human t lymphoblast cell line sup t1  (ATCC)

Journal: bioRxiv

Article Title: The stage-specific regulation imposed by Importinβ-1 on HIV-1 propagation and infectivity dynamics

doi: 10.1101/2024.06.10.598175

Figure Lengend Snippet: (A) Immunoblot showing the presence of Importinβ-1 in virus preparation from culture supernatants of pNL4.3 transfected HEK293T cells. The presence of virus was detected with anti-p24 antibody. GAPDH was used as the internal control. ( B) Immunoblot showing the presence of Importinβ-1 in virus preparation from culture supernatants of NL4.3 infected SUP-T1 cells. The presence of virus was detected with anti-p24 antibody. GAPDH was used as the internal control. (C) Immunoblot showing the presence of Importinβ-1 in virus preparation from culture supernatants of NL4.3 infected 1321N1 cells. The presence of virus was detected with anti-p24 antibody. GAPDH was used as the internal control. (D) Co-immune precipitation showing Importinβ-1 interaction with Gag and Capsid proteins upon pNL4.3 transfection in HEK293T cells. Mouse IgG was used as an isotypic control. (E) Confocal microscopy showing co-localisation of Importinβ-1 GFP and Gag/Capsid protein in the HEK293T cells. (F) The plot profiles showing the overlapping of Importinβ-1 GFP and Gag/Capsid protein in representative single cell generated using ImageJ software. All experiments were done at least 3 times.

Article Snippet: DMEM (HIMEDIA Cat: AL066A-500ML) was used for maintaining HEK293T, 1321N1, and TZM-bl cell lines, and RPMI-1640 (HIMEDIA Cat: AL162A-500ML) for SUP-T1 cell line.

Techniques: Western Blot, Virus, Transfection, Infection, Confocal Microscopy, Generated, Software

Journal: bioRxiv

Article Title: The stage-specific regulation imposed by Importinβ-1 on HIV-1 propagation and infectivity dynamics

doi: 10.1101/2024.06.10.598175

Figure Lengend Snippet: (A) Representative Immunoblot showing the levels of Importinβ-1 from 0hrs to 96hrs in SUP-T1 cells upon NL4.3 infection. GAPDH was used as a control. (B) Bar graph representing the quantification of Importinβ-1 for the experiments represented in A. (C) Levels of released virus determined by p24 ELISA in the supernatant of NL4.3 infected SUP-T1 cells from 0 hrs to 96 hrs. (D) Line graph representing the relationship of endogenous Importinβ-1 and HIV titers in SUP-T1 cells during the course of HIV infection from 0 hrs to 96 hrs. Endogenous GAPDH was used as a loading control. (E) Representative Immunoblot showing the levels of Importinβ-1 in SUP-T1 cells upon treatment with IFNγ, LPS, and IL4. Tubulin was used as a control. Bar graph showing immunoblot quantification of Importinβ-1 from three independent experiments. (F) Representative Immunoblot showing the levels of Importinβ-1 in HEK293T cells upon treatment with IFNγ, LPS, and IL4. Tubulin was used as a control. Bar graph showing immunoblot quantification of Importinβ-1 from three independent experiments. (G) Representative Immunoblot showing the levels of p55 in SUP-T1 cells upon treatment with IFNγ, LPS, and IL4. GAPDH was used as a control. Bar graph showing immunoblot quantification of Importinβ-1 from three independent experiments. All experiments were done at least 3 times. The significance is determined using an unpaired student’s t-test. The p values are denoted as *** p <0.001; * p <0.05, while non-significant values are denoted by n.s.

Article Snippet: DMEM (HIMEDIA Cat: AL066A-500ML) was used for maintaining HEK293T, 1321N1, and TZM-bl cell lines, and RPMI-1640 (HIMEDIA Cat: AL162A-500ML) for SUP-T1 cell line.

Techniques: Western Blot, Infection, Virus, Enzyme-linked Immunosorbent Assay

Journal: Nature

Article Title: HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes

doi: 10.1038/s41586-023-06762-6

Figure Lengend Snippet: Western blotting analysis for gp120, gp41 and p24 in the supernatant (S) and pellet (P) fractions of HIV-1 BaL viruses from ultracentrifugation after incubation with PBS, sCD4 alone, sCD4 in combination with 17b Fab, or MLV-CD4 VLPs. Residual 17b Fc fragments in 17b Fab were detected by anti-Human 2nd HRP-antibody (grey).

Article Snippet: Immature HIV-1 BaL viruses were produced by the HIV-1 BaL /SUP-T1-CCR5 cell line treated with a final concentration of 1 µM indinavir and 10 µM ritonavir.

Techniques: Western Blot, Incubation

Journal: Nature

Article Title: HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes

doi: 10.1038/s41586-023-06762-6

Figure Lengend Snippet: a , b , Representative cryo-tomograms of membrane–membrane interfaces (red boxes) between HIV-1 BaL viral particles and plasma membrane blebs generated from TZM-bl cells (bleb TZM-bl ). Scale bars, 100 nm. c , e , Top-down views of the membrane–membrane interfaces shown in a ( c ) and b ( e ), revealing Env clustering. Scale bars, 20 nm. d , f , Magnified images of the membrane–membrane interfaces shown in a ( d ) and b ( f ) depict Env–CD4 interactions in raw tomograms. Scale bars, 20 nm. g , Representative image of HIV-1 BaL virus with Env on its surface. The capsid is highlighted in green. HIV-1 BaL was inactivated with AT-2, resulting in less electron-dense capsids. Scale bars, 25 nm. h , Representative images of an MLV VLP carrying CD4 on its surface. The MLV capsid is highlighted in purple. Scale bars, 25 nm. i , A representative image of membrane–membrane interfaces (red boxes) in cryo-tomograms. Different capsid structures enable the identification of membrane–membrane interfaces as opposed to interfaces that do not have Env–CD4 interactions (blue box). Scale bar, 50 nm. j , A representative tomogram with the membrane–membrane interface highlighted in red. Scale bar, 50 nm. k , Magnified image of the membrane–membrane interface shown in j . Env–CD4 interactions are visible in raw tomograms. Scale bar, 10 nm.

Article Snippet: Immature HIV-1 BaL viruses were produced by the HIV-1 BaL /SUP-T1-CCR5 cell line treated with a final concentration of 1 µM indinavir and 10 µM ritonavir.

Techniques: Membrane, Generated, Virus

Journal: Nature

Article Title: HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes

doi: 10.1038/s41586-023-06762-6

Figure Lengend Snippet: a – c , Representative tomograms of small ( a ), large ( b ) and ring ( c ) Env cluster formations. The interfaces are indicated by red dashed ovals. Bottom, top-down views of each interface, revealing Env clustering. Scale bars, 50 nm. d , Three-dimensional representation of viral particles in a representative tomogram. The pink arrows represent Env trimers at membrane–membrane interfaces. The yellow arrows represent free Env trimers on the surface of HIV-1 BaL . e , The number of Env trimers present at interfaces in each clustering pattern. Mean ± s.d. = 3.0 ± 1.1 (small), 10.0 ± 3.4 (large) and 7.9 ± 3.4 (ring). * P = 0.0478, **** P < 0.0001. f , The distance between the membranes at the interfaces for each clustering pattern. Mean ± s.d. = 16.9 ± 3.8 nm (small), 15.1 ± 2.4 nm (large) and 11.9 ± 3.6 nm (ring). NS, P = 0.4178; *** P = 0.0002, **** P < 0.0001. g , Subtomogram-averaged interfaces from small, large and ring clusters. Individual subtomograms of the membrane–membrane interfaces from each class were aligned, and the coordinates of Env–CD4 complexes were overlaid and displayed as side and top-down views (middle and bottom, respectively). h , i , Clustering analysis of Env trimers on the surface of mature (black) and immature HIV-1 BaL particles alone (green, prepared by treating virus-producing cells with the protease inhibitors indinavir (IDV) and ritonavir (RTV)), and mature (red) and immature (purple) HIV-1 BaL particles mixed with MLV-CD4 VLPs. h , Histogram profile of the arc distances between Env trimers on the surface of particles. i , Multidistance spacial cluster analysis of the ratio of the largest number of Env trimers with increasing cluster radii to the total Env trimers on each viral particle. Env clustering (arrow) and dispersion (red asterisk) are indicated. Max., maximum.

