barasertib, 10 μM), LCK (LCK inhibitor, 100 μM), CDK1 (RO-3306, 20 μM) CDK9 (LDC000067, 10 μM), CDK13 (THZ531, 2 μM), DYRK1A (Harmine, 50 μM), ROCK1 (Y-27632, 50 μM) and WEE1 (MK1775, 20 μM) relative to untreated (UT) on cell viability (left panel) and TZM-bl cell activation (right panel) were assayed via a mitochondrial ATP assay (Cell titer-glo, Promega) and firefly luciferase (Bright-glo, Promega), respectively. (C) Dose response effects of inhibitors to selected kinases to cell-to-cell HIV spread and cell viability. (D) HIV expressing producer cells were pelleted and washed to remove extracellular VLPs. The cells were resuspended in media containing kinase inhibitor as described in (B) and incubated for 2hr. Treated cells were then pelleted and the VLP containing supernatants mixed with TZM-bl reporter cells, diluting the chemical 5-fold, and incubated for 48 hrs to allow for infection and reporter gene expression. (E) Western blot of HIV producer cells after 2-hour treatment with indicated inhibitors. Cells were mixed with target cells for two hours, then washed off the target cells, collected and processed for western blotting with the indicated antibodies. (A-D) The data shown are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three independent experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student’s t-test and significant changes relative to untreated are indicated.
Fig 5.
AURKB specifically regulates the fusion activity of the HIV envelope through the cytoplasmic tail domain of Env.
(A) The effects of kinase inhibitors targeting AURKB (Barasertib, 20 μM, and Hesperidin, 5 μM) and AURKA (TC-S 7010, 10 μM) was determined as described in Fig 4. (B) HIV-1 Jurkat producer cells treated with DMSO only, 20 μM barasertib and/or 500 nM AZT as indicated were mixed with TZM-bl cells as described in Fig 4 and then assayed for the TZM-bl-encoded firefly luciferase or the virally encoded GFP-NanoLuc (Promega) reporter gene. Barasertib treatment was conducted as described in Fig 4 and the associated figure legend. AZT was added with barasertib and maintained at 500 nM throughout the experiment. (C) HIV producer cells (Jurkat) with a fluorescent protein (CFP) fused to HIV-gag and WT HIV-1 Env were treated for 2h with DMSO or barasertib (20 μM) and mixed with target cells (SupT1) expressing fluorescent CD4 (CD4-YFP) for 20 minutes, fixed, mounted and imaged by confocal microscopy with a 60X objective. Inhibition of AURKB resulted in elongated cell-cell contacts and the frequent formation of syncytia. (D) The distribution of nuclei per cell after treatment with the indicated inhibitors and cell mixing was quantified and normalized as a percentage by counting a minimum of 120 cells from 3 fields from independent experiments are plotted. Mean and SEM are indicated. (E) Selected time frames from live cell images of AURKB inhibitor (Barasertib, 20 μM, 2 hr) -treated Jurkat cells transfected with HIV with a fluorescent protein (CFP) fused to HIV-gag and WT HIV-1 Env seeded on a monolayer of HeLa cells expressing CD4-YFP. (F) The distribution of nuclei per cell 12 hr after treatment and mixing was quantified and normalized as a percentage by counting a minimum of 120 cells from 3 fields from independent experiments. Mean and SEM are indicated in red. (G) Schematic of TZM-bl based syncytia/content mixing assay. Jurkat cells were co-transfected with a plasmid expressing the HIV-1 envelope protein and a second plasmid encoding the HIV transactivator TAT. Since no genome or other viral replication proteins are present, no virions can be formed. The cells were resuspended in media containing kinase inhibitor and incubated for 2hr. Treated cells were then mixed with TZM-bl reporter cells (HeLa cells that express the HIV CD4 receptor and co-receptors and have a TAT responsive promoter upstream of the firefly luciferase reporter). At 24 hours, cell viability and the activity of the TAT-responsive promoter were assayed. Since no virions were produced, the only activation of the TAT-responsive promoter can occur with cell-to-cell fusion and contact mixing. (H) Jurkat cells expressing TAT and WT NL43 Env were treated with an AURKB inhibitor (Hesperidin, 5 μM) as described in (G) and membrane fusion with TZM-bl cells was measured by TAT reporter gene activity. The data shown are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three independent experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student’s t-test and significant changes relative to DMSO treated controls are indicated.
Fig 6.
