Designed proteins induce the formation of nanocage-containing extracellular vesicles

Journal name:
Nature
Volume:
540,
Pages:
292–295
Date published:
DOI:
doi:10.1038/nature20607
Received
Accepted
Published online
Corrected online

Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids1, 2 or proteins3, 4, 5, but strategies for engineering hybrid biological materials are only beginning to emerge6, 7. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as ‘enveloped protein nanocages’ (EPNs). Robust EPN biogenesis requires protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the endosomal sorting complexes required for transport (ESCRT) machinery8. A variety of synthetic proteins with these functional elements induce EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical analyses and cryo-electron microscopy reveal that one design, EPN-01, comprises small (~100 nm) vesicles containing multiple protein nanocages that closely match the structure of the designed 60-subunit self-assembling scaffold9. EPNs that incorporate the vesicular stomatitis viral glycoprotein can fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These results show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells.

At a glance

Figures

  1. EPN release requires three functional elements.
    Figure 1: EPN release requires three functional elements.

    a, Schematic of the EPN-01 construct, with the membrane-binding N-myristoylation element from HIV-1 Gag in orange (underlined G denotes the myristoylated glycine residue), the I3-01 self-assembly domain in green (the L33R mutation has previously been shown to prevent assembly9), the Myc epitope in blue, and the ESCRT-recruiting HIV-1 p6Gag element in grey. The TSG101/ESCRT-I-, ALIX- and Vpr-binding sites are shown and key interaction residues mutated in this study are underlined. Residue numbers above the construct correspond to individual functional elements, numbers below correspond to the overall construct. b, Left, western blots showing EPN-01 proteins harvested from 293T cell culture supernatants (top blot), cellular EPN-01 proteins in the Triton-insoluble and Triton-soluble fractions (middle blots), and a soluble cellular GAPDH loading control (bottom). To the right, the percentage of EPN-01 and mutants released into the supernatant is plotted (error bars show standard deviations from three technical repetitions).

  2. EPNs comprise cell-derived membrane envelopes containing multiple protein nanocages.
    Figure 2: EPNs comprise cell-derived membrane envelopes containing multiple protein nanocages.

    a, Top, western blot showing EPN-01 in 293F cells (lane 1) or released from 293F cells and treated as indicated (lanes 2–4). Bottom, western blot showing equivalent samples of non-enveloped EPN-01* nanocages purified from E. coli. b, Representative cryo-EM images showing extracellular vesicles/EPNs in culture supernatants from 293T cells that expressed I3-01–Myc (left) or EPN-01 (right). c, Central slice from a cryo-EM tomographic reconstruction of a released EPN; two internal protein nanocages are marked with arrowheads. d, Isosurface model of the 3D cryo-EM reconstruction from c. The EPN membrane is green and individual protein nanocages are gold. e, Single-particle cryo-EM reconstruction of the nanocages released from EPNs following detergent treatment. Charge density from the 5.7 Å resolution electron microscopy reconstruction is shown in grey (contoured at 4.5σ). The I3-01 computational design model9 (green ribbon) was fitted into the density as a rigid body. Scale bars, 300 nm (b), 25 nm (c, d) and 5 nm (e).

  3. EPNs can package macromolecular cargoes and deliver them into new target cells.
    Figure 3: EPNs can package macromolecular cargoes and deliver them into new target cells.

    a, Western blots showing cellular expression and release of I3-01–Myc, EPN-01*, and Myc-tagged VSV-G and BlaM–Vpr constructs (see Supplementary Table 3 for sequence information). Panels are equivalent to those in Fig. 1b. b, Cytoplasmic delivery to target HeLa cells by the three released constructs from a, plotted as percentages of BlaM-positive cells following incubation with the designated quantities of the different EPNs (error bars show standard deviations from two technical repetitions). Inset, cryo-EM image of an EPN-01*/VSV-G EPN with internal protein nanocages (arrowheads) and apparent VSV-G envelope spikes (arrow). Scale bar, 25 nm.

  4. A variety of functional elements and protein architectures support EPN formation.
    Figure 4: A variety of functional elements and protein architectures support EPN formation.

    Schematic illustrations of constructs are shown, together with western blots and release quantification. a, Different membrane-binding domains support EPN release (lanes 1, 3, 5, 7). EPN point mutants shown in lanes 2, 4, 6 and 8 were designed to disrupt the membrane-binding interactions. b, The 24-subunit assembly O3-33 can function as an EPN self-assembly domain (lane 1). Mutants shown in lanes 2–4 were designed to disrupt the membrane-binding, self-assembly and ESCRT-recruiting elements. c, Different ESCRT-recruiting elements can support EPN release (lanes 1, 3, 5). ESCRT-dependent release is demonstrated by loss of EPN release upon co-expression of GFP–VPS4A(E228Q) (lanes 2, 4, 6). d, Membrane-binding, self-assembly and ESCRT-recruiting elements can function from different positions within EPN constructs (lanes 1, 3, 5, 7). ESCRT-dependent release is demonstrated by loss of EPN formation upon co-expression of GFP–VPS4A(E228Q) (lanes 2, 4, 6, 8). Error bars show standard deviations from three technical repetitions.

  5. EPN-01* is released from cells.
    Extended Data Fig. 1: EPN-01* is released from cells.

    a, Schematic of the EPN-01* constructs. Note that the EPN-01* constructs shown in this figure are analogous to the EPN-01 constructs shown in Fig. 1 except that EPN-01* ran as a doublet because a methionine codon at I3-01 position 3 (isoleucine in EPN-01) served as a second translation initiation codon. b, Western blots showing functional and mutant EPN-01* proteins harvested from 293T cell culture supernatants (top panel). The middle panels are western blots showing cellular EPN-01* proteins in the Triton-insoluble fraction (stably bound to membranes) and the Triton-soluble fraction (cytoplasm and weakly bound to membranes), respectively. The bottom panel is a western blot showing a GAPDH loading control from the Triton-soluble fraction. Right, the percentage of each protein released into the supernatant is plotted (error bars show standard deviation from three technical repetitions). In summary, EPN-01 and EPN-01* were functionally equivalent and were employed interchangeably in our studies. For western blot source data, see Supplementary Fig. 1.

  6. EPN-01* forms nanocages that resemble those of the I3-01 scaffold.
    Extended Data Fig. 2: EPN-01* forms nanocages that resemble those of the I3-01 scaffold.

    a, Size-exclusion chromatograms (Superose 6 10/300 GL) of EPN-01*, EPN-01(ΔM), EPN-01(ΔL1 + ΔL2), I3-01, and 1wa3-wt purified from E. coli. 1wa3-wt is the wild-type trimeric aldolase from which I3-01 was derived9. All of the proteins eluted as nanocages except for 1wa3-wt, which eluted at the volume expected for the trimer. b, SDS–PAGE of the peak fraction from each of the chromatograms in a. Molecular weight marker positions are indicated in kilodaltons. c, Negative stain electron microscopy image of purified E. coli EPN-01*, showing a homogeneous field of particles (~25 nm diameter).

  7. EPN-01 constructs are released within membrane vesicles.
    Extended Data Fig. 3: EPN-01 constructs are released within membrane vesicles.

    a, Detergent-dependent antibody accessibility of EPN-01 protein released from mammalian cells. Western blots of EPN-01 harvested from mammalian cell supernatants (top two blots) or non-enveloped EPN-01* nanocages purified from E. coli (bottom two blots) are shown. Samples were treated with 0.1% or 0.5% CHAPS, as indicated. Lane 1 shows input protein, and lanes 2–5 show bound and unbound fractions from immunoprecipitations with anti-Myc-agarose (lanes 2 and 3) or control anti-rabbit-IgG-agarose (lanes 4 and 5). b, Detergent-dependent aldolase substrate accessibility of EPN-01* protein released from mammalian cells. EPN-01* produced in 293F cells (top) or E. coli (bottom) were monitored for KPDG aldolase activity in the presence or absence of detergent. Note that the protein produced in mammalian cells only shows enzymatic activity in the presence of detergent, whereas the bacterially produced protein is equally active in both conditions. c, Size distribution of EPNs released into the culture supernatants of 293T cells expressing EPN-01*. n = 142 EPNs total; mean size, 107 ± 44. d, EPN-01* and associated cellular proteins are released from 293T cells. Western blots showing levels of I3-01–Myc, EPN-01*, ALIX and actin expressed in 293T cells (left) and released into the culture supernatant (right). e, Numbers of EPN-01* protein nanocages encapsulated within six different EPN-01* vesicles of varying sizes. Nanocage numbers were determined by visual counting of reconstructed EPNs from cryo-tomographic tilt series. An average EPN vesicle of diameter 110 nm has a volume of 7 × 105 nm3 and each nanocage has an estimated volume of approximately 8 × 103 nm3. A 110 nm diameter vesicle could therefore theoretically contain as many as 90 close-packed nanocages, but they actually contain, on average, around 14 nanocages.

  8. EPN-01* protein released from 293T cells assembles into nanocages that closely correspond to the designed I3-01 structure.
    Extended Data Fig. 4: EPN-01* protein released from 293T cells assembles into nanocages that closely correspond to the designed I3-01 structure.

    a, Cryo-EM image of extracellular EPN-01* nanocages released from vesicles by CHAPS detergent treatment. b, Cryo-EM 2D class averages showing the ten most prevalent classes, together with the numbers of particles in each class. c, Fourier shell correlations between the two half model charge density maps unmasked (blue curve) and masked (magenta curve). Orange curve shows Fourier shell correlations between the final charge density model and the I3-01 design model (with an additional residue built into the N terminus).

  9. Intracellular localization of EPN-01 and EPN-18.
    Extended Data Fig. 5: Intracellular localization of EPN-01 and EPN-18.

    a, Confocal fluorescence images of HeLa cells transfected with EPN-01 (left) and EPN-01(ΔM) (right) stained for Myc (green), DNA (blue) and actin (red). Note that EPN-01 is localized primarily in intracellular compartments and also at the plasma membrane (white arrow), whereas EPN-01(ΔM) is cytoplasmic. b, Confocal fluorescence images of HeLa cells transfected with EPN-18 (left) and EPN-18(ΔM) (right). Note that EPN-18 is predominantly localized at the plasma membrane (white arrow), whereas EPN-18(ΔM) concentrates at internal puncta.

  10. Intracellular localization and extracellular release of EPN-01.
    Extended Data Fig. 6: Intracellular localization and extracellular release of EPN-01.

    a, Immunogold labelling of thin sections from 293T cells expressing EPN-01. Right image shows the boundaries of plasma membrane (cyan), cytosolic (black) and intracellular compartments (red) used for quantification (see d). b, Immunogold labelling of thin sections from 293T cells expressing EPN-01(ΔM). Right image shows the boundaries used for quantification, colour-coded as in a, except that intracellular compartment(s) could not be identified in this case because the EPN-01(ΔM) mutant does not localize there. c, Immunogold labelling of thin sections from 293T cells expressing EPN-01 showing budding and released EPNs. Right image shows the boundaries used for quantification as in a, plus boundaries to quantify gold particle densities in released EPNs (green). Image to the left shows an expanded view of two EPNs that appear to be budding from the plasma membrane. d, Image quantification confirmed that EPN-01 is enriched at the plasma membrane, within intracellular compartments, and in released EPNs. The P value corresponding to the difference between plasma membrane localization of EPN-01 and EPN-01(ΔM) is 7 × 10−5, indicated by the three asterisks (unpaired t-test, n = 11 images for EPN-01 and n = 9 images for EPN-01(ΔM)).

  11. EPNs can package biological cargoes and deliver them to the cytoplasm of target HeLa cells.
    Extended Data Fig. 7: EPNs can package biological cargoes and deliver them to the cytoplasm of target HeLa cells.

    a, Schematic illustration showing the production, assembly, and release of EPNs incorporating BlaM–Vpr and VSV-G proteins (left), and detection of uptake and target cell membrane fusion using a BlaM colourimetric activity assay (right). b, Co-expression of VSV-G increases the number of vesicles that contain spikes, as evaluated by scoring >140 vesicles as either ‘containing’ or ‘not containing’ surface spikes (images like the one shown in the inset of Fig. 3b were scored independently by two different people, one blinded, and their counts were averaged). c, Western blots showing cellular expression and release of EPN-01 and Myc-tagged GFP constructs with or without fused Vpr (see Supplementary Table 3 for sequence information). Top blot shows released protein, middle and bottom blots show expression of Myc-tagged proteins and GAPDH in whole-cell lysates, respectively. Lane 1 shows co-expression of EPN-01 with Myc–GFP–Vpr, lane 2 shows co-expression of EPN-01 with Myc–GFP. d, Flow cytometric analyses of HeLa cells loaded with the fluorescent CCF2 β-lactamase substrate and incubated with increasing quantities of wild-type EPN-01*/VSV-G/BlaM–Vpr (top row), EPN-01*/VSV-G(P127D) mutant/BlaM–Vpr (middle row), and EPN-01*(LF45AA) mutant/VSV-G/BlaM–Vpr (bottom row).

