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

Nature Structural & Molecular Biology (2023)Cite this article

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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.

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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.

Data availability

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

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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

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  1. Qi Shen
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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.

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Correspondence to Chenxiang Lin or Yong Xiong.

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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.

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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.

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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.

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

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