Interaction of WT HIV Env-expressing cells with CD4+ cells relocalizes AURKB to nuclear puncta.
(A) The effect of AURKB inhibition (Hesperidin, 5 μM) on viral spread was determined as described in Fig 4, except the CCR5 tropic envelope from HIV-1 strain SF162 or the envelope from amphotropic MLV was used instead of the envelope from the CXCR4 HIV-1 strain NL4-3. (B) The effect of AURKB inhibition (Barasertib, 20 μM) on viral spread was determined as described in Fig 4 with cells expressing the NL43 genome and either the WT envelope or a variant lacking the cytoplasmic tail domain (ΔCTD) derived from the envelope of CXCR4 HIV-1 strain NL4-3. (C) The firefly luciferase activities from the experiment in (B) are reported as raw, unprocessed relative light units (RLU) instead of normalized to 100%. (D) The effect of AURKB inhibition (Barasertib, 20 μM) on syncytia formation was determined as described in Fig 5G with cells expressing HIV-1 TAT and either the WT envelope or a variant lacking the cytoplasmic tail domain (ΔCTD) derived from the envelope of CXCR4 HIV-1 strain NL4-3. The data shown in (A-D) are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three independent experiments. (E) HIV producer cells (Jurkat) were transfected with a plasmid encoding mCherry-AURKB and plasmids containing an HIV genome encoding fluorescent protein (CFP) fused to HIV-gag, and a plasmid WT HIV envelope and were allowed to settle to a poly-D-lysine slide for 20 minutes, fixed, mounted and imaged by confocal microscopy with a 60X objective. (F) HIV producer cells (Jurkat) were transfected with a plasmid encoding mCherry-AURKB and plasmids containing an HIV genome encoding fluorescent protein (CFP) fused to HIV-gag, and plasmids encoding either amphotropic MLV or WT HIV envelope and mixed with target cells (SupT1) expressing fluorescent CD4 (CD4-YFP) for 20 minutes, fixed, mounted and imaged by confocal microscopy with a 60X objective.(G) Total AURKB localization at nuclear puncta by WT HIV-1 or MLV envelope after mixing with target cells. 10 individual fields were counted and transfected cells with AURKB puncta were counted. The data shown are the average mean values from 10 independent fields and are representative of three independent experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student’s t-test and significant changes relative to DMSO treated or no target cell controls are indicated.
Fig 7.
HIV Env CTD relocalizes AURKB adjacent to inner centromeres.
(A) Jurkat cells expressing a fluorescent AURKB, fluorescent γ-tubulin and full length or ΔCTD variants of a CXCR4 HIV envelope (NL43), a CCR5 tropic HIV envelope (SF162) or amphotropic MLV envelope were mixed for 20 minutes with HeLa cells expressing fluorescent CD4-CFP, fixed, mounted and imaged at 60X by confocal microscopy. (B) HIV producer cells (Jurkat) were co-transfected with plasmids containing an HIV genome expressing the HIV matrix fused to a fluorescent protein (iRFP670) and plasmids encoding WT env or a variant lacking the cytoplasmic tail domain (CTD) and plasmids encoding a fluorescent (GFP) AURKB fusion and plasmids encoding fluorescently tagged (mCherry) variants of the inner centromere protein CENPB. Transfected cells were mixed with cells expressing the HIV receptor and co-receptors (TZM-bl) or receptor negative (HeLa) cells for 20 minutes, fixed, mounted and imaged at 60X by confocal microscopy. Consistent with relocalization to the CPC, AURKB relocalized to puncta adjacent to CENPB puncta. This relocalization requires the Env CTD and receptor on target cells. (C) Quantitation of AURKB relocalization. HIV producer cells (Jurkat) were co-transfected with plasmids containing an HIV genome and plasmids encoding X4 tropic (NL43) or R5 tropic (SF162) WT env, a variant lacking the cytoplasmic tail domain (CTD), or amphotropic MLV envelope along with plasmids encoding fluorescent fusion proteins GFP-AURKB and mCherry-CENPB. Transfected cells were mixed with cells expressing the HIV receptor and co-receptors (TZM-bl) or receptor free cells (HeLa) cells for 20 minutes, fixed, mounted and imaged at 60X by confocal microscopy. 10 individual fields were counted and transfected cells with AURKB puncta were counted. The data shown are the average mean values from 10 independent fields and are representative of three independent experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student’s t-test and significant changes between CD4 expressing HeLa cells relative to CD4 negative HeLa are indicated.
