human t lymphoblast cell line supt1  (ATCC)

Journal: PLoS ONE

Article Title: HIV-1 Vpr Triggers Mitochondrial Destruction by Impairing Mfn2-Mediated ER-Mitochondria Interaction

doi: 10.1371/journal.pone.0033657

Figure Lengend Snippet: A , HEK293 cells were transfected with GFP vector the plasmid encoding Vpr-GFP or Vpr 52–96- GFP, and harvested at different time (hours) post-transfection for PI staining. The percentage of dead cells among GFP-expressing cells was determined by flow cytometry. *** ( p <0.001) indicates significantly different from the GFP vector control. B , HEK293 cells were infected with lenti-vector (control) or Lenti-Vpr and harvested at different time (hours) post-infection for PI staining. The percentage of dead cells was determined by flow cytometry. *** ( p <0.001) indicates significantly different from the control. C, HEK293 and SupT1 cells were grown in 10% FBS or starved for 24 hours, and infected with Vpr-expressing lentivirus for 48 and 72 hours. The expression of Mfn2 was decreased in serum-starved HEK293 or SupT1 cells. The quantitative expression of Mfn2 was measured by Image J and normalized with the expression of β-actin. C indicates Vpr negative lentiviral control. D, MMP loss was determined after Vpr-expressing lentivirus infection. Vpr significantly impaired MMP in serum-starved HEK293 and SupT1 cells. E, Vpr expression led to cell death in serum-starved HEK293 and SupT1 cells. For panels D and E , results are the means ± S.D. of three independent experiments. * ( p <0.05), ** ( p <0.01) and ** ( p <0.001) indicate significantly higher than the Vpr negative lentiviral control (Con.). † (p<0.05) and †† ( p <0.01) indicate significantly different between 10% FBS and serum starvation. F, Human primary CD4 + T cells were isolated from peripheral blood mononuclear cell (PBMC) and infected with Vpr-expressing lentivirus for 72 hours. The expression of Mfn2 was decreased and the expression of nuclear DRP1 was increased in human primary CD4 + cells. The relative expression levels of Mfn2 and DRP1 were measured by Image J and normalized with the expression of β-actin. G, MMP loss was determined after Vpr-expressing lentivirus infection. Vpr led to a significant MMP loss in human primary CD4 + T cells. H, Vpr expression led to cell death in human primary CD4 + T cells. For panels G and H , results are the means ± S.D. of three independent experiments. ** ( p <0.01) and *** ( p <0.001) indicate significantly higher than control human primary CD4 + T cells. Band intensities were calculated using Image J. Relative intensities are shown at the bottom of each panel.

Article Snippet: Human CD4 + T lymphoblast cell line SupT1 was obtained from ATCC and grown in RPMI1640 medium supplemented with 10% FBS, 4 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Transfection, Plasmid Preparation, Staining, Expressing, Flow Cytometry, Infection, Isolation

Journal: PLoS ONE

Article Title: HIV-1 Vpr Triggers Mitochondrial Destruction by Impairing Mfn2-Mediated ER-Mitochondria Interaction

doi: 10.1371/journal.pone.0033657

Figure Lengend Snippet: After transient transfection of Mfn2, or DRP1 expression plasmid for 24 hours, cells were infected with Vpr-expressing lentivirus for 72 hours. A, In contrast to Lenti-Vpr negative (-) HEK293 cells (medium control and vector control), the 89 kDa cleavage fragment of PARP appeared exclusively in Lenti-Vpr positive (+) HEK293 cells. PARP cleavage was reduced in Mfn2-, DRP1- and Mfn2/DRP1-overexpressed Lenti-Vpr (+) HEK293 cells. B, PARP cleavage occurred only in Lenti-Vpr (+) SupT1 cells. The cleavage of PARP was reduced in Mfn2-, DRP1- and Mfn2/DRP1-expressed Lenti-Vpr (+) SupT1 cells. C, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 decreased the percentage of MMP loss in Lenti-Vpr (+) HEK293 and SupT1 cells. D, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 reduced Vpr-induced apoptosis. are the means ± S.D. of three independent experiments. For panels C and D , † (p<0.05) and †† ( p <0.01) indicate significantly lower than Lenti-Vpr (+) cells pretreated with mock (medium control) or vector transfection.

