Fig. 5: Nuclear import of E45A and E45A/R132T cores. | Nature Microbiology

Fig. 5: Nuclear import of E45A and E45A/R132T cores.

From: HIV-1 nuclear import is selective and depends on both capsid elasticity and nuclear pore adaptability

Fig. 5

a, Confocal microscopy images of permeabilized CEM cells incubated with E45A and E45A/R132T cores in the presence of RRL-ATP. Cores are labelled with mNeonGreen-IN (green) and nuclei are labelled with SiR-DNA (magenta). Representative single z-slice images (top) and MIPs of z slices (bottom) are shown. The arrows indicate mNeonGreen-IN signals inside the nucleus. Scale bar, 5 µm. b, Statistical analysis of the nuclear import of the mNeonGreen-IN puncta for brittle E45A revertant mutant E45A/R132T cores. The ratios represent the percentage of mNeonGreen-IN puncta localized inside the nuclei of permeabilized CEM cells under different conditions. WT cores, 6.2% ± 3.3% (n = 168); E45A cores, 1.3% ± 1.2% (n = 99); and E45A/R132T cores, 3.5% ± 2.4% (n = 111). The black lines represent medians. Significance was determined using a one-way ANOVA test for all; ****P < 0.0001. c, A representative tomographic slice of a correlatively acquired tomogram of E45A cores clashing on the NPC. Five E45A cores were identified and indicated by pink arrowheads and numbered as follows: numbers 1, 3 and 4 show clashing cone-shaped E45A cores; number 2 shows a clashing tube-shaped E45A core; and number 5 shows a docked cone-shaped E45A core with the narrow end on the NPC. The E45A cores labelled number 4 and number 5 are shown on other slices of the same tomogram (white framed). The NPC, ribosomes and nucleosomes are labelled. The nucleus, NE and membranes are annotated accordingly. Scale bar, 100 nm. d, The segmented volume of c, shown as an overview (left) and zoomed-in views of the clashed (top right; numbers 1–4) and docked (bottom right; number 5) E45A cores. E45A cores, NPCs, nucleosomes, ribosomes, NE and membranes are segmented and shown in the indicated colours. e, A representative tomographic slice of a correlatively acquired tomogram of HIV-1 E45A/R132T core nuclear import. Two E45A/R132T cores are identified and indicated by light purple arrowheads and numbered as follows: number 1 shows a docked cone-shaped E45A/R132T core with the wide end on the NPC; and number 2 shows an imported tube-shaped E45A/R132T core with discernible surrounding densities, also shown on another slice of the same tomogram (white framed). The NPC, ribosomes, nucleosomes and prominent surrounding nuclear factors are labelled. The nucleus, NE and membranes are annotated accordingly. Scale bar, 100 nm. f, The segmented volume of e, shown as an overview (left) and zoomed-in views of the docked (top right; number 1) and imported (bottom right; number 2) E45A/R132T cores. E45A/R132T cores, NPCs, nucleosomes, nuclear factors, ribosomes, NE and membranes are segmented and shown in the indicated colours. g, A bar chart showing the distribution of HIV-1 cores in each state across three samples: WT, E45A and E45A/R132T. The imported fraction in each case is annotated. Significance was determined using a two-sided Chi-square test for all; P < 0.0001. h, A violin plot of the statistical analysis of the size of E45A cores (width measured at the wide end) in each state. Imported E45A cores measure 44.80 ± 5.644 nm (s.e. = 1.262, n = 20), traversing cores measure 46.79 ± 6.658 nm (s.e. = 1.780, n = 14), docking cores measure 55.51 ± 6.993 nm (s.e. = 0.4135, n = 286), approaching cores measure 54.82 ± 7.250 nm (s.e. = 0.5842, n = 154) and input cores measure 55.17 ± 9.483 nm (s.e. = 0.6147, n = 238). The white lines represent the medians, black lines represent the quartiles and black dots represent individual E45A cores. Significance was determined using two-sided Brown–Forsythe and Welch ANOVA tests for all; **P < 0.01 (**P = 0.0038 for input versus traversing, **P = 0.0053 for approaching versus traversing, **P = 0.0028 for docking versus traversing), ****P < 0.0001 (only significant differences are shown). i, A violin plot of the statistical analysis on the size of E45A/R132T cores (width measured at the wide end) in each state. Imported E45A/R132T cores measure 44.84 ± 5.965 nm (s.e. = 1.369, n = 19), traversing cores measure 52.33 ± 6.670 nm (s.e. = 1.362, n = 24), docking cores measure 55.43 ± 9.228 nm (s.e. = 1.776, n = 27), approaching cores measure 53.88 ± 9.925 nm (s.e. = 1.754, n = 32) and the input cores measure 55.43 ± 10.53 nm (s.e. = 0.7763, n = 184). The white lines represent the medians, black lines represent the quartiles, and black dots represent individual E45A/R132T cores. Significance was determined using two-sided Brown–Forsythe and Welch ANOVA tests for all; **P < 0.01 (**P = 0.0018 for approaching versus imported, **P = 0.0038 for traversing versus imported), ***P = 0.0003, ****P < 0.0001 (only significant differences are shown).

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