  12. Aldolase and protease protection assays for EPNs with a variety of functional elements and protein architectures.
    Extended Data Fig. 8: Aldolase and protease protection assays for EPNs with a variety of functional elements and protein architectures.

    Schematic illustrations and analyses of the 16 EPN constructs that yielded robust EPN biogenesis are shown, as well as one negative control. Each panel shows the construct, a representative plot of aldolase activity in the presence (black line) and absence (grey line) of detergent, and a western blot analysis of the protease protection assay. Arrowheads next to each blot denote the full-length protein. Aldolase activity was monitored by disappearance of absorbance at 339 nm. a, Different membrane-binding elements support EPN formation. b, EPN-02, also referred to as EPN-01(ΔM), is a negative control construct in which the myristoylation site was inactivated by mutation. Both assays reveal that EPN-02 protein was not released from cells. c, EPN-51, which uses the designed 24-subunit protein assembly O3-33 as a self-assembly domain, forms an EPN with an intact membrane envelope. The aldolase assay was not included because O3-33 is not an aldolase. d, Different ESCRT-recruiting elements can support EPN formation. The asterisk next to the blot of EPN-36 signifies that the blot was overexposed: EPN-36 reproducibly yielded fainter bands on western blots than would be expected based on its aldolase activity and analyses of SDS–PAGE gels stained with Coomassie. e, Membrane-binding, self-assembly, and ESCRT-recruiting elements can function from different positions within EPN constructs. EPN-11 is a permutation of EPN-07, while EPN-23, EPN-24, and EPN-25 are permutations of EPN-18.

Videos

  1. Cryo-EM tomographic reconstruction of a released EPN
    Video 1: Cryo-EM tomographic reconstruction of a released EPN
    Shown is the raw charge density in different slices of the tomogram (first pass) and isosurface renderings (second pass) of individual protein nanocages (gold) and the limiting membrane of the EPN (green).
  2. Single particle cryo-EM reconstruction of released EPN-01* protein nanocage.
    Video 2: Single particle cryo-EM reconstruction of released EPN-01* protein nanocage.
    Shown are the raw charge density (grey, contoured at 4.5 σ) and a rigid body fitting of the I3-01 design model (dark green) plus one Nterminal amino acid (yellow) added to I3-01.

Change history

Corrected online 07 December 2016
Minor corrections were made to Fig. 4c and the legend of Fig. 3a.

References

  1. Rothemund, P. W. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297302 (2006)
  2. Ke, Y., Ong, L. L., Shih, W. M. & Yin, P. Three-dimensional structures self-assembled from DNA bricks. Science 338, 11771183 (2012)
  3. King, N. P. et al. Computational design of self-assembling protein nanomaterials with atomic level accuracy. Science 336, 11711174 (2012)
  4. Suzuki, Y. et al. Self-assembly of coherently dynamic, auxetic, two-dimensional protein crystals. Nature 533, 369373 (2016)
  5. Lai, Y. T. et al. Structure of a designed protein cage that self-assembles into a highly porous cube. Nat. Chem. 6, 10651071 (2014)
  6. Mou, Y., Yu, J. Y., Wannier, T. M., Guo, C. L. & Mayo, S. L. Computational design of co-assembling protein-DNA nanowires. Nature 525, 230233 (2015)
  7. Delebecque, C. J., Lindner, A. B., Silver, P. A. & Aldaye, F. A. Organization of intracellular reactions with rationally designed RNA assemblies. Science 333, 470474 (2011)
  8. Votteler, J. & Sundquist, W. I. Virus budding and the ESCRT pathway. Cell Host Microbe 14, 232241 (2013)
  9. Hsia, Y. et al. Design of a hyperstable 60-subunit protein icosahedron. Nature 535, 136139 (2016)
  10. Wills, J. W. & Craven, R. C. Form, function, and use of retroviral gag proteins. AIDS 5, 639654 (1991)
  11. Weissenhorn, W., Poudevigne, E., Effantin, G. & Bassereau, P. How to get out: ssRNA enveloped viruses and membrane fission. Curr. Opin. Virol . 3, 159167 (2013)
  12. Göttlinger, H. G., Sodroski, J. G. & Haseltine, W. A. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc. Natl Acad. Sci. USA 86, 57815785 (1989)
  13. Choi, D. S., Kim, D. K., Kim, Y. K. & Gho, Y. S. Proteomics, transcriptomics and lipidomics of exosomes and ectosomes. Proteomics 13, 15541571 (2013)
  14. Chertova, E. et al. Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J. Virol. 80, 90399052 (2006)
  15. Cavrois, M., De Noronha, C. & Greene, W. C. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat. Biotechnol. 20, 11511154 (2002)
  16. Tobiume, M., Lineberger, J. E., Lundquist, C. A., Miller, M. D. & Aiken, C. Nef does not affect the efficiency of human immunodeficiency virus type 1 fusion with target cells. J. Virol. 77, 1064510650 (2003)
  17. Kondo, E. & Göttlinger, H. G. A conserved LXXLF sequence is the major determinant in p6gag required for the incorporation of human immunodeficiency virus type 1 Vpr. J. Virol. 70, 159164 (1996)
  18. Fredericksen, B. L. & Whitt, M. A. Vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity. J. Virol. 69, 14351443 (1995)
  19. Strack, B., Calistri, A., Craig, S., Popova, E. & Göttlinger, H. G. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114, 689699 (2003)
  20. Martin-Serrano, J., Zang, T. & Bieniasz, P. D. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat. Med. 7, 13131319 (2001)
  21. Monroe, N. & Hill, C. P. Meiotic clade AAA ATPases: protein polymer disassembly machines. J. Mol. Biol. 428 (9 Pt B), 18971911 (2016)
  22. Parent, L. J. et al. Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins. J. Virol. 69, 54555460 (1995)
  23. Frank, G. A. et al. Maturation of the HIV-1 core by a non-diffusional phase transition. Nat. Commun. 6, 5854 (2015)
  24. Wang, X. et al. Hepatitis A virus and the origins of picornaviruses. Nature 517, 8588 (2015)
  25. Feng, Z. et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 496, 367371 (2013)
  26. Reynwar, B. J. et al. Aggregation and vesiculation of membrane proteins by curvature-mediated interactions. Nature 447, 461464 (2007)
  27. Perrault, S. D. & Shih, W. M. Virus-inspired membrane encapsulation of DNA nanostructures to achieve in vivo stability. ACS Nano 8, 51325140 (2014)
  28. Hu, C. M. et al. Nanoparticle biointerfacing by platelet membrane cloaking. Nature 526, 118121 (2015)
  29. EL Andaloussi, S., Mäger, I., Breakefield, X. O. & Wood, M. J. A. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12, 347357 (2013)
  30. György, B., Hung, M. E., Breakefield, X. O. & Leonard, J. N. Therapeutic applications of extracellular vesicles: clinical promise and open questions. Annu. Rev. Pharmacol. Toxicol. 55, 439464 (2015)
  31. Morita, E., Arii, J., Christensen, D., Votteler, J. & Sundquist, W. I. Attenuated protein expression vectors for use in siRNA rescue experiments. Biotechniques 0, 15 (2012)
  32. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343345 (2009)
  33. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671675 (2012)
  34. Thery, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Chapter 3, Unit 3 22 (2006)
  35. Griffiths, J. S. et al. Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase. Bioorg. Med. Chem. 10, 545550 (2002)
  36. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 3651 (2005)
  37. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 7176 (1996)
  38. Zheng, S. Q., Palovcak, E., Armache, J., Cheng, Y. & Agard, D. A. Anisotropic correction of beam-induced motion for improved single-particle electron cryo-microscopy. Preprint at http://dx.doi.org/10.1101/061960 (2016)
  39. de la Rosa-Trevínet al. Scipion: a software framework toward integration, reproducibility and validation in 3D electron microscopy. J. Struct. Biol . 195, 9399 (2016)
  40. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216221 (2015)
  41. Abrishami, V. et al. A pattern matching approach to the automatic selection of particles from low-contrast electron micrographs. Bioinformatics 29, 24602468 (2013)
  42. de la Rosa-Trevín, J. M. et al. Xmipp 3.0: an improved software suite for image processing in electron microscopy. J. Struct. Biol. 184, 321328 (2013)
  43. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519530 (2012)
  44. Vargas, J., Álvarez-Cabrera, A. L., Marabini, R., Carazo, J. M. & Sorzano, C. O. Efficient initial volume determination from electron microscopy images of single particles. Bioinformatics 30, 28912898 (2014)
  45. Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 16051612 (2004)
  46. Penczek, P. A. Resolution measures in molecular electron microscopy. Methods Enzymol . 482, 73100 (2010)

Download references

Author information

Affiliations

  1. Department of Biochemistry, University of Utah, Salt Lake City, Utah 84112, USA

    • Jörg Votteler,
    • David M. Belnap &
    • Wesley I. Sundquist
  2. Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA

    • Cassandra Ogohara,
    • Sue Yi,
    • Yang Hsia,
    • Una Nattermann &
    • Neil P. King
  3. Institute for Protein Design, University of Washington, Seattle, Washington 98195, USA

    • Cassandra Ogohara,
    • Sue Yi,
    • Yang Hsia,
    • Una Nattermann &
    • Neil P. King
  4. Graduate Program in Biological Physics, Structure and Design, University of Washington, Seattle, Washington 98195, USA

    • Yang Hsia &
    • Una Nattermann
  5. Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA

    • David M. Belnap

Contributions

J.V., N.P.K. and W.I.S. designed and coordinated the study. J.V. performed EPN and cellular protein release assays, IP assays, BlaM delivery assays, confocal fluorescence microscopy, and immunogold EM. J.V. and D.M.B. performed cryo-electron microscopy and tomography, including single-particle reconstruction. C.O. and S.Y. designed EPN constructs and produced, purified, and analysed EPN proteins expressed in E. coli. C.O. performed EPN release assays, protease protection assays, and aldolase assays. Y.H. performed aldolase assays and purified I3-01 from E. coli. U.N. performed negative stain EM on proteins purified from E. coli. J.V., C.O., D.M.B., N.P.K. and W.I.S. interpreted data. J.V., N.P.K. and W.I.S. wrote the manuscript.

Competing financial interests

J.V., Y.H., N.P.K. and W.I.S. co-authored a US patent application (62/126,331) with claims relating to Enveloped Protein Nanocages through the CoMotion office at the University of Washington. The authors declare no other competing financial interests.

Corresponding authors

Correspondence to:

Reviewer Information

Nature thanks O. Farokhzad, J. Hurley and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: EPN-01* is released from cells. (138 KB)

    a, Schematic of the EPN-01* constructs. Note that the EPN-01* constructs shown in this figure are analogous to the EPN-01 constructs shown in Fig. 1 except that EPN-01* ran as a doublet because a methionine codon at I3-01 position 3 (isoleucine in EPN-01) served as a second translation initiation codon. b, Western blots showing functional and mutant EPN-01* proteins harvested from 293T cell culture supernatants (top panel). The middle panels are western blots showing cellular EPN-01* proteins in the Triton-insoluble fraction (stably bound to membranes) and the Triton-soluble fraction (cytoplasm and weakly bound to membranes), respectively. The bottom panel is a western blot showing a GAPDH loading control from the Triton-soluble fraction. Right, the percentage of each protein released into the supernatant is plotted (error bars show standard deviation from three technical repetitions). In summary, EPN-01 and EPN-01* were functionally equivalent and were employed interchangeably in our studies. For western blot source data, see Supplementary Fig. 1.

  2. Extended Data Figure 2: EPN-01* forms nanocages that resemble those of the I3-01 scaffold. (185 KB)

    a, Size-exclusion chromatograms (Superose 6 10/300 GL) of EPN-01*, EPN-01(ΔM), EPN-01(ΔL1 + ΔL2), I3-01, and 1wa3-wt purified from E. coli. 1wa3-wt is the wild-type trimeric aldolase from which I3-01 was derived9. All of the proteins eluted as nanocages except for 1wa3-wt, which eluted at the volume expected for the trimer. b, SDS–PAGE of the peak fraction from each of the chromatograms in a. Molecular weight marker positions are indicated in kilodaltons. c, Negative stain electron microscopy image of purified E. coli EPN-01*, showing a homogeneous field of particles (~25 nm diameter).

  3. Extended Data Figure 3: EPN-01 constructs are released within membrane vesicles. (159 KB)

    a, Detergent-dependent antibody accessibility of EPN-01 protein released from mammalian cells. Western blots of EPN-01 harvested from mammalian cell supernatants (top two blots) or non-enveloped EPN-01* nanocages purified from E. coli (bottom two blots) are shown. Samples were treated with 0.1% or 0.5% CHAPS, as indicated. Lane 1 shows input protein, and lanes 2–5 show bound and unbound fractions from immunoprecipitations with anti-Myc-agarose (lanes 2 and 3) or control anti-rabbit-IgG-agarose (lanes 4 and 5). b, Detergent-dependent aldolase substrate accessibility of EPN-01* protein released from mammalian cells. EPN-01* produced in 293F cells (top) or E. coli (bottom) were monitored for KPDG aldolase activity in the presence or absence of detergent. Note that the protein produced in mammalian cells only shows enzymatic activity in the presence of detergent, whereas the bacterially produced protein is equally active in both conditions. c, Size distribution of EPNs released into the culture supernatants of 293T cells expressing EPN-01*. n = 142 EPNs total; mean size, 107 ± 44. d, EPN-01* and associated cellular proteins are released from 293T cells. Western blots showing levels of I3-01–Myc, EPN-01*, ALIX and actin expressed in 293T cells (left) and released into the culture supernatant (right). e, Numbers of EPN-01* protein nanocages encapsulated within six different EPN-01* vesicles of varying sizes. Nanocage numbers were determined by visual counting of reconstructed EPNs from cryo-tomographic tilt series. An average EPN vesicle of diameter 110 nm has a volume of 7 × 105 nm3 and each nanocage has an estimated volume of approximately 8 × 103 nm3. A 110 nm diameter vesicle could therefore theoretically contain as many as 90 close-packed nanocages, but they actually contain, on average, around 14 nanocages.

  4. Extended Data Figure 4: EPN-01* protein released from 293T cells assembles into nanocages that closely correspond to the designed I3-01 structure. (268 KB)

    a, Cryo-EM image of extracellular EPN-01* nanocages released from vesicles by CHAPS detergent treatment. b, Cryo-EM 2D class averages showing the ten most prevalent classes, together with the numbers of particles in each class. c, Fourier shell correlations between the two half model charge density maps unmasked (blue curve) and masked (magenta curve). Orange curve shows Fourier shell correlations between the final charge density model and the I3-01 design model (with an additional residue built into the N terminus).

  5. Extended Data Figure 5: Intracellular localization of EPN-01 and EPN-18. (1,140 KB)

    a, Confocal fluorescence images of HeLa cells transfected with EPN-01 (left) and EPN-01(ΔM) (right) stained for Myc (green), DNA (blue) and actin (red). Note that EPN-01 is localized primarily in intracellular compartments and also at the plasma membrane (white arrow), whereas EPN-01(ΔM) is cytoplasmic. b, Confocal fluorescence images of HeLa cells transfected with EPN-18 (left) and EPN-18(ΔM) (right). Note that EPN-18 is predominantly localized at the plasma membrane (white arrow), whereas EPN-18(ΔM) concentrates at internal puncta.

  6. Extended Data Figure 6: Intracellular localization and extracellular release of EPN-01. (718 KB)

    a, Immunogold labelling of thin sections from 293T cells expressing EPN-01. Right image shows the boundaries of plasma membrane (cyan), cytosolic (black) and intracellular compartments (red) used for quantification (see d). b, Immunogold labelling of thin sections from 293T cells expressing EPN-01(ΔM). Right image shows the boundaries used for quantification, colour-coded as in a, except that intracellular compartment(s) could not be identified in this case because the EPN-01(ΔM) mutant does not localize there. c, Immunogold labelling of thin sections from 293T cells expressing EPN-01 showing budding and released EPNs. Right image shows the boundaries used for quantification as in a, plus boundaries to quantify gold particle densities in released EPNs (green). Image to the left shows an expanded view of two EPNs that appear to be budding from the plasma membrane. d, Image quantification confirmed that EPN-01 is enriched at the plasma membrane, within intracellular compartments, and in released EPNs. The P value corresponding to the difference between plasma membrane localization of EPN-01 and EPN-01(ΔM) is 7 × 10−5, indicated by the three asterisks (unpaired t-test, n = 11 images for EPN-01 and n = 9 images for EPN-01(ΔM)).

