Fig. 4: The CA-N57A mutation impedes capsid passage through NPCs.

a, Targeting of capsid spheres (4 nM; wild type or CA-N57A mutant) to HeLa cell NPCs was tested as in Fig. 1. The mutation reduced the capsid signal at NPCs. b, The 40 nm capsid spheres were tested for partitioning into the GLFG phase as described in Fig. 3. The wild-type spheres entered the FG phase completely. CA-N57A mutant spheres, however, bound only to the surface of the phase, indicating that their FG interactions are not strong enough to melt cohesive, barrier-forming interactions in the FG phase. Numbers are means; bars indicate mean ± s.d. N = number of quantified FG particles and outside areas, respectively. c, The 40 nm capsid spheres (wild type or N57A) were injected (together with a tetrameric tCherry) into either the cytoplasm or the nucleoplasm of fully grown mouse oocytes. Images show confocal laser scans 25 min after injection, after reaching the endpoint distributions. Cytoplasmically injected wild- type capsids not only bound to the NE but also traversed NPCs and accumulated in intranuclear foci (indicated by white arrows); the tCherry injection marker remained cytoplasmic, indicating proper injections and intact NE and NPC barriers. N57A mutant spheres enriched only at the NE but failed to complete NPC passage and to reach the nuclear interior. The nuclear injection confirmed that the mutant would have bound to the intranuclear foci if it had reached the nucleoplasm. Images show the full nucleus and its surrounding cytoplasm. Nuclear (N) and cytoplasmic (C) regions can be distinguished by the injection marker (in magenta). NE and nucleolus are indicated by lines in the merged images. See Extended Data Fig. 7 for z-stacks and Extended Data Fig. 6 for a complete oocyte imaged after cytoplasmic injection of wild-type 40 nm spheres. Scan settings were identical for each wild type and N57A mutant pair. Experiments were repeated independently with identical outcomes (a,b,c: n = 3). Scale bars, 10 μm.

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