Fig. 4: Nuclear import of N74D cores.

a, Confocal microscopy images of permeabilized CEM cells incubated with N74D cores in the presence of RRL-ATP. N74D 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. Arrows indicate mNeonGreen-IN signals inside the nucleus. Scale bar, 5 µm. b, The nuclear import efficiency of mNeonGreen-IN puncta was analysed for WT and N74D cores. The percentage of mNeonGreen-IN puncta localized inside the nuclei of permeabilized CEM cells under different incubation times was as follows: WT 30 min, 6.2% ± 3.3% (n = 168); WT 1 h, 10.4% ± 3.7% (n = 117); WT 2 h, 11.5% ± 4.7% (n = 116); WT 4 h, 10.8% ± 4.5% (n = 98); N74D 30 min, 4.3% ± 2.2% (n = 97); N74D 1 h, 6.8% ± 3.4% (n = 72); N74D 2 h, 7.4% ± 3.1% (n = 80); and N74D 4 h, 7.4% ± 3.2% (n = 66). The black lines represent medians. Significance was determined using a one-way ANOVA test for all and two-sided Fisher’s exact test for each pair; ****P < 0.0001, ***P = 0.001 for WT 0.5 h versus N74D 0.5 h, ***P = 0.0004 for N74D 0.5 h versus N74D 1 h. c, The penetration depth of WT and N74D cores from the nuclear envelope were enumerated as follows: WT 30 min, 0.38 ± 0.31 µm (n = 3,010); WT 1 h, 0.50 ± 0.48 µm (n = 8,540); WT 2 h, 0.56 ± 0.53 µm (n = 8,713); WT 4 h, 0.57 ± 0.53 µm (n = 7,548); N74D 30 min, 0.29 ± 0.21 µm (n = 1,311); N74D 1 h, 0.35 ± 0.30 µm (n = 2,257); N74D 2 h, 0.39 ± 0.32 µm (n = 2,872); and N74D 4 h, 0.39 ± 0.33 µm (n = 2,413). The black lines represent medians. Significance was determined using a one-way ANOVA test for all and two-sided Fisher’s exact test for each pair; ****P < 0.0001 and ***P = 0.0009. d, A representative tomographic slice of a correlatively acquired tomogram of HIV-1 N74D core nuclear import. One just-imported cone-shaped N74D core with the wide end facing inwards is identified and indicated by the light blue arrowhead. No discernible surrounding densities are observed (enlarged in the inset). The NPC, ribosomes and nucleosomes are labelled. The nucleus, NE and membranes are annotated accordingly. Scale bar, 100 nm. e, The segmented volume of d, shown as an overview (left) and zoomed-in views of the just-imported N74D core from the top (top right) and side (bottom right). The N74D core, NPCs, nucleosomes, ribosomes, NE and membranes are segmented and shown in the indicated colours. f, A bar chart showing the distribution of N74D cores in each state; the import fraction is annotated above the imported bar. g, Right: a line chart depicting the change of density as a function of the distance from the surface of HIV-1 cores. The first solid arrow approximately indicates the surface of the core, and the second solid arrow approximately indicates the centre of the surrounding density. Left: the measurement of grey values along the normal lines (dashed arrows) extending from the core surface, including partial densities of CA (~3 nm) due to the resolution of images. Black density is assigned a value of zero, while white is assigned one. Higher density corresponds to lower numerical values. For all conditions, 20 lines are drawn for each core. The numbers of HIV-1 cores analysed are as follows: WT outside, n = 10; WT traversing, n = 10; WT imported, n = 10; E45A imported, n = 10; E45A/R132T imported, n = 10; and N74D imported, n = 5. h, A line chart illustrating the distribution of all HIV-1 cores in each state (in percent). Significance was determined using a two-sided Chi-square test for all; P < 0.0001. i, A bar chart depicting the distribution of all imported HIV-1 cores based on their distances from the nuclear envelope within the nucleus. The reference distance is calculated as the sum of the longest axis of the HIV-1 core (~120 nm) and the length of the nuclear basket (~80 nm). Significance was determined using a two-sided Fisher’s exact test for all; P = 0.8641.