p24 elisa kit  (Sino Biological)


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    Human Immunodeficiency Virus type 1 p24 Capsid Protein p24 ELISA Pair Set
    Description:
    Each vial contains 22 ng of recombinant Human Immunodeficiency Virus type 1 HIV 1 p24 Capsid Protein p24 Reconstitute with 1 mL Dilution Buffer After reconstitution store at 20℃ to 80℃ in a manual defrost freezer A seven point standard curve using 2 fold serial dilutions in Dilution Buffer and a high standard of 1200 pg mL is recommended
    Catalog Number:
    SEK11695
    Price:
    None
    Category:
    Elisa pair set
    Reactivity:
    HIV
    Product Aliases:
    Gag-p24 Matched ELISA Antibody Pair Set HIV
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    Structured Review

    Sino Biological p24 elisa kit
    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 <t>p24</t> <t>ELISA.</t> 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.
    Each vial contains 22 ng of recombinant Human Immunodeficiency Virus type 1 HIV 1 p24 Capsid Protein p24 Reconstitute with 1 mL Dilution Buffer After reconstitution store at 20℃ to 80℃ in a manual defrost freezer A seven point standard curve using 2 fold serial dilutions in Dilution Buffer and a high standard of 1200 pg mL is recommended
    https://www.bioz.com/result/p24 elisa kit/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p24 elisa kit - by Bioz Stars, 2021-05
    93/100 stars

    Images

    1) Product Images from "PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins"

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

    Journal: bioRxiv

    doi: 10.1101/2020.05.17.100875

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    2) Product Images from "PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins"

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

    Journal: Cell Discovery

    doi: 10.1038/s41421-020-0184-9

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    3) Product Images from "PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins"

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

    Journal: bioRxiv

    doi: 10.1101/2020.05.17.100875

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    4) Product Images from "PSGL-1 inhibits HIV-1 infection by restricting actin dynamics and sequestering HIV envelope proteins"

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

    Journal: Cell Discovery

    doi: 10.1038/s41421-020-0184-9

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

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    Article Title: PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production
    Article Snippet: + 0.0098)/0.2772 (3)where hSEAP is the estimated concentration of the hSEAP protein and R.A.U. is the measured hSEAP activity units in the samples ( ). .. Gag-eGFP Quantification using p24 Enzyme-Linked ImmunoSorbent Assay (ELISA) The intracellular concentration of Gag-eGFP in transfected High Five cells and in culture supernatants was determined with an HIV-1 p24 ELISA Kit (Sino Biological, Wayne, NJ, USA). ..

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    Sino Biological commercial p24 elisa kit
    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 <t>p24.</t> 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 <t>ELISA</t> 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.
    Commercial P24 Elisa Kit, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/commercial p24 elisa kit/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    94
    Sino Biological hiv 1 gag p24
    STAT1 or STAT3 inhibitors suppress <t>HIV-1</t> infection of MDM. MDM were pretreated with 100 nM Fludarabine (STAT1 inhibitor) or 50 nM LLL12 (STAT3 inhibitor) 24 h before infection and on PID 1, 4, and 7. ELISA was used to measure supernatant <t>p24</t> on PID 4, 7, and 10 in cultures treated with (A) Fludarabine and (B) LLL12. Bars represent mean ± SEM; * p
    Hiv 1 Gag P24, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    STAT1 or STAT3 inhibitors suppress HIV-1 infection of MDM. MDM were pretreated with 100 nM Fludarabine (STAT1 inhibitor) or 50 nM LLL12 (STAT3 inhibitor) 24 h before infection and on PID 1, 4, and 7. ELISA was used to measure supernatant p24 on PID 4, 7, and 10 in cultures treated with (A) Fludarabine and (B) LLL12. Bars represent mean ± SEM; * p

    Journal: AIDS Research and Human Retroviruses

    Article Title: HIV-1 Infection Primes Macrophages Through STAT Signaling to Promote Enhanced Inflammation and Viral Replication

    doi: 10.1089/aid.2016.0273

    Figure Lengend Snippet: STAT1 or STAT3 inhibitors suppress HIV-1 infection of MDM. MDM were pretreated with 100 nM Fludarabine (STAT1 inhibitor) or 50 nM LLL12 (STAT3 inhibitor) 24 h before infection and on PID 1, 4, and 7. ELISA was used to measure supernatant p24 on PID 4, 7, and 10 in cultures treated with (A) Fludarabine and (B) LLL12. Bars represent mean ± SEM; * p

    Article Snippet: HIV-1 Gag p24 in culture supernatants was measured by ELISA (Sino Biological, Daxing, China).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay

    STAT1 and STAT3 are involved in HIV-1-induced priming. MDM were treated with 100 nM Fludarabine (STAT1 inhibitor) or 50 nM LLL12 (STAT3 inhibitor) 24 h before infection and on PID 1, 4, and 7. Ten days postinfection, cells were activated with 1 μg/mL CL097 for 6 h. Intracellular p24 and TNF were measured by flow cytometry. Bars represent mean ± SEM MFI of TNF in uninfected cells and infected cells gated on either p24-negative or p24-positive populations; * p

    Journal: AIDS Research and Human Retroviruses

    Article Title: HIV-1 Infection Primes Macrophages Through STAT Signaling to Promote Enhanced Inflammation and Viral Replication

    doi: 10.1089/aid.2016.0273

    Figure Lengend Snippet: STAT1 and STAT3 are involved in HIV-1-induced priming. MDM were treated with 100 nM Fludarabine (STAT1 inhibitor) or 50 nM LLL12 (STAT3 inhibitor) 24 h before infection and on PID 1, 4, and 7. Ten days postinfection, cells were activated with 1 μg/mL CL097 for 6 h. Intracellular p24 and TNF were measured by flow cytometry. Bars represent mean ± SEM MFI of TNF in uninfected cells and infected cells gated on either p24-negative or p24-positive populations; * p

    Article Snippet: HIV-1 Gag p24 in culture supernatants was measured by ELISA (Sino Biological, Daxing, China).

    Techniques: Infection, Flow Cytometry, Cytometry

    Only productively infected (p24 + ) MDM are primed. (A) MDM were infected with 5000 TCID 50 HIV-1 NL4-3-BaL-HSA encoding the murine heat-stable antigen (HSA; CD24), which is expressed on the surface of productively infected cells. CD24 + and CD24 − cells were sorted using magnetic beads and expression of HIV-1 gag p24 was measured by western blot to confirm efficient sorting of infected cells. (B) Uninfected and infected (CD24 − and CD24 + ) MDM were replated and stimulated with 10 ng/mL LPS for 6 h. ELISA measured secreted TNF. The experiment was conducted in one donor. ND, not detectable.

    Journal: AIDS Research and Human Retroviruses

    Article Title: HIV-1 Infection Primes Macrophages Through STAT Signaling to Promote Enhanced Inflammation and Viral Replication

    doi: 10.1089/aid.2016.0273

    Figure Lengend Snippet: Only productively infected (p24 + ) MDM are primed. (A) MDM were infected with 5000 TCID 50 HIV-1 NL4-3-BaL-HSA encoding the murine heat-stable antigen (HSA; CD24), which is expressed on the surface of productively infected cells. CD24 + and CD24 − cells were sorted using magnetic beads and expression of HIV-1 gag p24 was measured by western blot to confirm efficient sorting of infected cells. (B) Uninfected and infected (CD24 − and CD24 + ) MDM were replated and stimulated with 10 ng/mL LPS for 6 h. ELISA measured secreted TNF. The experiment was conducted in one donor. ND, not detectable.

    Article Snippet: HIV-1 Gag p24 in culture supernatants was measured by ELISA (Sino Biological, Daxing, China).

    Techniques: Infection, Magnetic Beads, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    ( A – C ) The infection status of the three production systems over the course of the cultivations. ( D – F ) The hemagglutinin concentration. ( G – I ) The p24 concentrations measured with ELISA and standardised to 10 6 cells mL −1 for all three cultivation platforms. The error bars indicate the standard deviation (n = 3).

    Journal: Scientific Reports

    Article Title: Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells

    doi: 10.1038/s41598-020-57761-w

    Figure Lengend Snippet: ( A – C ) The infection status of the three production systems over the course of the cultivations. ( D – F ) The hemagglutinin concentration. ( G – I ) The p24 concentrations measured with ELISA and standardised to 10 6 cells mL −1 for all three cultivation platforms. The error bars indicate the standard deviation (n = 3).

    Article Snippet: Free soluble HIV-1 p24 and total HIV-1 p24 concentration, including VLP-incorporated p24, in the expression supernatant were determined by the HIV-1 p24 capsid protein p24 ELISA Kit (Sino Biological, Wayne, USA).

    Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation