Additional file 1 of P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

Additional file 1: Table S1. Amounts of plasmid DNA used to produce the BaL pseudovirus (PV) stocks. Table S2. Additional details regarding patient characteristics and the percentage of virion capture from Figure 5. Figure S1. PSGL-1 flow virometry staining quantification. Median PE MESF values from anti-PSGL-1 staining of viruses from Fig. 4. The MESF values from stained media controls, which demonstrate background levels of antibody fluorescence based on the detection limit for this instrument, were subtracted from all virus samples. Data shown are the mean + SD of three technical replicates. Figure S2. Determining the minimum threshold of detection for PSGL-1 staining on HIV-1 pseudoviruses using flow virometry. Staining of pseudoviruses produced through transfection with different amounts of PSGL-1 plasmid DNA (0–2.5 ng) with a PE-conjugated anti-PSGL-1 antibody. The horizontal dotted line on the virus dot plots denotes background fluorescence and the limit of instrument detection (~ 10 MESF). Figure S3. Detecting PSGL-1-incorporation on the surface of viruses and extracellular vesicles using flow virometry. A Staining of PSGL-1 positive viruses or matched cell culture supernatants (EV) from HEK293T cells transfected with different amounts of PSGL-1 pDNA (0 ng, 2.5 ng, 25 ng, 250 ng for negative, low, medium, high phenotypes respectively) with a PE-conjugated anti-PSGL-1 antibody. Virus and EV conditions are shown together to allow for comparison of the differential staining profiles. All conditions were transfected with an empty vector control to ensure the final amount of pDNA reached 3 μg. B PSGL-1 staining of viruses or vesicles from cell culture supernatants of infected (Virus) or mock infected (EV) T cell lines (H9, Jurkat, A3R5.7 and PM1). C PSGL-1 staining of infected or mock infected PBMC cell culture supernatants in two independent donors (D1–D2). The horizontal dotted line on the dot plots denotes background fluorescence and the limit of instrument detection (~ 10 MESF). Thick gates demarcate where the virus population is expected to be present based on light scattering. Events within the thin gate in the upper right quadrant were used to generate the histograms in each panel which display the comparison of the median light scattering properties of the EV and virus populations. Coloured histograms demonstrate the notable differences between virus (red) and EV (blue) populations and light scattering properties. Figure S4. Quality control assessment of the replication competent NL4-3 HIV produced through transfection with an infectious molecular clone. A Plasmid DNA concentrations used to produce viruses with distinct PSGL-1 phenotypes through transfection of HEK293T cells. B Both NL4-3 viruses (negative and high) were normalized based on viral p24 (displayed as 1:1 in graph) and were subjected to three-fold serial dilutions before incubation with the TZM-bl reporter cells for 48 h to measure viral infectivity. C Semi-quantitative comparisons of virion-incorporated PSGL-1 and gp120 on virus stocks using immunomagnetic virion capture assay as used in Fig. 3. D Staining of viruses for PSGL-1 incorporation using flow virometry with a PE-conjugated anti-PSGL-1 antibody. The horizontal dotted line on virus dot plots indicates background fluorescence and the limit of instrument detection (10 MESF). Results are representative of three independent experiments. For quantitative data, results shown are the mean ± SEM. Figure S5. PSGL-1+ virions can be captured by an anti-PSGL-1 mAb and transferred to HIV-permissive cells for infection. A Schematic depicting the experimental workflow: Viruses were added to wells pre-coated with monoclonal antibodies specific for either PSGL-1 or gp120, or with an isotype control (IgG) for two hours at room temperature to allow for virus binding. Post-incubation, wells were washed extensively to remove unbound virus and then were either assayed for the amount of captured virus using p24 detection (top workflow), or TZM-bl cells were overlayed onto each well containing captured virus, and trans-infection was measured via luminescence readout (bottom workflow). B Experimental results showing the levels of plate-based antibody-mediated virus capture and C viral infectivity from trans-infection assays. Only HIV IIIB viruses propagated in T cell lines (H9 in green or Jurkat in purple) were used for these proof-of-principle assays. Results are representative of three independent experiments and are displayed as mean ± SEM from three experiments with samples tested in duplicate.

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