DOI: 10.34810/data2398
title: Replication Data for: Early Steps of Individual Multireceptor Viral Interactions Dissected by High-Density, Multicolor Quantum Dot Mapping in Living Cells

description: &lt;b&gt;Primary Cell Culture&lt;/b&gt;&lt;br&gt;
Human immature dendritic cells (imDCs) were obtained from peripheral blood mononuclear cells (PBMC) from HIV-1 seronegative donors using a Ficoll-Hypaque gradient (Alere Technologies AS). The monocyte population was selected by adherence on a T75 cm2 flask for 1 h. imDCs were obtained by culturing the monocytes in complete RPMI with 1.000 IU/mL (granulocyte-macrophage colony-stimulating factor (GM-CSF)) and IL-4 (interleukin-4) both from R&D for 6 days. The medium was replaced every 2 days with fresh GM-CSF and IL-4. Experiments were performed at day 6 from the monocyte extraction.&lt;br&gt;&lt;br&gt;
&lt;b&gt;Antibodies and Reagents&lt;/b&gt;&lt;br&gt;
Monoclonal mouse antihuman CD44 (Clone G44–26) and monoclonal mouse anti-CD209 (Clone DCN46) were obtained from BD Biosciences. Recombinant human Gal-9 protein (Cat. number 9064-GA) was obtained from R&D systems. SARS-CoV-2 Spike Protein (RBD) Chimeric Recombinant Rabbit Monoclonal Antibody (P05DHuRb) tagged with Alexa Fluor 647 was obtained from eBioscience. Streptavidin QDs (565, 605, 655, and 705) were obtained from Thermo Fisher scientific. These QDs are based on a CdSe core nanocrystal surrounded by a ZnS shell to improve the brightness and stability of the QDs. Moreover they are functionalized with streptavidin enabling the attachment of the QDs to the biomolecules of interest (DC-SIGN, CD44 and Gal-9).&lt;br&gt;&lt;br&gt;
&lt;b&gt;Single Chain Antibody Generation&lt;/b&gt;&lt;br&gt;
All the experiments reported here have been performed using single chain antibodies to label DC-SIGN and CD44 in order to prevent antibody cross-linking (see Figure S2A for the general labeling strategy used for the generation of SPT and HiDenMaps). Both antihuman CD209 and CD44 single chain antibodies were generated using a similar protocol. First, the full chain antibodies were dialyzed using 10K dialysis devices (Thermo Scientific Slide-A-Lyzer MINI Dialysis Devices, 10 K MWCO) against PBS for 8 h at 4 °C. Second, we concentrated the dialyzed full chain antibodies to a concentration of 1 mg/mL. We then reduced the antibodies using DTT (1,4-dithiotrheritol, Sigma-Aldrich) at 1 mM, let the mixture to reduce at room temperature for 1 h while rotating and after that dialyzed overnight against PBS using the 10 K dialysis devices at 4 °C. We stabilized the broken sulfide-bonds with Iodoacetamide at 20 mM. We let the mix at room temperature for 1 h rotating gently and dialyzed to remove excess iodoacetamide overnight at 4 °C. Figure S2B shows the electrophoresis gel for the generated single chain antibodies together with the full chain antibodies as a control.&lt;br&gt;&lt;br&gt;
&lt;b&gt;
Biotinylation and Conjugation to Quantum Dots&lt;/b&gt;&lt;br&gt;
We performed the biotinylation of single chain antibodies and of Gal-9 with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher). For this, we added a 20× mol excess of biotin and let the mixture to shake for 1 h in ice. Then, we dialyzed using 10 K units overnight at 4 °C to remove excess of nonreacting biotin. To conjugate the biotinylated single chains and Gal-9 to the QDs, we mixed equal ratios of single-chain/Gal-9 and QDs in 5× excess of free biotin. The competition with excess of free biotin ensures a 1:1 labeling ratio between the target protein and the QD, as earlier reported. (15,35) To obtain a target concentration of 300 nM of stock conjugates, we first mixed 300 nM of QDs with 1.5 μM biotin and then added 300 nM of single-chain/Gal-9. Importantly, to avoid artifacts due to cross-linking of the recombinant added Gal-9 proteins, titrations were performed until single molecules of the conjugate Gal-9/QD were detected.&lt;br&gt;&lt;br&gt;&lt;b&gt;
Labeling Strategy&lt;/b&gt;&lt;br&gt;
For SPT experiments, we used conjugates of Gal-9/QD565, α-CD44/QD655, and α-DC-SIGN/QD705 at a concentration of 1 nM. For the HiDenMap experiments, we increased their concentration to 30 nM.&lt;br&gt;&lt;br&gt;&lt;b&gt;
Pseudovirus like Particle Generation&lt;/b&gt;&lt;br&gt;
The plasmid pr8ΔEnv.2 was obtained from Addgene, (Plasmid #12263). We generated VLPs as follows: 57 μL of Trans-IT reagent (Mirus) were added to 2 μg of pR8ΔEnv.2, 3 μg of iGFP-GagΔEnv, 1 μg of pcRev and 3 μg of NL4–3 Env to generate HIV-1 VLPs, or 0.5 μg of SARS-CoV-2-Spike [D614G] to generate SARS-CoV-2 VLPs. Importantly, both constructs contain full length Gag and Pol precursor proteins generating fully mature HIV-1 particles. pR8ΔEnv.2 contains WT, unlabeled Gag and HIV iGFP-GagΔEnv contains a GFP tag between the matrix (MA) and capsid subunits (CA). Of note, the GFP sequence is flanked by HIV protease recognition sites allowing dissociation of GFP from CA and MA upon virion maturation. To minimize production of nonfunctional viral particles, expression of labeled Gag has been complemented with unlabeled Gag using a 3:2 ratio. This approach has been demonstrated to retain high infectivity serving as a marker for viral core. (55,56) To generate mock viruses, no plasmid generating the Env or the Spike protein was added. We then added the mixture to 1.9 mL of OPTIMEM (Gibco) and incubated for 15 min at room temperature. We added the mixture to 18 mL of DMEM with FBS and l-Glut and without antibiotics. We added the medium to HEK-293T cells at 80–90% confluency and collected the supernatant at day 3. We first centrifuged briefly the supernatant (500g for 10 min) and filtered the supernatant through a 0.45 μm filter. We concentrated the supernatant with Lenti-X concentrator (Takara) following the manufacturer’s protocol and resuspend it in RPMI. Finally, the VLPs were aliquoted and kept under liquid nitrogen before storing them at −80 °C.&lt;br&gt;&lt;br&gt;
&lt;b&gt;Sample Preparation for HiDenMap Experiments&lt;/b&gt;&lt;br&gt;
We plated ∼50.000 cells on either glass coverslips (#1) coated with PLL (20 ng/mL) for control cells or on 35 mm Glass bottom dish with 10 mm microwell (#1, Cellvis) also coated with PLL. We seeded the cells for 1 h in RPMI without FBS, l-Glut or antibiotics. Viability of the cells was assessed by visual inspection using bright field imaging prior to the experiments. Only well-spread cells with defined dendrites (the normal phenotype of dendritic cells) were carefully chosen for subsequent imaging. Labeling using the single chain-QDs was performed sequentially by diluting 1 μL of DC-SIGN/QD655 and 3 μL of CD44/QD705 in 46 μL of PBS with 6% BSA. We incubated the conjugates and the cells for 5 min. After washing 3 times in RPMI, we took 5 μL of Gal-9/QD605 and 45 μL of PBS diluted in 6% BSA and incubated for 5 min. To avoid removal of the Gal-9 conjugate, we washed only once with RPMI. We then added RPMI to perform the imaging. For the experiments with VLPs (HIV-1, SARS-CoV-2 or mocks) we added the VLPs defrosted and added 10 ng/mL of LPS. The mixture was added to the cells and immediately imaged at 37 °C on the basal membrane for the next 30 min in order to preserve the functionality of the receptors and to avoid internalization.
description: MATLAB, R2020a
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