title: Generation and Characterization of Virus-Enhancing Peptide Nanofibrils Functionalized with Fluorescent Labels.
abstract: Retroviral gene transfer is the method of choice for the stable introduction of genetic material into the cellular genome. However, efficient gene transfer is often limited by low transduction rates of the viral vectors. We have recently described a 12-mer peptide, termed EF-C, that forms amyloid-like peptide nanofibrils (PNF), strongly increasing viral transduction efficiencies. These nanofibrils are polycationic and bind negatively charged membranes of virions and cells, thereby overcoming charge repulsions and resulting in increased rates of virion attachment and gene transfer. EF-C PNF enhance vector transduction more efficiently than other soluble additives and offer prospects for clinical applications. However, while the transduction-enhancing activity of PNF has been well-characterized, the exact mechanism and the kinetics underlying infection enhancement as well as the cellular fate of the fibrils are hardly explored. This is partially due to the fact that current labeling techniques for PNF rely on amyloid probes that cause high background staining or lose signal intensities after cellular uptake. Here, we sought to generate EF-C PNF covalently coupled with fluorescent labels. To achieve such covalent bioconjugates, the free amino groups of the EF-C peptide were coupled to the ATTO 495 or 647N NHS ester dyes. When small amounts of the labeled peptides were mixed with a 100- to 10 000-fold excess of the native peptide, PNF formed that were morphologically indistinguishable from those derived from the unlabeled peptide. The fluorescence of the fibrils could be readily detected using fluorescence spectroscopy, microscopy, and flow cytometry. In addition, labeled and nonlabeled fibrils captured viral particles and increased retroviral transduction with similar efficacy. These covalently fluorescence-labeled PNF are valuable tools with which to elucidate the mechanism(s) underlying transduction attachment and the fate of the fibrils in cells, tissues, and animal models.
authors.affiliation: Institute of Molecular Virology, Ulm University Medical Center , Meyerhofstraße 1, 89081 Ulm, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Rode
authors.affiliation: Institute of Molecular Virology, Ulm University Medical Center , Meyerhofstraße 1, 89081 Ulm, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Hayn
authors.affiliation: Institute of Molecular Virology, Ulm University Medical Center , Meyerhofstraße 1, 89081 Ulm, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Röcker
authors.affiliation: Max Planck Institute for Polymer Research , Ackermannweg 10, 55128 Mainz, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Sieste
authors.affiliation: Max Planck Institute for Polymer Research , Ackermannweg 10, 55128 Mainz, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Lamla
authors.affiliation: Institute of Protein Biochemistry, Ulm University , Helmholtzstraße 8/1, 89081 Ulm, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Markx
authors.forename: Christoph
authors.initials: C
authors.lastname: Meier
authors.affiliation: Institute of Molecular Virology, Ulm University Medical Center , Meyerhofstraße 1, 89081 Ulm, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Kirchhoff
authors.forename: Paul
authors.initials: P
authors.lastname: Walther
authors.affiliation: Institute of Protein Biochemistry, Ulm University , Helmholtzstraße 8/1, 89081 Ulm, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Fändrich
authors.affiliation: Max Planck Institute for Polymer Research , Ackermannweg 10, 55128 Mainz, Germany.
authors.forename: Tanja
authors.identifier: 0000-0002-5906-7205
authors.initials: T
authors.lastname: Weil
authors.affiliation: Institute of Molecular Virology, Ulm University Medical Center , Meyerhofstraße 1, 89081 Ulm, Germany.|Core Facility Functional Peptidomics, Ulm University Medical Center , Albert-Einstein-Allee 11, 89081 Ulm, Germany.
authors.forename: Jan
authors.identifier: 0000-0001-7316-7141
authors.initials: J
authors.lastname: Münch
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D018014:Gene Transfer Techniques; D057139:Nanofibers / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D012190:Retroviridae; D013050:Spectrometry, Fluorescence; D014161:Transduction, Genetic
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides