Journal: Cellular and Molecular Life Sciences

Article Title: Glucosylceramide in bunyavirus particles is essential for virus binding to host cells

doi: 10.1007/s00018-023-05103-0

Figure Lengend Snippet: Glucosylceramide (GlcCer) in viral particles promotes Uukuniemi virus (UUKV) binding. A UUKV particles derived from BHK-21 cells in the presence of DL- threo -phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, 2.5 µM) were bound to freshly seeded naïve BHK-21 cells for 2 h on ice before fixation and western blot analysis with an antibody recognizing the UUKV N protein. B N was semi-quantified from the cells described in A , and the value is presented as a percentage of the N level measured in the sample corresponding to virus binding in the absence of PPMP ( n = 8). C Alternatively, UUKV particles produced in the presence or the absence of PPMP were allowed to bind to BHK-21 cells for 2 h on ice, and binding was assessed by measuring the BHK-21 cell-associated S viral segment by RT-qPCR ( n = 6). D Fluorescently labeled UUKV particles (UUKV-ATTO488) were bound to BHK-21 cells [multiplicity of infection (MOI) ~ 4] on ice for 1 h, and viral binding was evaluated by flow cytometry analysis. E and F BHK-21 cells, A549 human lung epithelial cells, and BHK-21 cells expressing the UUKV receptor DC-SIGN (BHK-21 DC-SIGN +) were preincubated with varying amounts of soluble C6-GlcCer E or C6-Cer F for 2 h and then exposed to UUKV-ATTO488 (MOI ~ 4) on ice for 1 h. Virus binding was measured by flow cytometry, and the data were normalized to those in control samples processed in the absence of soluble C6-GlcCer or C6-Cer. An one-way ANOVA with Dunnett’s multiple comparison test was applied ( n ≥ 3). *, p < 0.05; **, p < 0.01; ns, not significant; RFI, relative fluorescence intensity. G BHK-21 cells were transduced with a retroviral vector system to express DC-SIGN (BHK-21 DC-SIGN +). DC-SIGN expression was measured by flow cytometry analysis using phycoerythrin-conjugated anti-DC-SIGN mAb. H Soluble C6-GlcCer was allowed to bind BHK-21 cells on ice for 2 h before exposure to UUKV for 1 h (MOI ~ 0.5). After virus binding on ice, unbound UUKV particles were washed away, and the cells were incubated at 37 ℃ for 8 h. Infection was quantified by flow cytometry after immunostaining for UUKV N protein. Values are presented as the percentage of the control sample without prebinding of soluble C6-GlcCer ( n = 4)

Article Snippet: The location of DC-SIGN was assessed at the surface of BHK-21 cells (not permeabilized) by flow cytometry using an anti-DC-SIGN phycoerythrin-conjugated antibody (FAB1621P R&D Systems) according to a standard procedure [ ].

Techniques: Virus, Binding Assay, Derivative Assay, Western Blot, Produced, Quantitative RT-PCR, Labeling, Infection, Flow Cytometry, Expressing, Control, Comparison, Fluorescence, Transduction, Retroviral, Plasmid Preparation, Incubation, Immunostaining

Journal: Cells

Article Title: SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Promote a Proinflammatory Activation Profile on Human Dendritic Cells

doi: 10.3390/cells10123279

Figure Lengend Snippet: Dendritic cell activation in response to Spike protein and the RBD is DC-SIGN independent. iDCs were preincubated with an anti-DC-SIGN antibody, stimulated as indicated for 24 h, and then stained and analyzed by flow cytometry. ( A ) CD83 and ( B ) CD86 expression in DCs with the indicated treatment are shown. Graphs show percentage of positivity (left) and GMFI (right) of each marker. Graphs show mean ± SEM; n = 5 different donors.

Article Snippet: Blocking assay was performed incubating iDCs (1 × 10 6 /mL) in complete medium with anti-DC-SIGN antibody (40 μg/mL) (Cat# MAB16211, RD Systems) at 37 °C, for 30 min. After two washes, cells were stimulated as indicated during 24 h and then analyzed.

