elisa  (Sino Biological)

Journal: bioRxiv

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1101/2020.05.17.100875

Figure Lengend Snippet: PSGL-1 inhibits R5-tropic virions infectivity and interacts with R5-tropic gp41. TZM-bl cells were infected with virions harvested from 293T cells transfected with two different R5-tropic HIV plasmids pYU2 ( a ) or pNL(AD8) ( c ) and different amounts of plasmids expressing PSGL-1. Empty vector was used to normalize the total transfected DNA. The virions were normalized by p24 ELISA. The infection rates were quantitated with luciferase assays. N=3. 293T cells transfected with pYU2 ( b ) or pNL(AD8) ( d ) and PSGL-1 or an empty vector were fixed and stained with anti-gp41 (red), anti-PSGL-1 (green) antibodies and DAPI (blue). Scale bar: 5µm.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Staining

Journal: bioRxiv

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1101/2020.05.17.100875

Figure Lengend Snippet: PSGL-1 increases F-actin intensity inside HIV-1 virions and affects virion infectivity. a , TZM-bl cells were infected with virions harvested from 293T 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 293T 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 293T cells with or without PSGL-1 overexpression were pre-incubated 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 293T 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 293T 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 293T 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 293T 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.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, FACS, Over Expression, Incubation, Generated, Staining, Imaging, Western Blot

Journal: bioRxiv

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1101/2020.05.17.100875

Figure Lengend Snippet: PSGL-1 stabilizes F-actin in HIV virions. a , Western blotting analysis shows nascent HIV-1 virions contain abundant cofilin and actin. b , Vpr-BlaM containing NL4-3 viruses generated from 283T cells with or without PSGL-1 overexpression were normalized with p24 ELISA. The viruses were used to infect cells treated with latrunculin A or cytochalasin D or DMSO control. The concentrations of the compounds are equal to the higher concentration of each drug in Fig.3d . Two hours after infection, the cells were incubated with β-lactamase substrate overnight before being fixed and analyzed by FACs. c , TZM-bl cells were treated with actin inhibitors latrunculin A or cytochalasin D or DMSO control at the concentration that is equal to the final concentration of each drug in the cell medium as in Fig.3e . NL4-3 viruses generated from 283T cells with or without PSGL-1 overexpression were normalized with p24 ELISA and used to infect the TZM-bl cells pretreated with the indicated compounds. Two days after infection, the infection rate was quantitated using luciferase assay.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Western Blot, Generated, Over Expression, Enzyme-linked Immunosorbent Assay, Concentration Assay, Infection, Incubation, FACS, Luciferase

Journal: bioRxiv

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1101/2020.05.17.100875

Figure Lengend Snippet: PSGL-1 binds HIV Env and sequesters Env at the plasma membrane. a , 293T cells were separately transfected with pNL4-3 proviral plasmid or plasmids expressing PSGL-1, PSGL-1 delCD, PSGL-1 LL/AA or an empty vector. One day after the transfection, PSGL-1 or gp41 were immunoprecipitated with protein A agarose beads with anti-PSGL-1 antibody or anti-gp41 antibody respectively and then the beads were mixed with cell lysates from transfection of the other protein in the IP. The cell lysates and the precipitated proteins were analyzed by Western blotting. b , 293T cells transfected with pNL4-3 and PSGL-1 or PSGL-1 delCD or PSGL-1 LL/AA or an empty vector were fixed and stained with anti-gp41 (red), anti-PSGL-1 (green) antibodies and DAPI (blue) and quantification of gp41 cellular localization of each sample was shown in ( c ). Scale bar: 5µm. N=40. d , Virions harvested from producer 293T cells transfected with PSGL-1 or PSGL-1 delCD or PSGL-1 LL/AA or an empty vector were pelleted through 20% sucrose cushion were analyzed by Western blotting. e , TZM-bl cells were infected with virions harvested from 293T cells transfected with pNL4-3 and different amounts of plasmids expressing PSGL-1, PSGL-1 delCD, PSGL-1 LL/AA. 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. f , Endocytosis of Env (gp160) protein in 293T cells as visualized by pulse labeling of FAP-tag in the Env protein. 293T cells were transfected with plasmids expressing PSGL-1, PSGL-1 delCD, PSGL-1 LL/AA and then stained with the fluorogen MG-11p (red) for 5 min. Cells were fixed after a 20-min chase period and stained with an anti-PSGL-1 (green) antibody and DAPI (blue). Scale bar: 5µm. g , Model of the mechanisms of the anti-HIV activity of PSGL-1. In the early stage of HIV life cycle, PSGL-1 restricts cortical actin filament to inhibit HIV reverse transcription. In the late stage of the life cycle, PSGL-1 prevents envelope incorporation into the nascent virions and blocks viral infectivity. PSGL-1 also stabilize F-actin inside of nascent virions to affect their infectivity.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Staining, Infection, Enzyme-linked Immunosorbent Assay, Luciferase, Labeling, Activity Assay

Journal: bioRxiv

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1101/2020.05.17.100875

Figure Lengend Snippet: PSGL-1 inhibits Env incorporation in virions and viral entry. a , Virions harvested from producer 293T cells transfected with two different doses of PSGL-1 or an empty vector were pelleted through 20% sucrose cushion were analyzed by Western blotting together with the cell lysates. b , PSGL-1-knockout Jurkat cells lines with two different sgRNAs or a control cell line with a non-targeting (NT) sgRNA were infected with NL4-3 virus or NL4-3 delVpu virus. The supernatants containing newly released viruses were concentrated and analyzed by Western blotting The cell lysates of the producer cells were also analyzed by Western blotting. c , Quantification of the band intensity of gp41 in ( b ) normalized to the intensity of p24. The fold of change between NL4-3 virus and NL4-3 delVpu virus is shown for each cell group. d , Virions harvested from infection experiments in ( b ) were normalized by p24 levels measured with ELISA and used to infected TZM-bl cells. The infectivity was measured by luciferase assays. The fold of change between NL4-3 virus and NL4-3 delVpu virus is shown for each cell group. e , Correlation analysis between ( c ) and ( d ). f-g , Virions from PSGL-1 knockout or control Jurkat cell lines were collected after 5 days post-infection with NL4-3 and pelleted through 20% sucrose cushion were fixed and stained for STORM imaging. Representative images are shown in ( f ). Scale bar: 100 nm. Quantification of STORM images results were showed in ( g ). The ratios between the average values of two groups and the p values were shown. h-i , Virions from producer 293T cells transfected with PSGL-1, PSGL-1 delCD or an empty vector were pelleted through 20% sucrose cushion were fixed and stained for STORM imaging. Representative images are shown in ( h ). Scale bar: 100 nm. Quantification of STORM images results were showed in ( i ). The ratios between the average values of two groups and the p values were shown. j-k , Concentrated virions were analyzed by cryo-EM analysis. Representative images were shown in ( j ) and quantification of images of virions shown in ( k ). Scale bar: 100nm.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Transfection, Plasmid Preparation, Western Blot, Knock-Out, Infection, Enzyme-linked Immunosorbent Assay, Luciferase, Staining, Imaging

Journal: bioRxiv

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1101/2020.05.17.100875

Figure Lengend Snippet: PSGL-1 stabilizes cellular F-actin to restrict HIV infection. a , Immunofluorescence staining of PSGL-1 in MAGI cells overexpressing PSGL-1 using anti-PSGL-1 antibody. Upper panel: PSGL-1 (red) colocalizes with LifeAct-GFP (green), which binds F-actin; Lower panel: PSGL-1 (red) colocalizes with phalloidin (green). Scale bar: 10 µm. b-c , Immunofluorescence staining of PSGL-1 using anti-PSGL-1 antibody (green) and phalloidin (red) in Jurkat T cells overexpressing luciferase or PSGL-1. Scale bar: 5 µm. The phalloidin intensity of cells in each group were shown in ( c ). d , Jurkat T cells overexpressing luciferase, PSGL-1 or PSGL-1 CD (cytoplasmic domain) alone were stained with phalloidin and analyzed with FACS. Relative MFIs were normalized to luciferase group. MFI: Mean Fluorescence intensity. N = 3. e-f , Activated primary CD4+ T cells were treated with IFN-γ or mock-treated for 12 h before being electroporated with two different siRNAs targeting PSGL-1 or non-targeting control siRNA (siNT) for 48 h. The cells were then either fixed for phalloidin staining and FACS quantification ( f ) or infected with HIV-1 NL4-3 for 72h before the supernatant being collected for p24 ELISA ( e ). N = 3 for ( e-f ). g , Correlation between cellular F-actin intensities and HIV-1 infection rates in ( e-f ). h-i , Activated primary CD4+ T cells from two healthy donors were incubated with PSGL-1 antibody or IgG for 2 h before being fixed for phalloidin staining ( h ) or infected with HIV-1 NL4-3 for 72 h before the supernatant being collected for p24 measurement ( i ). N = 3.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Infection, Immunofluorescence, Staining, Luciferase, FACS, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation

Journal: bioRxiv

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1101/2020.05.17.100875

Figure Lengend Snippet: PSGL-1’s dimerization-deficient mutation and gap co-clustering-deficient mutations have no effect on its anti-viral activity. a , TZM-bl cells were infected with virions harvested from 293T cells transfected with pNL4-3 plasmids and different amounts of plasmids expressing PSGL-1 or PSGL-1 C336A mutant (dimerization-deficient), PSGL-1 3A mutant (gap co-clustering-deficient ) . The virions were normalized by p24 ELISA before the infection. The infection rates were quantitated with luciferase assays. N=3.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Mutagenesis, Activity Assay, Infection, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Luciferase

Journal: Cell Discovery

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1038/s41421-020-0184-9

Figure Lengend Snippet: PSGL-1 inhibits Env incorporation in virions and viral entry. a Virions harvested from producer 293 T cells transfected with two different doses of PSGL-1 or an empty vector were pelleted through 20% sucrose cushion were analyzed by Western blotting together with the cell lysates. b PSGL-1-knockout Jurkat cells lines with two different sgRNAs or a control cell line with a non-targeting (NT) sgRNA were infected with NL4-3 virus or NL4-3 delVpu virus. The supernatants containing newly released viruses were concentrated and analyzed by Western blotting. The cell lysates of the producer cells were also analyzed by Western blotting. c Quantification of the band intensity of gp41 in b normalized to the intensity of p24. The fold of change between NL4-3 virus and NL4-3 delVpu virus is shown for each cell group. d Virions harvested from infection experiments in b were normalized by p24 levels measured with ELISA and used to infect TZM-bl cells. The infectivity was measured by luciferase assays. The fold of change between NL4-3 virus and NL4-3 delVpu virus is shown for each cell group. e Correlation analysis between c and d . f, g Virions from PSGL-1 knockout or control Jurkat cell lines were collected after 5 days post infection with NL4-3 and pelleted through 20% sucrose cushion were fixed and stained for STORM imaging. Representative images are shown in f . Scale bar: 100 nm. Quantification of STORM images results were showed in g . The ratios between the average values of two groups and the p values were shown. h, i Virions from producer 293 T cells transfected with PSGL-1, PSGL-1 delCD, or an empty vector were pelleted through 20% sucrose cushion were fixed and stained for STORM imaging. Representative images are shown in h . Scale bar: 100 nm. Quantification of STORM images were showed in i . The ratios between the average values of two groups and the p values were shown. j, k Concentrated virions were analyzed by cryo-EM analysis. Representative images were shown in j and quantification of images of virions shown in k . Scale bar: 100 nm.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Transfection, Plasmid Preparation, Western Blot, Knock-Out, Infection, Enzyme-linked Immunosorbent Assay, Luciferase, Staining, Imaging

Journal: Cell Discovery

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1038/s41421-020-0184-9

Figure Lengend Snippet: 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.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, FACS, Over Expression, Generated, Incubation, Staining, Imaging, Western Blot