Article Snippet: Human CD4 + T lymphoblast cell line SupT1 was obtained from ATCC and grown in RPMI1640 medium supplemented with 10% FBS, 4 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Transfection, Expressing, Plasmid Preparation, Infection, Over Expression

Journal: mBio

Article Title: Analysis of CA Content and CPSF6 Dependence of Early HIV-1 Replication Complexes in SupT1-R5 Cells

doi: 10.1128/mBio.02501-19

Figure Lengend Snippet: Kinetics of productive HIV-1 entry into SupT1-R5 cells. (A and B) Cells were incubated with HIV-1 NL4-3 for 90 min at 16°C. After adsorption, inoculum was changed for fresh medium and cells were shifted to 37°C to initiate virus entry. At the indicated time points after temperature shift, T-20 (A) or PF74, EFV, or Ral (B) was added. Infection was scored at 48 h p.i. by immunostaining for intracellular HIV-1 CA protein followed by flow cytometry. Noninfected cells, cells infected with equal amounts of HIV-1 NL4-3 lacking Env glycoprotein [Env(−)], and untreated cells were used as controls. Bars indicate mean values and standard deviations (SD) from one representative experiment performed in triplicate. (C) Quantitation of reverse transcription (RT) products using ddPCR. Cells were infected with HIV-1 NL4-3 or non-replication-competent HIV-1 CHIV particles and harvested at the indicated time points, and HIV-1 RT products were quantitated in cell lysates by ddPCR. Copy numbers of late RT products (C, left) or 2-LTR (C, right) circles were normalized to the copy numbers of the housekeeping gene RPP30. Graphs show mean values and SD from triplicates.

Article Snippet: The human T lymphoblast cell line SupT1-R5 (stably expressing exogenous CCR5 under puromycin selection; a kind gift from R. Doms, University of PA, USA; certified by Eurofins according to DAkkS ISO 9001:2008) was cultivated at 37°C in a humidified incubator with a 5% CO 2 atmosphere, using RPMI 1640 medium with GlutaMAX (ThermoFisher) supplemented with 10% fetal bovine serum (FBS; Merck), 50 U/ml of penicillin, 50 μg/ml of streptomycin (ThermoFisher), and 0.3 μg/ml puromycin (Merck).

Techniques: Incubation, Adsorption, Infection, Immunostaining, Flow Cytometry, Quantitation Assay

Journal: mBio

Article Title: Analysis of CA Content and CPSF6 Dependence of Early HIV-1 Replication Complexes in SupT1-R5 Cells

doi: 10.1128/mBio.02501-19

Figure Lengend Snippet: Effect of mCLING labeling on HIV-1 entry efficiency. (A and B) SupT1-R5 cells were incubated with HIV-1 NL4-3 Vpr.BlaM (0.2 μU of RT/cell) (A) or HIV-1 NL4-3 (2.8 μU of RT/cell) (B) for 120 min at 16°C. After adsorption, mCLING.Atto647N was added at the indicated concentration, and cells were incubated for an additional 10 min at 16°C and subsequently shifted to 37°C to initiate virus entry. (A) After 2 h at 37°C, cells were washed and processed to determine cytosolic entry efficiency using the BlaM assay. (B) Alternatively, T-20 was added after 2 h at 37°C, and cells were further incubated at 37°C and fixed 48 h after the shift to score infectivity. Noninfected cells and cells infected in the absence of the probe were used as controls. (C) Test for unspecific HIV-1 cytosolic entry via mCLING-mediated membrane fusion. SupT1-R5 cells were incubated with HIV-1 NL4-3 lacking or carrying Env (both at 2.8 μU RT/cell) for 90 min at 16°C. Cells were then stained with 2 μM mCLING.Atto647N for 10 min at 16°C and shifted to 37°C. T-20 was added after 2 h, and incubation was continued for 46 h before scoring infected cells by flow cytometry. (D) Contribution of endocytosis to HIV-1 infection in the presence of mCLING. SupT1-R5 cells were preincubated for 10 min at 22°C in the absence or presence of mCLING.Atto647N (2 μM). Subsequently, HIV-1 NL4-3 (6.9 μU of RT/cell) was added and cells were incubated for 4 h at 22°C to allow for endocytosis, but not fusion, prior to addition of T-20 to inhibit any fusion occurring from the plasma membrane. In parallel, infected cells were incubated for 4 h at 37°C as virus entry control. Cells were incubated at 22°C for one more hour and subsequently shifted to 37°C. To determine infection efficiency, cells were fixed at 48 h p.i., immunostained for intracellular CA, and scored using flow cytometry. The graph shows mean values and SD from one representative experiment performed in triplicate.

Article Snippet: The human T lymphoblast cell line SupT1-R5 (stably expressing exogenous CCR5 under puromycin selection; a kind gift from R. Doms, University of PA, USA; certified by Eurofins according to DAkkS ISO 9001:2008) was cultivated at 37°C in a humidified incubator with a 5% CO 2 atmosphere, using RPMI 1640 medium with GlutaMAX (ThermoFisher) supplemented with 10% fetal bovine serum (FBS; Merck), 50 U/ml of penicillin, 50 μg/ml of streptomycin (ThermoFisher), and 0.3 μg/ml puromycin (Merck).

Techniques: Labeling, Incubation, Adsorption, Concentration Assay, Infection, Staining, Flow Cytometry

Journal: mBio

Article Title: Analysis of CA Content and CPSF6 Dependence of Early HIV-1 Replication Complexes in SupT1-R5 Cells

doi: 10.1128/mBio.02501-19

Figure Lengend Snippet: Approach to identify HIV-1 postfusion complexes. (A) Application of mCLING to distinguish postfusion HIV-1 complexes from complete virions at the plasma membrane (PM) or within endosomes. PM and endosomes are stained by mCLING, whereas membrane-less viral complexes lack the mCLING signal. PM, plasma membrane; CC, clathrin-coated pit; ER, endoplasmic reticulum; NE, nuclear envelope; NPC, nuclear pore complex. (B to D) SupT1-R5 cells were incubated with IN.eGFP-carrying HIV-1 CHIV particles (0.5 μU of RT/cell) for 90 min at 16°C. Cells were then stained with mCLING.Atto647N for an additional 10 min at 16°C and shifted to 37°C to initiate virus entry. Cells were fixed with 4% FA–0.2% GA at 30 min (D) or 2 h (B and C) after temperature shift. IN.eGFP signals (green) were readily detected in association with mCLING-labeled (red) endosomes (B, I) or plasma membrane (B, II) and also inside the cytoplasm of cells without colocalization with mCLING (C and D). In panels B and C, STED microscopy was used to visualize the mCLING signal. White arrowheads point to the position of the selected IN.eGFP signals.

Article Snippet: The human T lymphoblast cell line SupT1-R5 (stably expressing exogenous CCR5 under puromycin selection; a kind gift from R. Doms, University of PA, USA; certified by Eurofins according to DAkkS ISO 9001:2008) was cultivated at 37°C in a humidified incubator with a 5% CO 2 atmosphere, using RPMI 1640 medium with GlutaMAX (ThermoFisher) supplemented with 10% fetal bovine serum (FBS; Merck), 50 U/ml of penicillin, 50 μg/ml of streptomycin (ThermoFisher), and 0.3 μg/ml puromycin (Merck).

Techniques: Staining, Incubation, Labeling, Microscopy

Journal: mBio

Article Title: Analysis of CA Content and CPSF6 Dependence of Early HIV-1 Replication Complexes in SupT1-R5 Cells

doi: 10.1128/mBio.02501-19

Figure Lengend Snippet: Validation of mCLING-based detection of HIV-1 postfusion complexes. (A) Experimental workflow. SupT1-R5 cells were infected with IN.eGFP-labeled HIV-1 particles in the presence of mCLING. Z-stacks were acquired using confocal microscopy and merged to cover a section of 8 μm through the cell body. 3D volumes were reconstructed from these image series and analyzed using the Imaris spot detection function that creates a 3D ellipsoid object (lower right, green) for each recognized IN.eGFP signal (upper right). Objects were classified as intracellular by manual assignment based on the 3D data. Intracellular objects displaying an mCLING signal intensity below the threshold (lower right, violet; see Materials and Methods) were classified as postfusion HIV-1 complexes. See also in the supplemental material. (B) Cells were incubated with IN.eGFP-labeled HIV-1 CHIV particles pseudotyped with wild-type Env glycoprotein (Env NL4-3 ) or with the fusion-incompetent Env mutant (EnvFusV2E) (both at 1.6 μU RT/cell) for 90 min at 16°C. After adsorption, cells were stained with mCLING.Atto647N (2 μM) for 10 min at 16°C and then shifted to 37°C for 90 min. Cells were transferred to 8-well chamber slides, fixed with 4% FA–0.2% GA, and analyzed as outlined for panel A. (C) Cells were incubated with IN.eGFP-carrying HIV-1 CHIV particles (0.6 μU RT/cell) for 90 min at 16°C. Incubation was continued in the absence or presence of T-20 for an additional 30 min at 16°C prior to shift to 37°C for 90 min. Cells were processed and fixed as described for panel B. (B and C) Top panels show 3D reconstructions of representative cells with IN.eGFP signals either colocalizing (green dots) or not colocalizing (violet dots) with mCLING. Bottom panels show selected IN.eGFP signals in corresponding confocal slices with corresponding orthogonal views. Arrowheads and arrows point to selected intracellular or extracellular mCLING-negative IN.eGFP signals, respectively. Results from quantitation are summarized in the tables on the right.

