Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Modeling HIV-1 nuclear entry with nucleoporin-gated DNA-origami channels

Nature Structural & Molecular Biology volume 30, pages 425–435 (2023)Cite this article

Subjects

Abstract

Delivering the virus genome into the host nucleus through the nuclear pore complex (NPC) is pivotal in human immunodeficiency virus 1 (HIV-1) infection. The mechanism of this process remains mysterious owing to the NPC complexity and the labyrinth of molecular interactions involved. Here we built a suite of NPC mimics—DNA-origami-corralled nucleoporins with programmable arrangements—to model HIV-1 nuclear entry. Using this system, we determined that multiple cytoplasm-facing Nup358 molecules provide avid binding for capsid docking to the NPC. The nucleoplasm-facing Nup153 preferentially attaches to high-curvature regions of the capsid, positioning it for tip-leading NPC insertion. Differential capsid binding strengths of Nup358 and Nup153 constitute an affinity gradient that drives capsid penetration. Nup62 in the NPC central channel forms a barrier that viruses must overcome during nuclear import. Our study thus provides a wealth of mechanistic insight and a transformative toolset for elucidating how viruses like HIV-1 enter the nucleus.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Design and assembly of DNA-origami channels as mimics of the NPC scaffold.
Fig. 2: Interactions between HIV-1 capsids and NuPODs containing copies of a single nup species.
Fig. 3: Nup153 prefers highly curved tip regions of the HIV-1 capsid for avid NuPOD association.
Fig. 4: Stable HIV-1 capsid binding requires multiple copies of Nup358 or Nup153 in a NuPOD.
Fig. 5: Penetration into multi-nup NuPODs is maximized when capsids travel along a built-in affinity gradient in the NPC.
Fig. 6: Nup62 acts as a barrier to block capsid entry.
Fig. 7: Model of HIV-1 nuclear entry.

Similar content being viewed by others

Data availability

All the data are available within this paper and its Supplementary Information. Source data are provided with this paper.

References

  1. Yamashita, M. & Emerman, M. Retroviral infection of non-dividing cells: old and new perspectives. Virology 344, 88–93 (2006).

  2. Strambio-De-Castillia, C., Niepel, M. & Rout, M. P. The nuclear pore complex: bridging nuclear transport and gene regulation. Nat. Rev. Mol. Cell Biol. 11, 490–501 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Beck, M. & Hurt, E. The nuclear pore complex: understanding its function through structural insight. Nat. Rev. Mol. Cell Biol. 18, 73–89 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Ganser, B. K., Li, S., Klishko, V. Y., Finch, J. T. & Sundquist, W. I. Assembly and analysis of conical models for the HIV-1 core. Science 283, 80–83 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Yamashita, M. & Engelman, A. N. Capsid-dependent host factors in HIV-1 infection. Trends Microbiol. 25, 741–755 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hulme, A. E., Kelley, Z., Okocha, E. A. & Hope, T. J. Identification of capsid mutations that alter the rate of HIV-1 uncoating in infected cells. J. Virol. 89, 643–651 (2015).

    Article  PubMed  Google Scholar 

  7. Temple, J., Tripler, T. N., Shen, Q. & Xiong, Y. A snapshot of HIV-1 capsid-host interactions. Curr. Res Struct. Biol. 2, 222–228 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Francis, A. C. & Melikyan, G. B. Single HIV-1 imaging reveals progression of infection through CA-dependent steps of docking at the nuclear pore, uncoating, and nuclear transport. Cell Host Microbe 23, 536–548 (2018).