  7. Extended Data Figure 7: EPNs can package biological cargoes and deliver them to the cytoplasm of target HeLa cells. (320 KB)

    a, Schematic illustration showing the production, assembly, and release of EPNs incorporating BlaM–Vpr and VSV-G proteins (left), and detection of uptake and target cell membrane fusion using a BlaM colourimetric activity assay (right). b, Co-expression of VSV-G increases the number of vesicles that contain spikes, as evaluated by scoring >140 vesicles as either ‘containing’ or ‘not containing’ surface spikes (images like the one shown in the inset of Fig. 3b were scored independently by two different people, one blinded, and their counts were averaged). c, Western blots showing cellular expression and release of EPN-01 and Myc-tagged GFP constructs with or without fused Vpr (see Supplementary Table 3 for sequence information). Top blot shows released protein, middle and bottom blots show expression of Myc-tagged proteins and GAPDH in whole-cell lysates, respectively. Lane 1 shows co-expression of EPN-01 with Myc–GFP–Vpr, lane 2 shows co-expression of EPN-01 with Myc–GFP. d, Flow cytometric analyses of HeLa cells loaded with the fluorescent CCF2 β-lactamase substrate and incubated with increasing quantities of wild-type EPN-01*/VSV-G/BlaM–Vpr (top row), EPN-01*/VSV-G(P127D) mutant/BlaM–Vpr (middle row), and EPN-01*(LF45AA) mutant/VSV-G/BlaM–Vpr (bottom row).

  8. Extended Data Figure 8: Aldolase and protease protection assays for EPNs with a variety of functional elements and protein architectures. (223 KB)

    Schematic illustrations and analyses of the 16 EPN constructs that yielded robust EPN biogenesis are shown, as well as one negative control. Each panel shows the construct, a representative plot of aldolase activity in the presence (black line) and absence (grey line) of detergent, and a western blot analysis of the protease protection assay. Arrowheads next to each blot denote the full-length protein. Aldolase activity was monitored by disappearance of absorbance at 339 nm. a, Different membrane-binding elements support EPN formation. b, EPN-02, also referred to as EPN-01(ΔM), is a negative control construct in which the myristoylation site was inactivated by mutation. Both assays reveal that EPN-02 protein was not released from cells. c, EPN-51, which uses the designed 24-subunit protein assembly O3-33 as a self-assembly domain, forms an EPN with an intact membrane envelope. The aldolase assay was not included because O3-33 is not an aldolase. d, Different ESCRT-recruiting elements can support EPN formation. The asterisk next to the blot of EPN-36 signifies that the blot was overexposed: EPN-36 reproducibly yielded fainter bands on western blots than would be expected based on its aldolase activity and analyses of SDS–PAGE gels stained with Coomassie. e, Membrane-binding, self-assembly, and ESCRT-recruiting elements can function from different positions within EPN constructs. EPN-11 is a permutation of EPN-07, while EPN-23, EPN-24, and EPN-25 are permutations of EPN-18.

Supplementary information

Video

  1. Video 1: Cryo-EM tomographic reconstruction of a released EPN (22.28 MB, Download)
    Shown is the raw charge density in different slices of the tomogram (first pass) and isosurface renderings (second pass) of individual protein nanocages (gold) and the limiting membrane of the EPN (green).
  2. Video 2: Single particle cryo-EM reconstruction of released EPN-01* protein nanocage. (10.12 MB, Download)
    Shown are the raw charge density (grey, contoured at 4.5 σ) and a rigid body fitting of the I3-01 design model (dark green) plus one Nterminal amino acid (yellow) added to I3-01.

PDF files

  1. Supplementary Information (934 KB)

    This file contains legends for Supplementary Videos 1 and 2, Supplementary Tables 1-4 and additional references.

  2. Supplementary Figures (908 KB)

    This file contains the uncropped scans with size marker indications for Figures 1b, 2a, 3a, 4a,b,c,d and Extended Data Figures 1, 2b, 3a,d, 7a and 8

Additional data