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Peptide nanofibrils as enhancers of retroviral gene transfer

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Amyloid fibrils are polypeptide‐based polymers that are typically associated with neurodegenerative disorders such as Alzheimer's disease. More recently, it has become clear that amyloid fibrils also fulfill functional roles in hormone storage and biosynthesis. Furthermore, it has been demonstrated that semen contains abundant levels of polycationic amyloid fibrils. The natural role of these seminal amyloids remains elusive. Strikingly, however, they drastically enhance HIV‐1 infection and may be exploited by the virus to increase its sexual transmission rate. Their strong activity in enhancing HIV‐1 infection suggests that seminal amyloid might also promote transduction by retroviral vectors. Indeed, SEVI (semen‐derived enhancer of virus infection), the best characterized seminal amyloid, boosts retroviral gene transfer more efficiently than conventional additives. However, the use of SEVI as laboratory tool for efficient retroviral gene transfer is limited because the polypeptide monomers are relatively expensive to produce. Furthermore, standardized production of SEVI fibrils with similar high activities is difficult to achieve because of the stochastic nature of the amyloid assembly process. These obstacles can be overcome by recently identified smaller peptides that spontaneously self‐assemble into nanofibrils. These nanofibrils increase retroviral gene transfer even more efficiently than SEVI, are easy to produce and to handle, and seem to be safe as assessed in an ex vivo gene transfer study. Furthermore, peptide‐based nanofibrils allow to concentrate viral particles by low‐speed centrifugation. Specific adaption and customization of self‐assembling peptides might lead to novel nanofibrils with versatile biological functions, e.g., targeted retroviral gene transfer or drug delivery. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology‐Inspired Nanomaterials > Peptide‐Based Structures
Classes of retroviral transduction enhancers. Currently used transduction enhancers are based on polycationic polymers [polybrene or diethylaminoethyl (DEAE)‐dextran], recombinant proteins (Retronectin), peptides (protamine and Vectofusin‐1), and polycationic peptide nanofibrils. The location of cationic charges in the transduction enhancers is highlighted in blue. (Protamine structure: Reprinted with permission from Ref . Copyright 1991 The Royal Society of Chemistry; Retronectin: Figure reproduced from Ref 27.)
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Schematic of retroviral gene transfer. Retroviral vectors are produced in packaging cells and consist of structural proteins (red) that encapsulate the viral genome encoding the transgene (green). The viral particles are released from the packaging cell and incorporate a viral glycoprotein (blue). These particles can be harvested and used to infect the target cells of choice. The viral glycoprotein mediates fusion of the viral particle with the cell membrane and the viral capsid is released into the cytoplasm. Thereafter, the viral RNA encoding the transgene is reverse transcribed into double‐stranded DNA that is integrated into the host cell chromosome where transgene expression takes place.
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Mechanism of nanofibril‐mediated enhancement of retroviral infection/transduction. (a) EF‐C nanofibrils promote retroviral infection more efficiently than SEVI (semen‐derived enhancer of virus infection) and polycationic polymers. (Reprinted with permission from Ref . Copyright 2013 Nature Publishing Group). (b) Simplified representation of the consequences of different bending rigidities on the interaction of nanofibrils and cationic polymers with virus. A single nanofibril interacts with multiple virions. Parts of the fibril do not interact with virions owing to steric constrains, which are available for interaction with the cell membrane. In contrast, flexible polymer chains absorb and wrap around virus particles until charge neutralization is achieved. (c) Tentative mechanism for transduction enhancement by nanofibrils. Cationic peptides self‐assemble into positively charged nanofibrils that form electrostatic complexes with viral particles. The complexes exhibit an excess cationic charge that facilitates attachment to the negatively charged cell membrane.
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Peptides derived from HIV‐1 gp41 form nanofibrils. (a) Electron micrographs of freshly dissolved P16 peptide indicating spontaneous fibril formation. (b) Viral infection‐enhancing activities of P16 and other additives. (Reprinted with permission from Ref . Copyright 2014 Wiley)
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EF‐C nanofibrils strongly increase retroviral transduction efficiencies. (a) Fibrils boost retroviral gene transfer into human cell lines and CD34+ stem cells. Numbers above the bars give the n‐fold enhancement of transduction. (b) Transduction of macrophages with a lentiviral vector in the absence and presence of EF‐C nanofibrils. (c) The transduction‐enhancing activity of EF‐C fibrils is independent of the glycoprotein used for pseudotyping. (Reprinted with permission from Ref . Copyright 2013 Nature Publishing Group)
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Prostatic acid phosphatase (PAP)248‐286 forms fibrils and enhances HIV‐1 infection. (a) Transmission electron microscopy (TEM) images of freshly dissolved PAP248‐286 and peptide that was agitated for 16 h. (b) Increase of Thioflavin T (ThT) fluorescence intensity over time indicates the formation of amyloid fibrils. (c) X‐ray powder diffraction of SEVI (semen‐derived enhancer of virus infection) indicating a typical amyloid cross β‐sheet structure. (d) SEVI increases HIV‐1 infection. UV microscopy images of cells expressing the Green Fluorescence Protein (GFP) upon HIV‐1 infection are shown. (Reprinted with permission from Ref . Copyright 2001 Elsevier)
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A peptide derived from the HIV‐1 gp120 enhances HIV‐1 infection and forms nanofibrils. (a) Peptide EF‐C enhances HIV‐1 infection more efficiently than SEVI. (b) Atomic force microscopy image of EF‐C nanofibrils that form instantaneously. (c) Top and side view of the elementary unit of the fibril comprising four β‐strands arranged into a stack of two antiparallel β‐sheets. (d) Refined model of an EF‐C nanofibril. (Reprinted with permission from Ref . Copyright 2013 Nature Publishing Group)
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A semenogelin 1‐derived peptide forms nanofibrils that interact with viral particles. (a) Transmission electron microscopy (TEM) images of SEM1(49‐107) that was agitated over night. (b) Interaction of SEM1(49‐107) fibrils (red) with retroviral particles (green). (Reprinted with permission from Ref . Copyright 2011 Elsevier)
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SEVI (semen‐derived enhancer of virus infection) captures virions and increases their attachment rates to target cells. Confocal microscopy image of SEVI fibrils (red), virions (white), and target cells (blue). Note that almost all virions are sequestered by fibrils. This image was generated by Walther Mothes, Joseph Luna, and Pradeep Uchil, Yale University.
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