Figure 3.

Intact CA-A77V capsids can deeply penetrate into the central channel of the NPC

SupT1-R5 cells were infected with IN.mScarlet carrying NNHIV-A77V CA mutant for 15 h at 37°C prior to high pressure freezing (for CLEM) or plunge freezing (for cryo-ET).

(A–A″) Plastic section CLEM of a cell stained with mCLING.Atto647N (magenta), post-stained with Hoechst (blue), and decorated with multi-fluorescent fiducials (Fd) for correlation. (A) Overlay of a fluorescence image with the correlated electron micrograph. The enlarged region displays the position of the IN.mScarlet signal (red) at the nuclear envelope (white arrow). (A′) Slice through a tomographic reconstruction at the correlated position. The boxed region is shown enlarged in (A″) highlighting an apparently intact capsid (black arrowhead) deep inside the central channel of the NPC (green arrowheads), exposing its narrow end toward the nucleoplasm. Cy, cytosol; Nu, nucleus; NE, nuclear envelope.

(B–D) Cryoelectron tomography (cryo-ET) of cryo-FIB milled SupT1-R5 cells infected with NNHIV-A77V.

(B) Slice through a tomographic reconstruction showing a capsid (black arrowhead) localized inside of the NPC (green arrowhead).

(B′) Same as in (B) but displayed segmented and isosurface rendered. Capsid, magenta; NE, yellow; NPC, cyan (cryo-EM map of NPC: this study).

(C) Enlarged and rotated view of the HIV-1 capsid (black arrowhead) shown in (B), dimensions are indicated.

(C′) Same as (C) but superimposed with structural models of capsid (magenta; cryo-EM density map of hexameric unit: EMD-3465 [Mattei et al., 2016]) and the NPC (cyan; cryo-EM map of NPC: this study). NE in yellow.

(D) Same as (C), but displayed in top view. The capsid is indicated by the black arrowhead. The NPC is highlighted by dashed circle.

(E) Distribution of CA hexamers along the surface of the same capsid as shown in (B) and (C) as identified by subtomogram averaging of 1,464 initial positions. Cross-correlation (cc) values are shown color-coded. The hexagonal lattice is clearly apparent.

(E′) Black lines highlight the regular arrangement of six CA hexamers surrounding a seventh, central CA hexamer.

See also Figures S2, S3B, S3C, S4, and S5 and Videos S4 and S5.