Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses

Journal name:
Nature Nanotechnology
Volume:
8,
Pages:
130–136
Year published:
DOI:
doi:10.1038/nnano.2012.248
Received
Accepted
Published online

Abstract

Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction1. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells2, 3, 4, 5, 6, 7, 8. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.

At a glance

Figures

  1. Peptides derived from the HIV-1 glycoprotein gp120 enhance virus infection.
    Figure 1: Peptides derived from the HIV-1 glycoprotein gp120 enhance virus infection.

    a, Overview of analysed gp120 fragments. Active peptides are shown in red. The localization in the HIV-1 Env precursor is indicated, and numbers correspond to the amino-acid positions in the HIV-1 HxB2 gp120 sequence. Superscript numbers indicate the net charge of the peptides. b, EF-A peptide enhances HIV-1 infection. The virus was treated with the peptide and mixtures were used to infect TZM-bl cells. The numbers above the bars give the n-fold enhancement of infection relative to the control without EF-A. RLU/s, relative light units per second; asterisk, over-infection. c, HIV-1 infection-promoting activity of the synthetic gp120 fragments outlined in a. d, EF-C enhances HIV-1 infection more efficiently than SEVI. e,f, Congo Red staining (e) and ThT fluorescence (f) of the tested peptides. OD, optical density; c.p.s., counts per second.

  2. Structural characterization and molecular modelling of EF-C fibrils.
    Figure 2: Structural characterization and molecular modelling of EF-C fibrils.

    a, AFM image of EF-C fibrils. b, Profile along the indicated line in a. Autocorrelation of the profile plot gives a mean half pitch of the fibrils of 26 ± 2 nm. c, FTIR spectrum fitted with Gaussians to determine the secondary structure. The peaks at 1,630 cm−1 and 1,693 cm−1 indicate an antiparallel β-sheet arrangement. d, Molecular model of the EF-C peptide. e, Top and side view of the elementary unit of the fibril comprising four β-strands arranged into a stack of two antiparallel β-sheets. f, Refined molecular model of a fibril exhibiting a helical pitch of 28 nm. C, grey; N, blue; O, red; S, yellow.

  3. EF-C fibrils bind, precipitate and concentrate virions.
    Figure 3: EF-C fibrils bind, precipitate and concentrate virions.

    a, Fibrils formed by Rho-labelled EF-C peptide efficiently boost HIV infection. b, Confocal microscopy images (×630 magnification) of MLV–YFP virions alone, Rho–EF-C alone or mixtures thereof, including a ×5 magnification of the latter. Scale bars, 15 μm. c, Schematic of the experimental procedure to concentrate virions. A 10 ml volume of virus stock containing used-up medium (1) is treated with nanofibrils (2) and subjected to low-speed centrifugation. After removal of the supernatant (3), the pelleted virions are resuspended in fresh medium (4) or buffer (5). Reducing the volume of resuspension medium allows virus concentration (6). d, Infectivity of the resulting solutions. e, p24 ELISA of the original virus stock (1) and nanofibril-treated virus after resuspension in one-tenth of the original volume of medium (6). f, p24 ELISA of the original virus stock (1), the supernatants (sup) derived after centrifugation of PBS or EF-C treated virus, and the pellets (pel) dissolved in the original volume.

  4. EF-C fibrils are broad-based transduction enhancers.
    Figure 4: EF-C fibrils are broad-based transduction enhancers.

    a, Nanofibrils enhance virion attachment. Analysis of HeLa cells (blue) inoculated with either MLV–YFP (green) alone or in the presence of Rho–EF-C (red) (×630 magnification). Scale bars, 15 μm. b, Fibrils increase fusion rates of virions with cells. c, EF-C fibrils increase lentiviral transduction of 293T cells independently of the viral glycoprotein. d, Effect of EF-C on lentiviral gene transfer into human glioblastoma (U87MG), endocrine pancreatic tumour (BON), myeloid KG-1, peripheral blood mononuclear (PBL) and haematopoetic CD34+ stem cells. e, Analysis of human foreskin fibroblasts (HFF; upper panel) or monocyte-derived macrophages after transduction with fibril-treated or untreated γ-retroviral (RV) or lentiviral (LV) particles. Percentages of transduced (GFP+) cells are indicated. Scale bars, 50 μm. f, Comparison of fibrils and other transduction enhancers on HIV-1 infection. Numbers above the bars in c, d and f give n-fold enhancement of transduction relative to the control without fibrils.

  5. EF-C fibrils can be immobilized (a-c) and allow efficient gene transfer into mice (d-f).
    Figure 5: EF-C fibrils can be immobilized (a–c) and allow efficient gene transfer into mice (d–f).

    a, Z-stack images of immobilized Rho-EF-C fibrils only (top left), MLV–YFP only (bottom left) and immobilized Rho-EF-C fibrils exposed to virus (right; the upper image shows both channels, merged, and the lower images, separately). b, EF-C coated on microtitre plates facilitates lentiviral infection. c, Coated EF-C enhances lentiviral transduction with a similar efficiency as RetroNectin. d, Additive effects of RetroNectin and EF-C fibrils. Virions were added to RetroNectin-coated plates. On removal of the inoculum, cells in the presence or absence of fibril-treated virus were added. e, EF-C fibrils increase lentiviral gene transfer into mouse cells. Bone marrow cells were transduced with a lentiviral vector in the absence of enhancer (control, ctr.), using a multistep RetroNectin-based spin-infection protocol (RN), or by brief treatment with EF-C. For details see Supplementary Information. f, The transduced cells were transplanted into recipient mice and analysed by determining the percentage of GFP-positive cells in peripheral blood. Asterisks indicate statistical significance (P < 0.01). n.s.; not statistically significant.

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Author information

  1. These authors contributed equally to this work

    • Maral Yolamanova &
    • Christoph Meier

Affiliations

  1. Institute of Molecular Virology, Ulm University Medical Center, Meyerhofstrasse 1, 89081 Ulm, Germany

    • Maral Yolamanova,
    • Franziska Arnold,
    • Onofrio Zirafi,
    • Shariq M. Usmani,
    • Janis A. Müller,
    • Daniel Sauter,
    • Christine Goffinet,
    • David Palesch,
    • Frank Kirchhoff &
    • Jan Münch
  2. Institute of Organic Chemistry III/Macromolecular Chemistry, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany

    • Christoph Meier &
    • Tanja Weil
  3. Institute of Polymer Science, Ulm University, Albert-Einstein-Allee 47, 89069 Ulm, Germany

    • Alexey K. Shaytan,
    • Pavel G. Khalatur &
    • Alexei R. Khokhlov
  4. Faculty of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, 119991 Moscow, Russia

    • Alexey K. Shaytan
  5. Department of Dermatology and Allergic Diseases, Ulm University Medical Center, Meyerhofstrasse 1, 89081 Ulm, Germany

    • Virag Vas &
    • Hartmut Geiger
  6. ICREA and Joint BSC-IRB Research Programme in Computational Biology, Institute for Research in Biomedicine, Parc Científic de Barcelona c/ Baldiri Reixac 10, 08028 Barcelona, Spain

    • Carlos W. Bertoncini &
    • Xavier Salvatella
  7. Central Electron Microscopy Facility, Ulm University, Albert-Einstein-Allee 11, 89069 Ulm, Germany

    • Paul Walther
  8. Department of Urology, Gladstone Institute of Virology and Immunology, University of California at San Francisco, 1650 Owens St, San Francisco, California 94158, USA

    • Nadia R. Roan
  9. Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Helmholtzstrasse 20, 89081 Ulm, Germany

    • Oleg Lunov &
    • Thomas Simmet
  10. Institute for Virology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

    • Jens Bohne
  11. Institute for Transfusion Medicine, Ulm University and Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, DRK Blood Service Baden-Württemberg – Hessen, gemeinnützige GmbH, Helmholtzstrasse 10, 89081 Ulm, Germany

    • Hubert Schrezenmeier &
    • Klaus Schwarz
  12. Peptide Research Group, Clinic for Immunology, Hannover Medical School, Feodor-Lynen Str. 31, 30625 Hannover, Germany

    • Ludger Ständker &
    • Wolf-Georg Forssmann
  13. A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilova St. 28, 119991 Moscow, Russia

    • Pavel G. Khalatur
  14. Faculty of Physics, Lomonosov Moscow State University, 1-2 Leninskie Gory, 119991 Moscow, Russia

    • Alexei R. Khokhlov
  15. Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, UK

    • Tuomas P. J. Knowles

Contributions

M.Y. generated virus stocks and performed most transductions. C.M. and T.W. assisted in writing the manuscript and were responsible for the structural elucidation of EF-C fibrils. A.K.S., P.G.K. and A.R.K. performed molecular simulations. V.V. and H.G. conducted the ex vivo gene transfer study. C.W.B. and X.S. performed CD analysis. F.A. and D.P. performed coating experiments and measured zeta potentials. O.Z. and J.M. performed Congo Red and ThT assays, S.M.U. was responsible for the analysis of immobilized EF-C by confocal microscopy. D.S. and C.G. performed the fusion assay and flow cytometry assays. P.W. assisted in microscopy. O.L. and T.S. studied the EF-C interaction with virions and cells. J.B. provided retro- and lentiviral vectors and knowhow. H.S. and K.S. isolated and provided human stem cells and performed colony-forming unit assays. L.S. and W.G.F. synthesized and provided peptides. N.R.R. assisted in planning and writing. T.K. analysed AFM data. F.K. and J.M. planned research, analysed data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing financial interests

M. Yo., F. Ki. and J. Mü. filed for a patent to use EF-C fibrils as tranduction and infection enhancer.

Corresponding author

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Supplementary information

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