Techniques: Activation Assay, Staining, Flow Cytometry, Expressing, Marker

Journal: mSphere

Article Title: Term Human Placental Trophoblasts Express SARS-CoV-2 Entry Factors ACE2, TMPRSS2, and Furin

doi: 10.1128/mSphere.00250-21

Figure Lengend Snippet: The ACE2-dependent entry of SARS-CoV-2 pseudovirus into cultured PHT cells. (A) PHT (five donors) and 293T-ACE2 cells inoculated with SARS-CoV-2 protein pseudotyped lentiviruses containing SMNE proteins (spike, membrane, nucleocapsid, and envelope) or MNE proteins, as detailed in Materials and Methods. Cells, harvested on day 2 postinoculation with the virus, were washed three times with PBS and trypsinized to remove adherent virus, and the cell pellet was lysed to release intracellular p24. Shown is p24 content (in picograms per milliliter) in cell lysates. *, P < 0.01 (paired t test). (Inset) 293T cells expressing ACE2, assessed at 2 days or 5 days ( n = 2 for each) and serving as a positive control. (B) PHT were preincubated or not with 20 μg/ml of anti-ACE-2 or anti-DC-SIGN antibody (Ab) and then inoculated with pvSARS-CoV-2 S+. The PHT cells were also inoculated with pseudovirus lacking S protein (pvS-) or with inactivated HIV-1 as negative controls. Cells were harvested on day 2 postinoculation, and p24 content (in picograms per milliliter) was measured in cell lysates, as detailed in Materials and Methods. *, P < 0.05 (ANOVA and Tukey test, n = 5).

Article Snippet: For antibody inhibition experiments, PHT cells were preexposed to 20 μg/ml anti-ACE2 (catalog no. AF933; R&D Systems, Minneapolis, MN ) or anti-DC-SIGN (R&D catalog no. MAB161) for 30 min at 37°C before inoculation with the viruses ( , ).

Techniques: Cell Culture, Expressing, Positive Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Dendritic Cell Maturation Regulates TSPAN7 Function in HIV-1 Transfer to CD4 + T Lymphocytes

doi: 10.3389/fcimb.2020.00070

Figure Lengend Snippet: Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4 + T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4 + T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living cells, CD3 + T cells (top and middle panel), or DC-SIGN + MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A) . (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH , 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7 . NS, not significant. **** p < 0.0001.

Article Snippet: For flow cytometry analysis, the staining was performed with anti-CD3 AF700 (for T cells) (eBioscience; 56-0038-82), anti–DC-SIGN PE (for MDDCs) (Clone, 120507; R&D systems, catalog number: FAB161P), anti-CD169 APC (for CD169/Siglec-1; Biolegend; 346007), and DAPI for live/dead gating and in some cases anti-P24 PE (Beckman Coulter, 6604667).

Techniques: Expressing, Flow Cytometry, Infection, Co-Culture Assay, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Dendritic Cell Maturation Regulates TSPAN7 Function in HIV-1 Transfer to CD4 + T Lymphocytes

doi: 10.3389/fcimb.2020.00070

Figure Lengend Snippet: CD169, as an HIV-1 receptor, mostly impacts transfer from mature MDDCs rather than immature MDDCs. (A) Flow cytometry plots showing CD86, DC-SIGN, and CD169 expression levels on MDDCs (pre-gated on SSC FSC, living cells, CD3 − cells and singlets). Panels show the expression of these proteins in iMDDCs (left panel) and MDDCs with LPS pretreatment at 100 ng/ml for 48 or 24 h before co-culture (middle and right panels, respectively). (B) Percentage of variation of HIV-1 transfer when using iMDDCs or LPS-treated MDDCs (100 ng/ml LPS for different lengths of time) incubated with a blocking antibody against CD169 as compared to an isotype control for each condition. Results are displayed for 4 different blood donors with the mean ± SD of technical triplicates. (C) Percent of variation in HIV-1 transfer to assess the impact of blocking CD169 and TSPAN7 knockdown as compared to scramble shRNA on MDDCs matured with LPS for 48 h treated by an isotype control. Mean ± SD of seven different blood donors in 4 experiments. (B,C) NS, not significant. ** p < 0.01; *** p < 0.001. (D) Confocal microscopy images of iMDDCs (left panel) and mature MDDCs (mMDDCs) right panel, to assess the degree of colocalization between CD169 (magenta) and incoming X4–HIV-1–Gag-iGFP (green). Actin filaments and nuclei were stained with phalloidin (red) and DAPI (blue). Four hundred nanometers of Z-stacks were taken 40 h after the start of the co-culture with CD4 + T cells and X4–HIV-1–Gag-iGFP. The pictures presented here are from a representative donor from four unrelated blood donors.

Article Snippet: For flow cytometry analysis, the staining was performed with anti-CD3 AF700 (for T cells) (eBioscience; 56-0038-82), anti–DC-SIGN PE (for MDDCs) (Clone, 120507; R&D systems, catalog number: FAB161P), anti-CD169 APC (for CD169/Siglec-1; Biolegend; 346007), and DAPI for live/dead gating and in some cases anti-P24 PE (Beckman Coulter, 6604667).

Techniques: Flow Cytometry, Expressing, Co-Culture Assay, Incubation, Blocking Assay, shRNA, Confocal Microscopy, Staining