Fig. 3: PSGL-1 increases F-actin intensity inside HIV-1 virions and affects virion infectivity.

a TZM-bl cells were infected with virions harvested from 293 T cells transfected with pNL4-3 and different amounts of plasmids expressing PSGL-1 and PSGL-1 delCD. Empty vector was used to normalize the total transfected DNA. The virions were normalized by p24 ELISA. The infection rates were quantitated with luciferase assay. n = 3. b, c Jurkat cells were infected with Vpr-BlaM virions harvested from 293 T cells transfected with pNL4-3 and different amounts of plasmids expressing PSGL-1 and PSGL-1 delCD. Empty vector was used to normalize the total transfected DNA. The virions were normalized by p24 ELISA. The infection rates were measured by FACS. n = 3. d NL4-3 virions packaged from 293 T cells with or without PSGL-1 overexpression were preincubated with indicated doses of F-actin inhibitors, latrunculin A, or cytochalasin D. TZM-bl cells were infected with treated virions for 48 h before the infection units were measured with luciferase assay. n = 3. e Vpr-BlaM containing virions generated from 293 T cells with or without PSGL-1 overexpression were preincubated with latrunculin A or cytochalasin D at indicated concentrations for 2 h. Jurkat cells were infected with the treated virions for 2 h and incubated with β-lactamase substrate overnight before being fixed and analyzed by FACS. n = 3. f Virions harvested from producer 293 T cells transfected with PSGL-1 or PSGL-1 T393A or an empty vector were pelleted through 20% sucrose cushion, fixed by 4% PFA and stained with antibodies and phalloidin before STORM imaging. Scale bar: 100 nm. g Quantification of the intensities of F-actin or PSGL-1 staining in f shows that PSGL-1 but not PSGL-1 T393A significantly stabilizes F-actin in virions. h Virions containing PSGL-1 or PSGL-1 T393A were pelleted through 20% sucrose cushion before being sedimented for fractions containing G-actin or F-actin and analyzed by Western blotting. i TZM-bl cells were infected with virions harvested from 293 T cells transfected with pNL4-3 and different amounts of plasmids expressing PSGL-1 and PSGL-1 T393A. Empty vector DNA was used to normalize the total DNA transfected. The infection rates were quantitated by luciferase assay. n = 3. j Vpr-BlaM containing virions generated from 293 T cells transfected with pNL4-3, Vpr-BlaM plasmid, and different amounts of PSGL-1 or PSGL-1 T393A plasmids. Empty vector was used to normalize the total DNA transfected. The viruses harvested were normalized by p24 ELISA and used to infect Jurkat cells for 2 h. The cells were then incubated with β-lactamase substrate overnight before being fixed and analyzed by FACS. n = 3.