Fig. 1: Nuclear import of HIV-1 WT cores. | Nature Microbiology

Fig. 1: Nuclear import of HIV-1 WT cores.

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

Fig. 1

a, Confocal microscopy images of permeabilized CEM cells incubated with WT cores in the absence (P-CEM) and presence (PS-CEM) of RRL-ATP. WT cores are labelled with mNeonGreen-IN (green) and nuclei are labelled with SiR-DNA (magenta). Representative single z-slice images (top) and maximum intensity projections (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. The ratios represent the percentage of mNeonGreen-IN puncta localized inside the nuclei of permeabilized CEM cells under different conditions. Without RRL-ATP, 0.9% ± 1.2% (n = 61) for WT cores. With RRL-ATP for WT cores with an incubation time of 30 min, 6.2% ± 3.3% (n = 168); 1 h, 10.4% ± 3.7% (n = 117); 2 h, 11.5% ± 4.7% (n = 116); and 4 h, 10.8% ± 4.5% (n = 98). 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 (only significant differences are shown). c, A representative tomographic slice of a correlatively acquired tomogram of WT core nuclear import. Three WT cores are identified and indicated by the numbered purple arrowheads. Number 1 highlights an imported tube-shaped core with discernible surrounding densities (enlarged in the inset); number 2 shows a docked cone-shaped core with the wide end on the NPC; and number 3 shows a cone-shaped core traversing through the NPC with the narrow end facing inwards. The NPC, ribosomes, nucleosomes and prominent surrounding nuclear factors are labelled. The nucleus, nuclear envelope (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 imported, docked (top right; numbers 1 and 2) and traversing (bottom right; number 3) WT cores. WT cores, NPCs, nucleosomes, ribosomes, nuclear factors, NE and membranes are segmented and shown in the indicated colours. e, A bar chart illustrating the distribution of HIV-1 cores in each state of two groups: WT cores incubated with P-CEM cells (WT + P-CEM) and WT cores incubated with PS-CEM cells (WT + PS-CEM). Imported fractions are indicated. Significance was determined using a two-sided Chi-square test for all; P = 0.0464. f, A violin plot of the statistical analysis on the width of WT cores (width measured at the wide end) in each state. The imported WT cores measure 44.84 ± 6.519 nm (s.e. = 0.7429, n = 77), the traversing cores measure 52.97 ± 6.875 nm (s.e. = 0.6199, n = 123), the docking cores measure 54.53 ± 7.350 nm (s.e. = 0.4989, n = 217), the approaching cores measure 53.55 ± 7.432 nm (s.e. = 0.4540, n = 268) and the input cores measure 54.93 ± 7.442 nm (s.e. = 0.6578, n = 128). The white lines represent the medians, black lines represent the quartiles and black dots represent individual WT cores. Significance was determined using two-sided Brown–Forsythe and Welch ANOVA tests for all; ****P < 0.0001 (only significant differences are shown). g, A bar chart illustrating the composition of WT core shapes in each state. Cone-shaped WT cores are shown in purple and tube-shaped cores are in blue. Significance was determined using a two-sided Chi-square test for all; P < 0.0001. h, A bar chart showing the orientation distribution of cone-shaped WT cores in docking and traversing states, with the wide end in first (grey) and narrow end in first (purple). Significance was determined using a two-sided Fisher’s exact test for 1 comparison; P < 0.0001.

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