Journal: Journal of Virology
Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells
doi: 10.1128/jvi.00363-24
Figure Lengend Snippet: LPS binds to HIV-1 in a CD14-specific manner and does not passively associate with the lipid envelope. ( A ) Optimized LPS-bio (300 ng/mL, overnight, 4°C) and PE-SA (0.2 µg/mL, 4 h, 4°C) staining on CD14 + virions. Particle counts in PE + gates are enumerated on each plot. ( B ) Virion capture assay using immobilized untagged LPS. Ninety-six-well plates were coated with buffer (PBS) or LPS and virions with (CD14 + , orange bars) or without CD14 (WT, teal bars) were added. Captured virions were quantified by Gag p24 ELISA. Bars are mean ± SD of duplicate capture assays and are representative of three independent experiments. ( C ) FVM evaluating the contribution of recombinant LPS-binding protein (LBP) on LPS-bio binding to CD14 + virions. Varying amounts of recombinant LBP (rLBP) were mixed with LPS-bio prior to incubating with WT (top row) and CD14 + (bottom row) virus samples and staining with PE-SA. Gates define PE + events based on negative control (PE-SA only). WT and CD14 + virus stocks shown in FVM plots for ( A ) and ( C ) had a respective titer of 76 and 57 ng/mL p24 as measured by ELISA. ( D ) Particle counts in PE + gates from ( C ) were plotted as a function of PE-SA concentrations for WT (teal triangles) and CD14 + virions (orange circles). Data are mean ± SD of two independent virus stocks. ( E ) FVM measuring neutralization of LPS-bio binding to virions. Anti-CD14 (M5E2) was added to WT (top row) and CD14 + (bottom row) virions before incubation with LPS-bio and PE-SA. Gates are as described for ( C ). WT and CD14 + virus stocks shown in FVM plots had a respective titer of 368 and 141 ng/mL p24 as measured by ELISA. ( F ) Particle counts in PE + gates from ( E ) were plotted over the range of anti-CD14 concentrations tested for WT (teal triangles) and CD14 + (orange circles) virions. Data are representative of two independent virus stocks.
Article Snippet: Where indicated, LPS-bio was pre-incubated with increasing concentrations of rLBP (1 μL; R&D Systems #870-LP-025) or purified anti-CD14 (1 μL; mAb M5E2, BD Biosciences #557152) before adding to samples overnight and subsequently developing with PE-SA the next day.
Techniques: Staining, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Virus, Negative Control, Neutralization, Incubation
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