Article Snippet: Immature HIV-1 BaL viruses were produced by the HIV-1 BaL /SUP-T1-CCR5 cell line treated with a final concentration of 1 µM indinavir and 10 µM ritonavir.

Techniques: Membrane, Virus, Dispersion

Journal: Nature

Article Title: HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes

doi: 10.1038/s41586-023-06762-6

Figure Lengend Snippet: a A representative tomogram showing immature HIV-1 BaL particles and MLV-CD4 VLPs. HIV-1 capsids are noted in green and MLV capsids are noted in purple. Scale bar = 50 nm. b Three-dimensional representation of immature HIV-1BaL particles and MLV-CD4 VLPs in a representative tomogram. Pink arrows represent Env trimers at membrane-membrane interfaces. Yellow arrows represent free Env trimers on the surface of the HIV-1BaL particles. c The number of Env trimers present at membrane-membrane interfaces for mature and immature HIV-1 BaL particles mixed with MLV-CD4 VLPs. Mature HIV-1 BaL mean ± s.d. = 7.5 ± 4. Immature HIV-1 BaL mean ± s.d. = 4.6 ± 2.5. P-value **** = <0.0001. d The distance between membranes at membrane-membrane interfaces with mature and immature HIV-1 BaL particles mixed with MLV-CD4 VLPs. Mature HIV-1 BaL mean ± s.d. = 14.3 nm ± 3.8 nm. Immature HIV-1 BaL mean ± s.d. = 13.5 nm ± 4.6 nm. * = 0.0420.

Article Snippet: Immature HIV-1 BaL viruses were produced by the HIV-1 BaL /SUP-T1-CCR5 cell line treated with a final concentration of 1 µM indinavir and 10 µM ritonavir.

Techniques: Membrane

Journal: Nature

Article Title: HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes

doi: 10.1038/s41586-023-06762-6

Figure Lengend Snippet: a Representative images of the HIV-1 viral particle produced from the cell line expressing Env ADA.CM.755* . The capsid is highlighted in green. Scale bar = 25 nm. b Representative images of the MLV VLP carrying CD4. The capsid is highlighted in purple. Scale bar = 25 nm. c A representative image of membrane-membrane interfaces in cryo-tomograms. HIV-1 and MLV capsids are highlighted in green and purple, respectively. Scale bar = 50 nm. d Violin plot of the number of Env trimers present at membrane-membrane interfaces for HIV-1 BaL (blue), mature HIV-1 ADA.CM.755* (brown), and immature HIV-1 ADA.CM.755* (beige) with MLV-CD4 VLPs. P-values ns = 0.0779, ** = 0.0028, **** = <0.0001.

Article Snippet: Immature HIV-1 BaL viruses were produced by the HIV-1 BaL /SUP-T1-CCR5 cell line treated with a final concentration of 1 µM indinavir and 10 µM ritonavir.

Techniques: Produced, Expressing, Membrane

Journal: Nature

Article Title: HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes

doi: 10.1038/s41586-023-06762-6

Figure Lengend Snippet: a Individual subtomograms of all the membrane-membrane interfaces were aligned and averaged (left panel). In the middle panel, the coordinates of Env bound to one (purple), two (cyan) and three (green) CD4 molecules are overlaid onto the averaged volumes. The top-down view is shown to the right. b , c Gallery of HIV-1 Env trimers bound to CD4 receptors on biological membranes. Representative images from cryo-tomograms of the HIV-1 Env trimers bound to one CD4 (b) or two and three CD4 (c) molecules at the membrane-membrane interfaces between HIV-1 and MLV-CD4 particles. Scale bar = 20 nm.

Article Snippet: Immature HIV-1 BaL viruses were produced by the HIV-1 BaL /SUP-T1-CCR5 cell line treated with a final concentration of 1 µM indinavir and 10 µM ritonavir.

Techniques: Membrane