Figure 4.

CA-A77V structures accumulated at the nuclear basket are morphologically altered

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

(A–C′) Capsid-related structures at the nuclear basket region visualized by CLEM. Dashed lines outline the nuclear envelope.

(A and A′) CLEM overlay (A) with enlargement (A′) showing positions of IN.mScarlet signals (red; white arrows) at the nuclear envelope in an EM section stained with mCLING.Atto647N (magenta), post-stained with Hoechst (blue) and decorated with multi-fluorescent fiducials (Fd) for correlation.

(A″) Slice through a tomographic reconstruction at the correlated position shown in (A) and (A′). The features i–iii that are shown enlarged in the bottom panel are framed in black and contain three different capsid-related structures at the nuclear basket region.

(B–C′) Same as (A)–(A″) showing two capsid-related structures from two different cells. Capsids appear tube-shaped and empty.

(D–G″) Cryo-ET and subtomogram averaging of NNHIV-A77V.

(D and E) Slices through tomographic reconstructions showing capsid-related structures localized at the nuclear basket region. Black, green and white arrowheads in (A)–(E) indicate nuclear capsid-related structures, NPCs and a microtubule, respectively. Cy, cytosol; Nu, nucleus; NE, nuclear envelope.

(F) Surface area of the HIV-1 capsids identified in the cytosol (Cyt) (n = 4) and inside the nucleus (Nu) (n = 5) in the cryoelectron tomograms. Statistical significance was assessed by unpaired two-tailed Student’s t test; ^∗∗p = 0.0064. Representative models for three shape-based classes of nuclear structures, such as conical (magenta), tubular (salmon), and spherical (pink) are shown in the graph.

(G) Same as in Figure 3E but for the capsid-like structure shown in (D), identified by subtomogram averaging of 1,197 initial positions.

(G′ and G″) CA pentamer (G′) and two incomplete consecutive CA hexamers (G″).

See also Figures S2, S3B, S3C, S4, and S5 and Video S6.