Article Snippet: The human T lymphoblast cell line SupT1-R5 (stably expressing exogenous CCR5 under puromycin selection; a kind gift from R. Doms, University of PA, USA; certified by Eurofins according to DAkkS ISO 9001:2008) was cultivated at 37°C in a humidified incubator with a 5% CO 2 atmosphere, using RPMI 1640 medium with GlutaMAX (ThermoFisher) supplemented with 10% fetal bovine serum (FBS; Merck), 50 U/ml of penicillin, 50 μg/ml of streptomycin (ThermoFisher), and 0.3 μg/ml puromycin (Merck).

Techniques: Infection, Labeling, Confocal Microscopy, Incubation, Mutagenesis, Adsorption, Staining, Quantitation Assay

Journal: mBio

Article Title: Analysis of CA Content and CPSF6 Dependence of Early HIV-1 Replication Complexes in SupT1-R5 Cells

doi: 10.1128/mBio.02501-19

Figure Lengend Snippet: CA content of subviral HIV-1 complexes drops by 50% after fusion and appears to be lost upon nucleoplasmic entry. (A and B) SupT1-R5 cells were incubated with IN.eGFP-carrying HIV-1 NL4-3 virions (2 μU of RT/cell) for 90 min at 16°C. To visualize postfusion HIV-1 complexes, cells were stained with mCLING.Atto647N for 10 min at 16°C. Subsequently, the inoculum was changed for medium with (A) or without (B) mCLING, and cells were transferred to PEI-coated 8-well chamber slides, shifted to 37°C for the indicated time, and fixed with 4% FA–0.2% GA (A) or 4% FA (B). See schematic illustrations of the experiment above fluorescent panels. Samples were immunostained for HIV-1 CA (A and B) and NPC (B). DNA was stained with Hoechst. Confocal sections of cells with enlarged details are shown. In panel A, the arrowhead indicates an mCLING-negative HIV-1 complex inside the cytosol, and arrows indicate HIV-1 virions at the plasma membrane. In panel B, the arrowhead indicates a viral complex at the NPC and arrows point to IN.eGFP-positive complexes in the nucleus. Scale bars, 5 μm. (A and B) The graphs on the right show distribution and intensities of CA signals detected at the indicated time points (TP) for virions at the plasma membrane (PM), subviral complexes in the cytosol (A) or in association with the NPC (B), and for IN.eGFP-labeled complexes inside the nucleus (NU). 3D reconstructions based on z-stacks of cells were analyzed using the Imaris spot detection function applied on the CA and IN.eGFP signals. The median of CA signal intensity in the detected objects was then measured. For intranuclear complexes, only IN.eGFP spots were analyzed. At least 40 cells were analyzed per TP. CA intensities were lower in mCLING-stained samples (A, right), possibly due to a negative effect of the required treatment with 0.2% glutaraldehyde on antigenicity. a.u., arbitrary units; N/A, not applicable. (C) Detection of CA on nuclear HIV-1 complexes after copper-based extraction of infected cells. MDM or SupT1-R5 cells were infected with R5-tropic, IN.eGFP-carrying HIV-1 NL4-3 (6.7 × 10 5 μU of RT/well) at 37°C for 48 h or 5.5 h, respectively. Samples were fixed with 4% FA in PBS, permeabilized with 0.5% Triton X-100, and then extracted using the Click-IT reaction mixture usually employed for copper-catalyzed EdU click labeling. Cells were immunostained for HIV-1 CA and lamin A/C (MDM) (left) or Nup358 (SupT1-R5) (right). Z-sections through the nuclear regions of representative cells are shown. Arrowheads indicate IN.eGFP-positive viral complexes inside the nucleus.

Article Snippet: The human T lymphoblast cell line SupT1-R5 (stably expressing exogenous CCR5 under puromycin selection; a kind gift from R. Doms, University of PA, USA; certified by Eurofins according to DAkkS ISO 9001:2008) was cultivated at 37°C in a humidified incubator with a 5% CO 2 atmosphere, using RPMI 1640 medium with GlutaMAX (ThermoFisher) supplemented with 10% fetal bovine serum (FBS; Merck), 50 U/ml of penicillin, 50 μg/ml of streptomycin (ThermoFisher), and 0.3 μg/ml puromycin (Merck).