Fig 8.
HIV Env CTD relocalizes AURKB and SGO1 to the chromosomal passenger complex (CPC) adjacent to inner centromeres.
HIV producer cells (Jurkat) were co-transfected with plasmids containing an HIV genome expressing the HIV gag fused to a fluorescent protein (iRFP670) and plasmids expressing WT env or a variant lacking the cytoplasmic tail domain (CTD) along with plasmids expressing the indicated fluorescent fusion proteins (A) GFP-AURKB and mCherry-INCENP fusion and (B) GFP-AURKB and mCherry-SGO1 or (C) GFP-AURKB and mCherry-AURKA. (D) GFP-AURKC and mCherry-CENPB, (E) GFP-AURKC and mCherry-INCENP, (F) GFP-AURKC and mCherry-SGO1 fusion. Transfected cells were mixed with cells expressing the HIV receptor CD4 and its co-receptors (TZM-bl cells) for 20 minutes, fixed, mounted and imaged at 60X by confocal microscopy. (G) Percentage of cells transfected with the indicated florescent protein showing that protein localized in nuclear puncta. The data shown are the average mean values from 10 independent fields and are representative of three independent experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student’s t-test and significant changes between CD4 expressing HeLa cells relative to CD4 negative HeLa are indicated. Consistent with relocalization to the CPC, AURKB, AURKC and SGO1 relocalized to puncta adjacent to CENPB puncta. This relocalization requires the Env CTD and receptor on target cells.
Fig 9.
Interaction with soluble CD4 HIV Env is sufficient to relocalize AURKB.
(A) Jurkat cells were co-transfected with plasmids encoding the indicated HIV envelope, GFP-AURKB and mCherry-CENBP. 24 hours post transfection, cells were incubated with purified IgG or soluble IgG-CD4 fusion protein for 20 minutes, fixed, mounted and imaged at 60X by confocal microscopy. Treatment with the soluble CD4 fusion protein caused HIV to relocalized to CENBP adjacent foci. (B) Schematic for quantitation of CD4 induced AURKB localization and cell cycle changes. Jurkat cells were co-transfected with plasmids encoding the indicated HIV envelope, along with mAzurite-Histone H2B to mark the transfected cells and a plasmid that encodes the 2-color PIP-Fucci system than uses a YFP-PIP protein to mark G1 cells, an mCherry-Geminin to mark S-phase cells. G2/M phase cells are dual positive. 24 hours post transfection, cells were incubated with purified IgG or soluble IgG-CD4 fusion protein for 20 minutes, fixed, stained for endogenous AURKB with a far-red secondary (alexafluor 647), mounted and imaged at 60X by confocal microscopy. 10 independent fields were counted, and transfected cells were scored for CD4 induced changes to the cell cycle stage with (C) WT or (D) ΔCTD envelope. (E) Total AURKB localization at nuclear puncta by WT or (F) ΔCTD envelope. The data shown are the average mean values obtained from three independent experiments with a minimum of 550 cells counted per condition between experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student’s t-test and significant changes of IgG treated control cells to soluble CD4 treated cells are indicated.
Fig 10.
Interaction with CD4+ T cells relocalizes AURKB to nuclear puncta in primary T cells expressing WT but not ΔCTD HIV Env.
(A) Primary human T cells were transfected with plasmids expressing mCherry-AURKB, an HIV genome, Blue fluorescent histone H2B, and either WT or ΔCTD HIV envelope. These cells then were mixed with target cells (SupT1) expressing fluorescent CD4 (CD4-YFP) for 20 minutes, fixed, mounted and imaged by confocal microscopy with a 60X objective. (B) Percentage of primary T-cells transfected with mCherry AURKB and WT HIV or ΔCTD HIV envelope as indicated showing AURKB localization at nuclear puncta after mixing with target cells. For each condition the data shown are the average mean values from 10 independent fields and are representative of three independent experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student’s t-test and significant changes relative to WT HIV Env expressing cells are indicated.
Fig 11.
AURKB regulation of HIV spread through virological synapse requires the C-terminal domain of Env.
Schematic of HIV-1 virological synapse. Cytoplasmic AURKB exerts an unknown negative effect on HIV-1 spread through the virological synapse which reduces the fusion activity of the HIV-1 Env protein. HIV-1 overcomes this through the CTD of HIV-1 Env which induces premature nuclear localization of AURKB to the CPC.