    Article  Google Scholar 

  9. Francis, A. C., Marin, M., Shi, J., Aiken, C. & Melikyan, G. B. Time-resolved imaging of single HIV-1 Uncoating in vitro and in living cells. PLoS Pathog. 12, e1005709 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Burdick, R. C. et al. HIV-1 uncoats in the nucleus near sites of integration. Proc. Natl Acad. Sci. USA 117, 5486–5493 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zila, V. et al. Cone-shaped HIV-1 capsids are transported through intact nuclear pores. Cell 184, 1032–1046 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Muller, T. G. et al. HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells. eLife 10, e64776 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Li, C., Burdick, R. C., Nagashima, K., Hu, W. S. & Pathak, V. K. HIV-1 cores retain their integrity until minutes before uncoating in the nucleus. Proc. Natl Acad. Sci. USA 118, e2019467118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Dharan, A., Bachmann, N., Talley, S., Zwikelmaier, V. & Campbell, E. M. Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus. Nat. Microbiol. 5, 1088–1095 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Selyutina, A., Persaud, M., Lee, K., KewalRamani, V. & Diaz-Griffero, F. Nuclear import of the HIV-1 core precedes reverse transcription and uncoating. Cell Rep. 32, 108201 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Guedan, A. et al. HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration. PLoS Pathog. 17, e1009484 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kane, M. et al. Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2. eLife 7, e35738 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Di Nunzio, F. et al. Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration. PLoS ONE 7, e46037 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Ebina, H., Aoki, J., Hatta, S., Yoshida, T. & Koyanagi, Y. Role of Nup98 in nuclear entry of human immunodeficiency virus type 1 cDNA. Microbes Infect. 6, 715–724 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Lee, K. et al. Flexible use of nuclear import pathways by HIV-1. Cell Host Microbe 7, 221–233 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang, R. N., Mehla, R. & Chauhan, A. Perturbation of host nuclear membrane component RanBP2 impairs the nuclear import of human immunodeficiency virus-1 preintegration complex (DNA). PLoS ONE 5, e15620 (2010).

  22. Matreyek, K. A. & Engelman, A. The requirement for nucleoporin NUP153 during human immunodeficiency virus type 1 infection is determined by the viral capsid. J. Virol. 85, 7818–7827 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dharan, A. et al. KIF5B and Nup358 coperatively mediate the nuclear import of HIV-1 during infection. PLoS Pathog. 12, e1005700 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Guo, J. et al. The transmembrane nucleoporin Pom121 ensures efficient HIV-1 pre-integration complex nuclear import. Virology 521, 169–174 (2018).

    Article  CAS  PubMed  Google Scholar 

  25. Lin, D. H. & Hoelz, A. The structure of the nuclear pore complex (an update). Annu. Rev. Biochem. 88, 725–783 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. von Appen, A. et al. In situ structural analysis of the human nuclear pore complex. Nature 526, 140–143 (2015).

    Article  Google Scholar 

  27. Zimmerli, C. E. et al. Nuclear pores dilate and constrict in cellulo. Science 374, eabd9776 (2021).

    Article  CAS  PubMed  Google Scholar 

  28. Schuller, A. P. et al. The cellular environment shapes the nuclear pore complex architecture. Nature 598, 667–671 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Matreyek, K. A., Yucel, S. S., Li, X. & Engelman, A. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity. PLoS Pathog. 9, e1003693 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Price, A. J. et al. Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly. PLoS Pathog. 10, e1004459 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Bhattacharya, A. et al. Structural basis of HIV-1 capsid recognition by PF74 and CPSF6. Proc. Natl Acad. Sci. USA 111, 18625–18630 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bichel, K. et al. HIV-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein NUP358. Retrovirology 10, 81 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Di Nunzio, F. et al. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication. Virology 440, 8–18 (2013).

    Article  PubMed  Google Scholar 

  34. Buffone, C. et al. Nup153 unlocks the nuclear pore complex for HIV-1 nuclear translocation in nondividing cells. J. Virol. 92, e00648-18 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Lin, D. H., Zimmermann, S., Stuwe, T., Stuwe, E. & Hoelz, A. Structural and functional analysis of the C-terminal domain of Nup358/RanBP2. J. Mol. Biol. 425, 1318–1329 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Schaller, T. et al. HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency. PLoS Pathog. 7, e1002439 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Seeman, N. C. & Sleiman, H. F. DNA nanotechnology. Nat. Rev. Mater. 3, 17068 (2018).

  39. Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into twisted and curved nanoscale shapes. Science 325, 725–730 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dey, S. et al. DNA origami. Nat. Rev. Methods Prim. 1, 13 (2021).

    Article  Google Scholar 

  41. Fisher, P. D. E. et al. A programmable DNA origami platform for organizing intrinsically disordered nucleoporins within nanopore confinement. ACS Nano. 12, 1508–1518 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ketterer, P. et al. DNA origami scaffold for studying intrinsically disordered proteins of the nuclear pore complex. Nat. Commun. 9, 902 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Shen, Q. et al. DNA-origami nanotrap for studying the selective barriers formed by phenylalanine-glycine-rich nucleoporins. J. Am. Chem. Soc. 143, 12294–12303 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Huang, G. X. Y. et al. Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex by cryo-electron microscopy single particle analysis. Cell Res. 30, 520–531 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Lin, C., Perrault, S. D., Kwak, M., Graf, F. & Shih, W. M. Purification of DNA-origami nanostructures by rate-zonal centrifugation. Nucleic Acids Res. 41, e40 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Ori, A. et al. Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines. Mol. Syst. Biol. 9, 648 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Carmofonseca, M., Kern, H. & Hurt, E. C. Human nucleoporin P62 and the essential yeast nuclear-pore protein Nsp1 show sequence homology and a similar domain organization. Eur. J. Cell Biol. 55, 17–30 (1991).

    CAS  PubMed  Google Scholar 

  48. Selyutina, A., Bulnes-Ramos, A. & Diaz-Griffero, F. Binding of host factors to stabilized HIV-1 capsid tubes. Virology 523, 1–5 (2018).

    Article  CAS  PubMed  Google Scholar 

  49. Zhao, G. et al. Rhesus TRIM5α disrupts the HIV-1 capsid at the inter-hexamer interfaces. PLoS Pathog. 7, e1002009 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhang, Z. et al. T = 4 icosahedral HIV-1 capsid as an immunogenic vector for HIV-1 V3 loop epitope display. Viruses 10, 667 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Shah, V. B. & Aiken, C. In vitro uncoating of HIV-1 cores. https://doi.org/10.3791/3384 (2011).

  52. Liu, J., Sadre-Marandi, F., Tavener, S. & Chen, C. Curvature concentrations on the HIV-1 capsid. https://doi.org/10.1515/mlbmb-2015-0003 (2015).

  53. Shen Q, et al. A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid. Preprint at bioRxiv https://doi.org/10.1101/2020.08.10.245522 (2020).

  54. Labokha, A. A. et al. Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes. EMBO J. 32, 204–218 (2013).

    Article  CAS  PubMed  Google Scholar 

  55. Patel, S. S., Belmont, B. J., Sante, J. M. & Rexach, M. F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex. Cell 129, 83–96 (2007).

    Article  CAS  PubMed  Google Scholar 

  56. Chug, H., Trakhanov, S., Hulsmann, B. B., Pleiner, T. & Gorlich, D. Crystal structure of the metazoan Nup62*Nup58*Nup54 nucleoporin complex. Science 350, 106–110 (2015).

    Article  CAS  PubMed  Google Scholar 

  57. Hu, T. H., Guan, T. L. & Gerace, L. Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins. J. Cell Biol. 134, 589–601 (1996).

    Article  CAS  PubMed  Google Scholar 

  58. Fahrenkrog, B. et al. Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex. J. Struct. Biol. 140, 254–267 (2002).

    Article  CAS  PubMed  Google Scholar 

  59. Wente, S. R. & Rout, M. P. The nuclear pore complex and nuclear transport. Cold Spring Harb. Perspect. Biol. 2, a000562 (2010).

  60. Link, J. O. et al. Clinical targeting of HIV capsid protein with a long-acting small molecule. Nature 584, 614–618 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Bester, S. M. et al. Structural and mechanistic bases for a potent HIV-1 capsid inhibitor. Science 370, 360–364 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Busnadiego, I. et al. Host and viral determinants of Mx2 antiretroviral activity. J. Virol. 88, 7738–7752 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Price, A. J. et al. CPSF6 defines a conserved capsid interface that modulates HIV-1 replication. PLoS Pathog. 8, e1002896 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Bejarano, D. A. et al. HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex. eLife 8, e41800 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Fernandez, J. et al. Transportin-1 binds to the HIV-1 capsid via a nuclear localization signal and triggers uncoating. Nat. Microbiol. 4, 1840–1850 (2019).

    Article  CAS  PubMed  Google Scholar 

  66. Song, Y. et al. Importin KPNA2 confers HIV-1 pre-integration complex nuclear import by interacting with the capsid protein. Antivir. Res. 200, 105289 (2022).

    Article  CAS  PubMed  Google Scholar 

  67. Shen, Q., Wu, C., Freniere, C., Tripler, T. N. & Xiong, Y. Nuclear import of HIV-1. Viruses 13, 2242 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Hoelz, A., Debler, E. W. & Blobel, G. The structure of the nuclear pore complex. Annu. Rev. Biochem. 80, 613–643 (2011).

    Article  CAS  PubMed  Google Scholar 

  69. Fay, N. & Pante, N. Nuclear entry of DNA viruses. Front. Microbiol. 6, 467 (2015).

  70. Gallucci, L. & Kann, M. Nuclear import of hepatitis B virus capsids and genome. Viruses 9, 21 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Douglas, S. M. et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001–5006 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Pauwels, R. et al. Sensitive and rapid assay on Mt-4 cells for detection of antiviral compounds against the AIDS virus. J. Virol. Methods 16, 171–185 (1987).

    Article  CAS  PubMed  Google Scholar 

  73. Gallay, P., Hope, T., Chin, D. & Trono, D. HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway. Proc. Natl Acad. Sci. USA 94, 9825–9830 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Wehrly, K. & Chesebro, B. p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents. Methods 12, 288–293 (1997).

    Article  CAS  PubMed  Google Scholar 

  75. Chesebro, B., Wehrly, K., Nishio, J. & Perryman, S. Macrophage-tropic human-immunodeficiency-virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates—definition of critical amino-acids involved in cell tropism. J. Virol. 66, 6547–6554 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Xiong and Lin labs for discussion. This work was supported by NIH grants P50AI150481 (Y. X., C. A. and A. N. E.), U54AI170791 (Y.X., C. A. and A. N. E.), R01AI052014 (A. N. E.), T32AI007386 (G. J. B.), R21GM109466 (C. L. and C. P. L.), R01GM105672 (C. P. L.), R01AI162260 (Y. X. and C. L.), a Collaboration Development Award Program from the Pittsburgh Center for HIV Protein Interactions (C.L.) and a Singapore Agency for Science, Technology and Research Graduate Scholarship (Q. X.)

Author information

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Qi Shen, Chunxiang Wu, Shuai Yuan, Ran Yang & Yong Xiong

  2. Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA

    Qi Shen, Qingzhou Feng, Qiancheng Xiong, C. Patrick Lusk & Chenxiang Lin

  3. Nanobiology Institute, Yale University, West Haven, CT, USA

    Qi Shen, Qingzhou Feng, Qiancheng Xiong, Taoran Tian & Chenxiang Lin

  4. Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

    Jiong Shi & Christopher Aiken

  5. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA

    Gregory J. Bedwell & Alan N. Engelman

  6. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Gregory J. Bedwell & Alan N. Engelman

  7. Department of Biomedical Engineering, Yale University, New Haven, CT, USA

    Chenxiang Lin

Authors
  1. Qi Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qingzhou Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chunxiang Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qiancheng Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Taoran Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shuai Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jiong Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gregory J. Bedwell
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ran Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Christopher Aiken
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Alan N. Engelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. C. Patrick Lusk
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Chenxiang Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Yong Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Q. S., C. L. and Y. X. conceived and designed the project. Q. S., Q. F., C. W., T. T., S. Y., J. S., G. J. B. and R. Y. performed the experimental work. Q. S. and T. T. performed the experimental analysis. Q. S. wrote the original draft. Q. S, Q. X., Q. F., C. A., A. N. E., C. P. L., C. L. and Y. X. reviewed and edited the manuscript. All authors participated in the discussions.

Corresponding authors

Correspondence to Chenxiang Lin or Yong Xiong.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks Grigory Tikhomirov and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Editor recognition statement (if applicable to your journal): Florian Ullrich, Carolina Perdigoto, Beth Moorefield and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 DNA-origami designs rendered in caDNAno.

a, Cross-sections (left) and strand diagrams (right) of the 45-nm channel design: (1) 0–32 inner handle version; (2) 48 outer handle version. The scaffold strand is in gray. Staples carrying inner handles and outer handles are shown in red and orange, respectively. Handles (see Methods for sequences, not shown here for clarity) were extended from the 3′ end (marked by a triangle) of staple strands. The rest of the staple strands are in black. b, Cross-sections (left) and strand diagrams (right) of the 40-nm channel design: (P1) top monomer; (P2) bottom monomer. The scaffold strand is in gray. Inner handles and sticky ends are shown in red and blue, respectively. Handles (see Methods for sequences, not shown here for clarity) were extended from the 3′ end of staple strands. The rest of the staple strands are in black. c, Same as (b) but showing the 60-nm channel design.

Extended Data Fig. 2 DNA-origami assembly and purification.

a, Agarose gel electrophoreses showing the dimerization yields of the 40-nm and 60-nm wide DNA channels at different MgCl2 concentrations. Channel (dimer) bands are denoted by asterisks. The experiment was repeated three times with similar results. b, Different sizes of DNA channels purified by rate-zonal centrifugation. Agarose gel (1.5%) electrophoreses show the enrichment of DNA channel in fractions 11–13 (40 nm), 9–10 (60 nm), and 10–12 (45 nm), respectively. Well-folded nanostructure bands are denoted by asterisks. This experiment was repeated three times with similar results.

Source data

Extended Data Fig. 3 Nup purification and CA nanotube co-pelleting assays.

a, Size exclusion chromatography analysis showing that the DNA-conjugated of Nup358RBD4-CycH, MBP-Nup62FL, and MBP-Nup153CTD are mainly monodispersed monomers in solution. Right panels: SDS-PAGE analysis of protein purity. The experiment was repeated three times with similar results. b, Left: co-pelleting assay of purified nups using A14C/E45C disulfide crosslinked CA nanotubes. Soluble (S) and pellet (P) denote fractions of co-pelleting assays. Total (T) denotes the nup-CA tube mixture before fractionation. Right: percentages of various nups bound to CA tubes in the co-pelleting assays in PBS buffer. Data are plotted as mean±SD of three independent experiments (n = 3), with individual data points shown as black circles. Differences were determined by one-way ANOVA and Tukey’s multiple comparisons test. Unless marked in the graph, comparisons were made with MBP-Nup153CTD. Full statistical results are provided in source data.

Source data

Extended Data Fig. 4 Characterizing 45-nm NuPODs and their interactions with CA assemblies and virus cores.

a, The NuPODs assembled with 32 copies of nups are characterized by SDS–agarose gel electrophoresis. The experiment was repeated three times with similar results. b, Negative-stain electron micrographs of CA assemblies and purified virus cores. The negative-stain EM experiments were repeated three times with similar results. Scale bar, 100 nm. c, Quantification of the interactions between HIV-1 capsid assemblies and NuPODs with various nups. Percentage values in the "CA tube" and "CA sphere" rows describe the population of NuPODs bound to CA assemblies. As the concentration of purified natural HIV-1 cores is low, percentage values in the "Virus core" row describe the population of cores occupied by NuPODs. n = 121–223. d, Schematics and negative-stain electron micrographs of CA nanotubes threading through the Nup153 NuPOD. The negative-stain EM results were repeated three times with similar results. Scale bar: 100 nm. Bottom: quantification of all binding events between Nup153 NuPODs and CA tubes. n = 447.

Source data

Extended Data Fig. 5 Nup358 or Nup153 NuPODs interacting with CA assemblies.

a, Negative-stain electron micrographs of Nup358 NuPODs (top) or Nup153 NuPODs (bottom) mixed with in vitro assembled WT (left), P90A (middle), or N57D (right) CA tubes. All negative-stain EM experiments were repeated three times with similar results. Scale bar, 50 nm. b, Schematics and negative-stain electron micrographs of the Nup153 NuPOD bound to in vitro assembled capsid cones. The experiment was repeated three times with similar results. Scale bar, 50 nm.

Extended Data Fig. 6 Co-pelleting assays of NuPODs with variants of CA tubes.

a, Schematic of the NuPOD-CA nanotube co-pelleting assay. b, Top: Supernatant (S) and pellet (P) fractions collected after co-pelleting Cy3-labelled NuPODs and A14C/E45C disulfide crosslinked CA tubes were analyzed on a 0.05% SDS–agarose gel. Fluorescence signals were used to detect the Cy3-labelled NuPODs. Black arrowheads indicate the NuPOD bands. Bottom: a plot showing the populations of Cy3-labelled NuPODs in the supernatant and pellet fractions. Data are plotted as mean±SD of three independent experiments (n = 3), with individual data points shown as black circles. c, Supernatant (S) and pellet (P) fractions from the co-pelleting assays analyzed by SDS-PAGE to detect the protein component of the NuPODs. The experiment was repeated three times with similar results. d, Negative-stain electron micrographs of supernatant (S) and pellet (P) samples from the co-pelleting assays. The experiment was repeated three times with similar results. Scale bar, 100 nm.

Source data

Extended Data Fig. 7 Nup153 prefers highly-curved regions of HIV-1 capsid.

a, Left: supernatant (S) and pellet (P) fractions of CA spheres, nanotubes, or their mixture after centrifugation at 8,000 × g, analyzed by SDS-PAGE. Right: negative-stain electron micrographs of supernatant (S) and pellet (P) fractions of the CA sphere/nanotube mixture. The CA spheres remained in the supernatant, while the CA tubes were completely pelleted. Scale bar, 50 nm. This experiment was repeated three times with similar results. b, Schematics and negative-stain electron micrographs of Nup153 NuPODout (top left) binding to CA nanotubes (top right), CA spheres (bottom left), and a 1:1 mixture of CA spheres and nanotubes (bottom right). Note the lack of NuPOD binding to the flat tube surface in the competition binding experiment. All negative-stain EM experiments were repeated three times with similar results. Scale bar, 50 nm.

Source data

Extended Data Fig. 8 Negative-stain electron micrographs of CA spheres mixed with NuPODs.

a, 1 to 32 copies of Nup358RBD4-CycH. b, 1 to 32 copies of Nup153CTD. All negative-stain EM experiments were repeated three times with similar results. Scale bar, 50 nm.

Extended Data Fig. 9 Penetration assay for multilayer NuPODs and controls.

a, The 40-nm and 60-nm wide NuPODs containing multiple layers of nups analyzed by SDS–agarose gel electrophoresis. The experiment was repeated three times with similar results. b, Schematics and negative-stain electron micrographs of the 40-nm (right) and 60-nm (left) wide two-layer Nup358-Nup153 NuPODs (32 nups per layer). Direction indexes on NuPODs are marked by black arrowheads. The experiment was repeated three times with similar results. Scale bar, 50 nm. c, Left: cartoon models and representative negative-stain electron micrographs of purified virus cores inserted into single-layer Nup358 or Nup153 NuPODs. Scale bar, 50 nm. Right: Quantification of capsid insertion depths. Data are plotted as mean±SD, The HIV-1 capsid-NuPOD binding events (n) were captured over three independent experiments: Nup358 group (n = 50), Nup153 group (n = 52). d, Capsid insertion depths into NuPODs, measured from the tip of the capsid to the point it enters the DNA channel (entry point). Direction indexes on NuPODs are marked by black arrowheads. The experiment was repeated three times with similar results. Scale bar, 50 nm. e, Negative-stain electron micrographs of 32 copies of Nup62 grafted on the exterior of the 45-nm DNA-origami channel. This experiment was repeated three times with similar results. Scale bar, 50 nm.

Source data

Extended Data Fig. 10 Galleries of representative negative-stain electron micrographs of HIV-1 core-NuPOD interactions.

The purified virus cores bound to NuPODs loaded with different nups, with schematics on the left. All negative-stain EM experiments were repeated three times with similar results. Scale bar, 50 nm. More high-magnification images of NuPOD-virus core interactions are included in Supplementary Fig. 1–7.

Supplementary information

Supplementary Information

Supplementary Figures 1–7.

Reporting Summary

Source data

Source Data Fig. 3

Unprocessed gels.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 2

Unprocessed gels.

Source Data Extended Data Fig. 3

Unprocessed gels.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed gels.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed gels.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Unprocessed gels.

Source Data Extended Data Fig. 9

Unprocessed gels.

Source Data Extended Data Fig. 9

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shen, Q., Feng, Q., Wu, C. et al. Modeling HIV-1 nuclear entry with nucleoporin-gated DNA-origami channels. Nat Struct Mol Biol 30, 425–435 (2023). https://doi.org/10.1038/s41594-023-00925-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-023-00925-9

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing