Anti dc sign

For cell surface flow cytometric analysis, cells were harvested, washed, and stained for 30 min at 4°C in FACS buffer (PBS, 0.5% heat inactivated NHS, 1% BSA, 0.02% NaN₃) with anti‐CD14 MΦ P9 (BD Biosciences, San Diego, CA, USA) or anti‐DC‐SIGN (R&D Systems, Wiesbaden, Germany). Non‐conjugated antibodies were detected with PE‐conjugated goat‐anti‐mouse Ig (Dako, Glostrup, Denmark). Isotype matched control antibodies were used to determine the level of background staining. The fluorescence was measured on an FACS Calibur flow cytometer, and data were analyzed with Cell Quest Software (BD Biosciences, San Diego, CA, USA) and FlowJo Software (Tree Star, USA).

The proviral plasmids pNL4.3 and pNLAD8 were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, while the plasmid encoding the VSV-G envelope glycoprotein (pVSVg) has been described previously (64 (link)). The following antibodies were used: phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (clone ICRF44; BD Biosciences), PE-Cy7-conjugated anti-CD11b (clone ICRF44; Biolegend), RD1- or FITC-conjugated anti-Gag (clone KC57; Beckman Coulter), anti-CD4 (clone Leu3a; Biolegend), anti-CD3 (clone UCHT1; Biolegend), anti-DC-SIGN (clone 120507; R&D Systems), FITC-conjugated anti-CD11c (clone B-ly6; BD Biosciences), anti-CD14 (clone M5E2; BD Biosciences), allophycocyanin (APC)-conjugated anti-CD83 (clone HB15; Miltenyi), Alexa Fluor 647-conjugated anti-CCR5 (clone HEK/1/85a; Biolegend), PE-conjugated anti-CXCR4 (clone 12G5; Biolegend), anti-CD2 (clone TS2/18; a gift from Andres Alcover, Paris, France [65 (link)]), and Alexa Fluor 647- or Alexa Fluor 555-conjugated phalloidin (Life Technologies). The following reagents were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID: HIV-1 CAp24 hybridoma (183-H12-5C), HIV-IG, Maraviroc, T20, and AZT.

PHT cells and 293T-ACE2 (Integral Molecular, Philadelphia, PA) were seeded in 12-well plates at a density of 1.3 × 10⁶ cells/well for PHT (five donors) and 3 × 10⁵ cells/well for 293T-ACE2. Cells were infected with pvSARS-CoV-2 lentiviruses (100 ng/ml based on the p24 content) (Virongy, Manassas, VA) containing SMNE proteins (spike, membrane, nucleocapsid, and envelope), MNE proteins, S protein only, or no SARS-CoV-2 proteins, as well as aldrithiol-inactivated HIV-1 (86 (link)) as a negative control. 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 (1 (link), 7 (link)). Cells were harvested at day 2 and day 5 postinfection, and the cells were washed three times with 2 ml of PBS followed by 0.25% trypsin-EDTA for 10 min to release the cells from plastic and to remove any virus adhering to them. The cells were centrifuged at 400 × g for 5 min to pellet cells, which was lysed with 1% Triton X-100 for 15 min at 37°C and centrifuged at 2,000 × g for 5 min. The lysate supernatant was collected, and p24 was measured in the cell lysate using a cytometric bead assay (87 (link)). Values were adjusted to normalize for volume differences (1 ml for each fraction).