Figure 2.
HIV-1 capsids are intact in the cytosol and adjacent to the NPC but morphologically altered inside the nucleus of T cells
(A) Schematic illustration of the experiment. SupT1-R5 cells were infected with IN.mScarlet carrying NNHIV for 90 min at 16°C. To identify post-fusion subviral complexes, cells were stained with mCLING.Atto647N for additional 10 min at 16°C. Cells were subsequently incubated in the presence of mCLING.Atto647N for 3 h at 37°C prior to cryo-immobilization by high pressure freezing (HPF) and freeze substitution.
(B) CLEM overlay of a 250-nm thick resin section of the cell stained with mCLING.Atto647N (far-red; magenta), post-stained with Hoechst (blue) and decorated with multi-fluorescent fiducials (Fd) for correlation. Enlarged regions indicate the presence of mCLING-negative IN.mScarlet signals (red) in two positions (i and ii) within the cytosol (white arrows).
(C–D′) Slices through a tomographic reconstruction at the correlated positions i (C) and ii (D), with rotated and enlarged views highlighting cone-shaped capsids (black arrowheads) in the cytosol (C′) and in proximity to MTs (D and D′; white arrowheads).
(D″) Same as in (D′) but displayed segmented and isosurface rendered. MT red; capsid, magenta; NE, yellow; NPC, cyan (cryo-EM map of NPC: this study).
(E) CLEM overlay enlarged in the inset shows mCLING-negative signal of IN.mScarlet (red) located at the nuclear envelope (white arrow). (E′ and E″) Correlated position within an electron tomogram with enlarged inset (E′) and slice through a tomographic reconstruction in different orientation (E″) showing a MT-associated capsid docking to the NPC. Black, white, and green arrowheads indicate the capsid, microtubule cross section, and the NPC, respectively. Dashed lines outline the nuclear membrane.
(E′″) Same as in (E″) but segmented and isosurface rendered. Color code as in (D″).
(F–F″) Same as in (E–E″) but for NNHIV complexes captured inside the nucleus, highlighting the morphology of four clustered, capsid-related structures. In (F′), two of these structures visible in the tomographic slice are enlarged in the inset (black arrowheads).
(G) Further representative examples of nuclear NNHIV complexes captured in different cells. Cy, cytosol; Nu, nucleus; NE, nuclear envelope; NPC, nuclear pore complex; MT, microtubule.