Techniques: Incubation, Staining, Labeling, Infection

Journal: mBio

Article Title: Analysis of CA Content and CPSF6 Dependence of Early HIV-1 Replication Complexes in SupT1-R5 Cells

doi: 10.1128/mBio.02501-19

Figure Lengend Snippet: CPSF6 binding-defective mutant A77V accumulates at the NPC without major loss of infectivity. (A) SupT1-R5 cells were infected with the indicated amounts of HIV-1 NL4-3 wild type (WT) or A77V. At 48 h p.i., cells were immunostained for intracellular HIV-1 CA and infection was scored by flow cytometry. The graph shows mean values and SD from three independent experiments performed in triplicates. (B) SupT1-R5 cells were incubated with IN.eGFP-carrying HIV-1 NL4-3 WT or A77V virions (2 μU of RT/cell) for 90 min at 16°C. After adsorption, inoculum was removed and cells were shifted to 37°C to initiate virus entry. Cells were fixed 8 h after temperature shift and immunostained for HIV-1 CA and NPC. DNA was stained with Hoechst. Deconvolved confocal sections through the nuclear region of representative cells infected with WT or A77V HIV-1 are shown. Arrowheads in enlargements indicate CA + and IN.eGFP + complexes associated with NPC (filled arrowheads) and IN-eGFP + complexes inside the nucleus (open arrowheads). Scale bars, 5 μm. See Fig. S5 for enlarged regions 1 to 9 separated by fluorescence channels. (C) Numbers of IN.eGFP + complexes in nuclei of HIV-1 WT- or A77V-infected cells. Mean values and standard errors of the means (SEM) for n cells from at least 8 randomly chosen fields of view are shown. (D) Distribution and intensities of CA signals detected 8 h after the temperature shift for WT or A77V extracellular virions (adhered to the 8-well chamber slides) or subviral complexes associated with the NPC. Z-stacks of cells were analyzed using the Imaris spot detector applied to the IN.eGFP signals, and the median of CA signal intensity of these objects was determined as described in Materials and Methods. Objects in more than 10 cells per condition were analyzed. Mean intensity values are indicated by black lines. Statistical significance was assessed by a nonpaired two-tailed Student's t test. *, P < 0.05; ***, P < 0.0001; n.s., not significant.

Article Snippet: The human T lymphoblast cell line SupT1-R5 (stably expressing exogenous CCR5 under puromycin selection; a kind gift from R. Doms, University of PA, USA; certified by Eurofins according to DAkkS ISO 9001:2008) was cultivated at 37°C in a humidified incubator with a 5% CO 2 atmosphere, using RPMI 1640 medium with GlutaMAX (ThermoFisher) supplemented with 10% fetal bovine serum (FBS; Merck), 50 U/ml of penicillin, 50 μg/ml of streptomycin (ThermoFisher), and 0.3 μg/ml puromycin (Merck).

Techniques: Binding Assay, Mutagenesis, Infection, Flow Cytometry, Incubation, Adsorption, Staining, Fluorescence, Two Tailed Test

Journal: mBio

Article Title: Analysis of CA Content and CPSF6 Dependence of Early HIV-1 Replication Complexes in SupT1-R5 Cells

doi: 10.1128/mBio.02501-19

Figure Lengend Snippet: Visualization of HIV-1 wild-type and A77V complexes at the NPC using STED nanoscopy. SupT1-R5 cells were infected with HIV-1 NL4-3 wild-type (WT) (A) or A77V (B) virions (2 μU of RT/cell) for 8 h at 37°C and immunostained for HIV-1 CA protein (magenta) and NPC (FG repeats, green). DNA was stained with Hoechst (blue). (A and B) Confocal images (upper) and STED images (lower) of the nucleus of a representative infected cell. Arrowheads indicate CA-positive objects colocalizing with NPC proteins. Enlargements of the boxed regions are shown below. Scale bars, 2 μm (confocal images), 500 nm (STED images), and 100 nm (enlarged regions). (C) Averaged line profiles from panel B of selected CA-positive objects ( n = 30). Error bars represent SD.

Article Snippet: The human T lymphoblast cell line SupT1-R5 (stably expressing exogenous CCR5 under puromycin selection; a kind gift from R. Doms, University of PA, USA; certified by Eurofins according to DAkkS ISO 9001:2008) was cultivated at 37°C in a humidified incubator with a 5% CO 2 atmosphere, using RPMI 1640 medium with GlutaMAX (ThermoFisher) supplemented with 10% fetal bovine serum (FBS; Merck), 50 U/ml of penicillin, 50 μg/ml of streptomycin (ThermoFisher), and 0.3 μg/ml puromycin (Merck).

Techniques: Infection, Staining