Journal: EMBO Reports

Article Title: Tissue‐like environments shape functional interactions of HIV ‐1 with immature dendritic cells

doi: 10.15252/embr.202356818

Figure Lengend Snippet: Representative spinning disc confocal microscopy images of HIV‐1 NLENG R5 Vpr‐mRuby2 particles stained with anti‐HIV‐1 p24 antibody. Vpr‐mRuby2 is shown in red, while p24 is shown in green. Scale bar = 5 μm. Relative infectivity of NLENG‐1 R5 particles in the presence or absence of Vpr‐mRuby2 produced in 293T cells. Particle infectivity was measured by infecting TZM‐bl reporter cells. The number of blue cells present was normalized to the RT activity of the respective virus stock determined by SG‐PERT assay. Bars represent mean ± SD of four technical replicates. Representative spinning disc microscopy images of iDCs cultured in 2D suspension (top) or 3D collagen (bottom) at 48 h. p.i. GAPDH staining is shown in magenta, Vpr‐mRuby particles are shown in red, and GFP expression in productively infected iDCs is shown in green. Scale bar = 20 μm. Imaris quantification workflow. The first left panels show an example of rendered particles created with the spot finder wizard using the signal from mRuby2. The second panels show an example of segmented cells (in green, purple, and violet) obtained through the cell finder wizard using the staining of cytoplasmic GAPDH. Phalloidin‐atto 390 signal, staining F‐actin, was used to find cell surface (represented in glass transparent color) and, finally, distance of each particle with respect to the cell cytoplasm, and surface was calculated. On the top, raw data are shown, while on the bottom, the rendering is shown. The last panel shows an example of cytoplasmic, surface‐associated, and extracellular particles in blue, yellow, and red, respectively. Scale bar = 4 μm. Subcellular localization of Vpr‐mRuby2 particles in iDCs cultured in 2D suspension (left) or 3D collagen (right) for a representative donor, calculated following the workflow described in (D). For each time point, two to four pictures were analyzed and the graphs show mean values ± SD. Source data are available online for this figure.

Article Snippet: The plasmid pmRuby2.Vpr, encoding Vpr fused to mRuby2, was generated by excision of the eGFP coding region of peGFP.Vpr (McDonald et al , , a kind gift of Tom Hope) using AgeI and BsrGI restriction enzymes, followed by insertion of a PCR fragment encoding mRuby2 (Lam et al , ); amplified from pcDNA3‐mRuby2, a gift from Michael Lin (Addgene plasmid # 40260) and flanked by the same restriction sites.

Techniques: Confocal Microscopy, Staining, Infection, Produced, Activity Assay, Virus, Microscopy, Cell Culture, Suspension, Expressing

Journal: EMBO Reports

Article Title: Tissue‐like environments shape functional interactions of HIV ‐1 with immature dendritic cells

doi: 10.15252/embr.202356818

Figure Lengend Snippet: A Experimental workflow. iDC differentiated from peripheral blood monocytes for 5 days were spin transduced with VLPs carrying Vpxmac239. One day later, iDCs were infected with HIV‐1 NLENG‐1 R5‐harboring Vpr‐mRuby2 or HIV‐1 R5‐containing Vpr‐Blam and cultured in 2D suspension or cultured in 3D collagen. At the indicated time points, cells were either fixed and imaged with the spinning disc confocal microscope or used for Blam fusion assays. B Representative spinning disc confocal microscopy images of iDC cultured in 2D suspension (left) or 3D collagen (right) at 4 h.p.i. Cells were stained with Phalloidin‐Atto390 to visualize F‐actin. Vpr‐mRuby2 particles are shown in red. Scale bar = 20 μm. Arrowheads highlight virus particles in 3D collagen. The dotted lines indicate which areas are shown in higher magnification. Scale bars = 2 μm. C Vpr signal per cell. Graphs depict means values ± SD for cells from four donors. *** P < 0.001 calculated with two‐way ANOVA test. D–G Cells were infected with HIV‐1 R5 Vpr‐Blam for 4 or 48 h and then incubated with CCF2 for 5 h at 11°C to prevent particle fusion during the staining process. For the indicated sample, T20 was added at the moment of infection. (D) Representative dot plots of FACS analysis to quantify the formation of the CCF2 product generated by B‐lactamase enzymatic cleavage at 4 h.p.i. The fluorescence intensity of the CCF2 substrate is plotted against the fluorescence intensity of the CCF2 product. Gates show the percentage of CCF2 product‐positive cells. (E) Percentage of positive cells for the presence of the CFF2 product at 4 h.p.i. ** P < 0.01 calculated with two‐way ANOVA test. (F, G) Percentage of positive cells for the presence of the CFF2 product at 4 h.p.i. (left) and 48 h.p.i. (right). * P < 0.05 calculated with two‐way ANOVA test. The graphs show mean values ± SD for cells from five and three donors, respectively. Source data are available online for this figure.

Article Snippet: The plasmid pmRuby2.Vpr, encoding Vpr fused to mRuby2, was generated by excision of the eGFP coding region of peGFP.Vpr (McDonald et al , , a kind gift of Tom Hope) using AgeI and BsrGI restriction enzymes, followed by insertion of a PCR fragment encoding mRuby2 (Lam et al , ); amplified from pcDNA3‐mRuby2, a gift from Michael Lin (Addgene plasmid # 40260) and flanked by the same restriction sites.

Techniques: Transduction, Infection, Cell Culture, Suspension, Microscopy, Confocal Microscopy, Staining, Virus, Incubation, Generated, Fluorescence