Journal: Cell Discovery

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1038/s41421-020-0184-9

Figure Lengend Snippet: PSGL-1 stabilizes cellular F-actin to restrict HIV infection. a Immunofluorescence staining of PSGL-1 in HeLa-based MAGI cells overexpressing PSGL-1 using anti-PSGL-1 antibody. Upper panel: PSGL-1 (red) colocalizes with LifeAct-GFP (green), which binds F-actin; Lower panel: PSGL-1 (red) colocalizes with phalloidin (green). Scale bar: 10 μm. b, c Immunofluorescence staining of PSGL-1 using anti-PSGL-1 antibody (green) and phalloidin (red) in Jurkat T cells overexpressing luciferase or PSGL-1. Scale bar: 5 μm. The phalloidin intensity of cells in each group is shown in c . d Jurkat T cells overexpressing luciferase, PSGL-1 or PSGL-1 CD (cytoplasmic domain) alone were stained with phalloidin and analyzed with FACS. Relative MFIs were normalized to luciferase group. MFI mean fluorescence intensity. n = 3. e, f Activated primary CD4 + T cells were treated with IFN-γ or mock-treated for 12 h before being electroporated with two different siRNAs targeting PSGL-1 or non-targeting control siRNA (siNT) for 48 h. The cells were then either fixed for phalloidin staining and FACS quantification ( f ) or infected with HIV-1 NL4-3 for 72 h before the supernatant being collected for p24 ELISA ( e ). n = 3 for e, f . g Correlation between cellular F-actin intensities and HIV-1 infection rates in e, f . h, i Activated primary CD4 + T cells from two healthy donors were incubated with PSGL-1 antibody or IgG for 2 h before being fixed for phalloidin staining ( h ) or infected with HIV-1 NL4-3 for 72 h before the supernatant being collected for p24 measurement ( i ). n = 3.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Infection, Immunofluorescence, Staining, Luciferase, FACS, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Cell Discovery

Article Title: PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins

doi: 10.1038/s41421-020-0184-9

Figure Lengend Snippet: PSGL-1 binds HIV Env and sequesters Env at the plasma membrane. a 293 T cells were separately transfected with pNL4-3 proviral plasmid or plasmids expressing PSGL-1, PSGL-1 delCD, PSGL-1 LL/AA, or an empty vector. One day after the transfection, PSGL-1 or gp41 were immunoprecipitated with protein A agarose beads with anti-PSGL-1 antibody or anti-gp41 antibody respectively and then the beads were mixed with cell lysates from transfection of the other protein in the IP. The cell lysates and the precipitated proteins were analyzed by Western blotting. b 293 T cells transfected with pNL4-3 and PSGL-1 or PSGL-1 delCD or PSGL-1 LL/AA or an empty vector were fixed and stained with anti-gp41 (red), anti-PSGL-1 (green) antibodies and DAPI (blue), and quantification of gp41 cellular localization of each sample is shown in c . Scale bar: 5 μm. n = 40. d Virions harvested from producer 293 T cells transfected with PSGL-1 or PSGL-1 delCD or PSGL-1 LL/AA or an empty vector were pelleted through 20% sucrose cushion were analyzed by Western blotting. e TZM-bl cells were infected with virions harvested from 293 T cells transfected with pNL4-3 and different amounts of plasmids expressing PSGL-1, PSGL-1 delCD, PSGL-1 LL/AA. Empty vector was used to normalize the total transfected DNA. The virions were normalized by p24 ELISA. The infection rates were quantitated with the luciferase assay. n = 3. f Endocytosis of Env (gp160) protein in 293 T cells as visualized by pulse labeling of FAP-tag in the Env protein. 293 T cells were transfected with plasmids expressing PSGL-1, PSGL-1 delCD, PSGL-1 LL/AA and then stained with the fluorogen MG-11p (red) for 5 min. Cells were fixed after a 20-min chase period and stained with an anti-PSGL-1 (green) antibody and DAPI (blue). Scale bar: 5 μm. g Model of the mechanisms of the anti-HIV activity of PSGL-1. In the early stage of HIV life cycle, PSGL-1 restricts cortical actin filament to inhibit HIV reverse transcription. In the late stage of the life cycle, PSGL-1 prevents envelope incorporation into the nascent virions and blocks viral infectivity. PSGL-1 also stabilize F-actin inside of nascent virions to affect their infectivity.

Article Snippet: Two days post transfection, the supernatant was collected for p24 measurement using a commercial p24 ELISA kit (Sinobiological).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Staining, Infection, Enzyme-linked Immunosorbent Assay, Luciferase, Labeling, Activity Assay