Overview of the CG molecular model of the NPC, HIV-1 capsid, and LEN.

(A) The left panel shows the composite membrane-embedded CG model of human NPC. The NPC is shown in gray spheres. The disordered NUP98 chains are shown in red chains. The nuclear membrane is modeled with a 4-site CG lipid bilayer model. The CG bead representing the lipid headgroups are shown in pink spheres, interfacial and tail beads are shown in white spheres. In the right panel, the cross-section of the NPC is shown. The CR and NR are shown in silver to distinguish from the IR (shown in gray spheres). The FG-NUP62 is modeled as a heterotrimeric subcomplex along with NUP54 and NUP58 is shown in cyan spheres (occluded by the FG-NUP98 mesh). The diameter of the central channel is 59 nm. (B) The left panel shows the CG representation of LEN with CG sites 1-8 labeled on the LEN chemical representation. The center panel shows the CG-mapped representation of LEN bound to a CA hexamer. The CG-mapped structure was generated from the X-ray crystal structure PDB: 6VKV. A CG LEN molecule is shown in orange beads. The CG sites of adjoining CA monomers CA1 and CA2 that are in contact with LEN are shown in red and green, respectively. In the right panel, the CG representation of the CA monomer is shown. The CANTD and CACTD domains are shown in cyan and silver spheres, respectively. In the snapshot of the cone-shaped capsid, the CANTD of the hexamer is shown in cyan, and the pentamer is shown in red. The CACTD domain of both hexamer and pentamer is shown in silver.

Competition between NUP98 and LEN during docking of LEN-treated capsid.

(A) The time series of the distance (DCap-NPC) between the geometric center of the LEN-bound capsid and equatorial midplane of the NPC inner ring along the channel axis (upper panel), the number of FG sites of NUP98 (fFG-NUP98) directly in contact with the CA monomers (middle panel), and the number of LEN molecules bound to the capsid (NLEN) (lower panel) are plotted. Here, fFG-NUP98 denotes the total number of FG sites that are in the vicinity of the CA hydrophobic FG-binding pocket (within 4 nm radius). The solid lines are the mean values calculated from the time series of two independent replicas, and the shaded region is the standard deviation at each timestep. (B) The upper panel shows the initial configuration and the final configuration of the capsid at the end of 200 × 106 τCG. In the snapshot at 200 × 106 τCG, one RNP chain (blue spheres) partly extrudes out concomitant to the first appearance of defects at the pentamer-hexamer interface. The color scheme is the same as in Figure 1. In the lower panel, only the CTD domain is shown for each CA monomer. The CA monomers to which at least one LEN molecule is bound are shown in white spheres. The CA monomers to which no LEN molecule is bound are shown in orange spheres. Note the high density of orange spheres located at the narrow end of the capsid in the right panel (docked capsid). This indicates that some LEN molecules are displaced by the NUP98 mesh to allow capsid docking. A cutaway view of the nuclear membrane is also shown to represent the degree of capsid docking from the cytoplasmic side toward the nuclear side. The NPC, LEN, and NUP98 are not shown for figure clarity.

Stepwise rupture of capsid treated with LEN during NPC docking.

(A) The NTD domain of CA hexamer and pentamer are shown in cyan and red spheres, respectively. The CTD domain of all CA monomers is shown in white spheres. The RNP chains are shown in blue. The NPC (cutaway view) is shown in gray, and the nuclear membrane is shown in pink (head group) and white (other groups). The initial events of CA-CA contact disruption at the hexamer-pentamer interface (CAHex − CAPen) at the narrow end are labeled with arrow. In the leftmost snapshot, the RNA extrudes out of the defect as a result of partial dissociation of the pentamer. In the final stage, nucleation of cracks occurs at the hexamer-hexamer interface (CAHex − CAHex) and extends from the narrow to the wide tip. (B) Snapshots of the defects at the hexamer-pentamer interface and hexamer-hexamer interface. CACTD domains of the hexamers that constitute the defect site are highlighted in orange spheres. Note that the CG beads constituting the CACTD domains at the defect site are disconnected from at least one nearest neighbor, leading to the locally cracked lattice (marked with red arrows). (C) Time series of the degree of defects at the pentamer-hexamer (NPen-Hex) and hexamer-hexamer (NHex-Hex). Note that in NPen-Hex and NHex-Hex are calculated by normalizing by total number of CA pentamer (12) and hexamer rings (209) respectively. The left panel shows the time series of the undercoordinated CA monomers at the pentamer-hexamer interface. The right panel shows the time series of the undercoordinated CA monomers at the hexamer-hexamer interface. (D) Time series of the radius of gyration (Rg) of the two RNP chains. The solid lines are the mean values calculated from the time series of two independent replicas, and the shaded region is the standard deviation at each timestep.

