PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells

Significance PSGL-1 and CD43 are surface glycoproteins expressed on blood CD4 T cells to bind to selectins for T cell tethering, rolling, and migration into inflamed tissues. The PSGL-1 level is greatly up-regulated during inflammation. Here we found that PSGL-1 and CD43 expression inhibits HIV spreading infection. Mechanistically, PSGL-1 blocks the binding of virus particles to target cells. PSGL-1–mediated suppression of virus infectivity extends to another retrovirus—murine leukemia virus—and to influenza A virus. These results further our understanding of virus–host interactions and help elucidate mechanisms by which cellular host factors regulate viral infection and pathogenesis.


Viral attachment assay.
Virion particles produced in the presence of PSGL-1 or the empty vector were incubated with HelaJC.53 cells (pre-chilled at 4°C for 1 hour) at 4°C for 2 hours. The cells were then washed extensively (5 times) with cold PBS buffer and then lysed with LDS lysis buffer (Invitrogen) for analysis by Western blot.
For luciferase-based, single-cycle infectivity assays, RT-normalized virus stocks were used to infect the CD4 + /CXCR4 + /CCR5 + HeLa derivative TZM-bl. This indicator cell line contains integrated copies of the β-galactosidase and luciferase genes under the control of the HIV-1 LTR. Infection efficiency was determined by measuring luciferase activity 2 days postinfection. For infectivity assays in HeLa JC53-PSGL-1 and HeLa-JC53-empty cell lines, the cells were seeded in 6-well plates at a density of 0.2 x 10 6 /well 24 hours prior to infection. Cells were infected with the indicated p24 equivalents of either WT NL4-3, NL43ΔVpu, or NL43ΔNef. Viral replication was quantified by virion p24 released into the medium by p24 ELISA. For MLV (murine leukemia virus) virion infectivity, MLV-GFP reporter virus was assembled by co-transfecting HEK293T cells (in 6-well plate) with pSV-Ψ-MLV-env -(0.375 µg), pRetroQ-AcGFP1-N1 (0.5 µg), pHCMV-G (0.125 µg), and pCMV3-PSGL1 or an empty vector at the indicated dosages. An equal amount of DNA was used all transfections. Viral supernatants were harvested 48h post transfection and used to infect HEK293T cells for 6 hours in the presence of Infectin (Virongy, Manassas, VA). Cells were washed to remove virus and Infectin, and cultured for 48 hours for flowcytometry analyses.
To determine the effect of PSGL-1 on influenza A virus replication, HEK293T and MDCK cells were co-cultured at approximately 70 % confluence in 6-well plates and transfected with either empty vector or pCMV3-PSGL-1 (both vectors at 1.0 or 3.0 µg), together with an eight-plasmid influenza A/WSN/33 reverse genetics system (RGS) (1.0 µg of each plasmid). Transfection reaction was prepared with PEI ( polyethylenimine). Culture supernatants were collected at 16 and 24 h post-transfection and titrated in MDCK cells to determine end-point titers (TCID 50 per ml).
shRNA knockdown of PSGL-1. Lentiviral vectors carrying shRNAs against PSGL-1 or nontarget control (NTC) (Sigma MISSION shRNA, PSGL-1 TRCN0000436811 or shRNA NTC) were purchased from Sigma. Virion particles were assembled by cotransfecting HEK293T cells with 0.5 µg of pHCMV-G, 1.5 µg pCMV-ΔR8.2, and 2 µg of lentiviral vectors using Lipofectamine 3000 (Invitrogen). Supernatant was collected at 48 hours post co-transfection, and then filtered through 0.45um filter. Virion particles were used to transduce Jurkat T cells for 6 hours. Cells were then washed twice and cultured in fresh media for 3 days, and then selected in puromycin (4 µg /ml) for one to two weeks. PSGL-1 knockdown was confirmed by surface staining with an anti-PSGL-1 antibody (KPL-1) (BD Pharmingen) followed by staining with Alexa Fluor 488conjugated goat anti-mouse secondary antibody (Invitrogen). PSGPL-1 knockdown or NTC control cells (2 x 10 6 ) were also transfected with 2 µg of HIV-1(NL4-3) DNA by electroporation using a T cell electroporation kit (Lonza). Viruses were harvested and used for the infection of Rev-A3R5-GFP cells (20 ng p24 per infection). Flow cytometry analysis of GFP expression was performed on the indicated days. Lentiviral vector-mediated ShRNA knockdown of PSGL-1 in primary CD4 T cells was performed as described previously (1). Briefly, blood resting CD4 T cells were purified by negative depletion, transiently stimulated with anti-CD3/CD28 beads (1 -2 beads per cell) for 12 hours, and then transduced with the lentiviral vectors carrying shRNAs against PSGL-1 or non-target control (NTC) (Sigma MISSION shRNA, PSGL-1 TRCN0000436811 or shRNA NTC). Following transduction, the beads were removed at 12 hours, and cells were cultured for 3 days, and then analyzed from surface PSGL-1 expression. Cells were also subsequently transfected with HIV-1(NL4-3) DNA by electroporation using a T cell electroporation kit (Lonza). HIV-1 viral replication was monitored by harvesting cell culture supernatant, and HIV p24 was detected by an in-house p24 ELISA kit.     One day posttransfection, virus supernatants were harvested, and virus release efficiency (VRE) was quantified by western blot as the amount of virion-associated p24 (CA) relative to total Gag in cell and virus lysates. VRE was set to 100% for WT HIV-1 in the absence of PSGL-1. B), At 30 hours posttransfection, virus supernatants were harvested, and an aliquot was used for reverse transcriptase (RT) and infectivity assays. RT-normalized virus was used to infect TZM-bl cells for 2 days, and luciferase activity was measured. Infectivity of WT HIV-1 in the absence of PSGL-1 was set to 100%. Data shown are ± SD from three independent experiments. Data were evaluated for statistical significance using the unpaired t test.     Supernatants were harvested at 48 hours, filtered, concentrated, and purified by ultra-speed centrifugation through a 6%-18% OptiPrep gradient. PSGL-1 and viral p24 proteins in each fraction were analyzed by western blot using antibodies against PSGL-1 (polyclonal) or HIV-1 p24 (anti-p24). For comparison, an anti-CD63 antibody was used to identify the fractions also containing exosomes. D and E) HEK293T cells were cotransfected with HIV-1(NL4-3) DNA plus PSGL-1 DNA at the indicated ratios. Virus particles were harvested at 48 hours and purified by ultraspeed centrifugation through an OptiPrep gradient (6%-18%). The presence of viral p24 in each fraction was directly analyzed by p24 ELISA (D). Expression of viral proteins in cotransfected cells was analyzed by western blot using antibodies against HIV-1 proteins (anti-HIV serum); virion proteins were also analyzed by harvesting and pelleting the peak virion fraction, and performing western blot using antibodies against PSGL-1 (polyclonal) or HIV-1 proteins (anti-HIV serum). Positions of Gag precursor protein (pr55) and p24 (CA) are indicated (E). F) HEK293T cells were cotransfected with varying amounts of PSGL-1 DNA plus 1 µg HIV-1(NL4-3) or HIV(∆Vpu) DNA at the indicated ratios. Virus particles were harvested at 48 hours and purified by ultra-speed centrifugation through an OptiPrep gradient (6%-18%). Intracellular and virion proteins were similarly analyzed by western blots for PSGL-1 and HIV-1 proteins.

Fig. S14. CD43 inactivates HIV-1 virion infectivity. A)
HEK293T cells were cotransfected with various amounts of CD43 DNA (0.5 -400 ng) plus 1 µg HIV(NL4-3) DNA. Virions were harvested at 48 hours and normalized for p24. Viral infectivity was quantified by infecting Rev-A3R5-GFP indicator cells. HIV-1 replication was quantified by GFP expression. Shown are the percentages of GFP+ cells at 48 hours postinfection. B) The CD43 dose-dependent inhibition curve was plotted. C) Downregulation of CD43 from the cell surface by Vpu and Nef. HEK293T cells were cotransfected with CD43 (100 ng) and a Vpu or Nef expression vector (4 µg). Surface CD43 expression was quantified and shown as the percentage of cells expressing CD43. For controls, an empty vector was used (+vector). The same amount of DNA was used in all transfections.