Live-cell imaging of LEN-induced rupture of HIV-1 cores docked at nuclear envelope.

(A) Dual-labeled viral cores: the capsid lattice is labeled with GFP-CA, and the capsid content is labeled with content marker HALO (cmHALO; JF646 dye). (B) Representative GFP-CA labeled viral core in a cell expressing POM121-HALO (JF549 dye) that was stably associated with the nuclear envelope, retaining cmHALO in a DMSO-treated control cell (top) or losing cmHALO within 1 min of 100 nM LEN addition (bottom). (C) Number of GFP-CA labeled viral cores per cell that remained at the nuclear envelope during the 15-minute observation period. A total of 237 DMSO-treated cells and 192 LEN-treated cells were analyzed. (D) Percentage of GFP-CA labeled viral cores that were cmHALO+ at the start of imaging. GFP-CA labeled viral cores were analyzed for DMSO-treated (29 total) and nm LEN-treated (37 total) cells. (E) Percentage of GFP-CA labeled viral cores that lost cmHALO during the 15-minute observation period. (F) Time (minutes) of cmHALO disappearance following DMSO or LEN addition. P values were calculated using Fisher’s exact test; ns, not significant (P > 0.05).

Molecular view of defects and LEN-induced rupture of free capsids.

The color scheme of the capsid and RNP is the same as in Figure 3. CACTD domains of the hexamers that constitute the defect site are highlighted in orange spheres. The snapshots in the inset show zoomed-in view of representative defects. For the uncondensed RNP, snapshot 1 shows partial dissociation of pentamers at the narrow end, and RNP chains extrude out of the capsid interior. Snapshot 2 shows a ruptured narrow end. For the condensed RNP, snapshots 3 and 4 show defects arising from the partial dissociation of pentamers at the wide end and the narrow end. Condensed RNP localizes at the capsid wide end. In snapshot 3, the RNP extrudes out of the defect at the wide end. Finally, snapshot 5 shows rupture of the narrow end.

Molecular details of LEN binding and alteration of capsid microstructure.

(A) The left panel shows the time series of the number of LEN molecules (normalized by the number of CA hexamer rings) bound to a CA monomer which is either part of the ordered lattice (LENo) or distorted lattice (LENd). The snapshot in the left-center panel shows LEN molecules bound to regions of the lattice that are ordered. The CTD domain of the CA monomers with ⟨q6 ⟩neigh < 0.4 (distorted lattice) is colored in magenta. The CTD domain of the rest of the CA monomers (ordered lattice) is colored in white. The NTD domain of the CA monomer of all the hexamers is represented as cyan spheres. The highlighted region (orange box) shows 5 LEN molecules bound to 2 adjoining CA hexamers that are classified as distorted lattice. The snapshot in the right-center panel shows LEN bound to CA that are classified as ordered lattice. The right panel shows the schematic of LEN molecule (represented as orange circle) binding to ordered and distorted CA hexamer ring. (B) The probability distribution of the CA-LEN potential energy calculated for all LEN molecules bound to CA sites that are part of the ordered (LENo) and distorted (LENd) lattice. The CA-LEN potential energy calculations were performed for the final 250 × 106 τCG. (C) Deviation of the kCA-CA(i) for each CA monomer i relative to the value is calculated for the free capsid. The CANTD domain of all CA monomers for which the kCA-CA(i) increase and decrease relative to the free capsid is shown in red and blue, respectively. The red patches indicate effective stabilization relative to free capsid.