Vectofusin-1, a potent peptidic enhancer of viral gene transfer forms pH-dependent α-helical nanofibrils, concentrating viral particles.

Gene transfer using lentiviral vectors has therapeutic applications spanning from monogenic and infectious diseases to cancer. Such gene therapy has to be improved by enhancing the levels of viral infection of target cells and/or reducing the amount of lentivirus for greater safety and reduced costs. Vectofusin-1, a recently developed cationic amphipathic peptide with a pronounced capacity to enhance such viral transduction, strongly promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium. To clarify the molecular basis of its action the peptide was investigated on a molecular and a supramolecular level by a variety of biophysical approaches. We show that in culture medium vectofusin-1 rapidly forms complexes in the 10 nm range that further assemble into annular and extended nanofibrils. These associate with viral particles allowing them to be easily pelleted for optimal virus-cell interaction. Thioflavin T fluorescence, circular dichroism and infrared spectroscopies indicate that these fibrils have a unique α-helical structure whereas most other viral transduction enhancers form β-amyloid fibrils. A vectofusin-1 derivative (LAH2-A4) is inefficient in biological assays and does not form nanofibrils, suggesting that supramolecular assembly is essential for transduction enhancement. Our observations define vectofusin-1 as a member of a new class of α-helical enhancers of lentiviral infection. Its fibril formation is reversible which bears considerable advantages in handling the peptide in conditions well-adapted to Good Manufacturing Practices and scalable gene therapy protocols.

IntroductionWith the recent success in clinical trials using lentiviral vectors (LVs), gene therapy has seen a renaissance [1]. Its efficiency can be improved by enhancing transduction levels of target cells and its safety optimized by reducing the amount of viral vectors. One way to promote viral infectivity is the addition of culture additives such as the cationic polymer polybrene [2], a compound mainly used for laboratory research. The mechanism of action of this cationic additive is based on its ability to neutralize membrane charges and promote virus aggregation [3].
For clinical applications in gene therapy, transduction protocols include the polypeptide CH-296 (also called retronectin), a fragment of the human fibronectin [4][5][6]. Retronectin enhances transduction by promoting the co-localization of viruses and cells and rendering the target cell more permissive to the vector [7]. However, its use requires the surface coating of culture dishes which is not optimal both practically and for precise dosage of the additive relative to the vector. More importantly, retronectin is weakly efficient on the broadly used lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G-LV) [8,9]. Identification of new additives that are easy to manipulate and capable of enhancing the infectivity of a broad spectrum of LV pseudotypes is therefore required.
Peptide additives are interesting for their biodegradability, reduced size, simplicity of characterization (as compared to polymers), and the ease of production of a large range of different derivatives. In the last few years, new peptides with transduction enhancing activity have been identified and were shown to self-assemble into β-amyloid nanofibrils [10]. Examples of such peptides are the semen-derived enhancer of viral infection (SEVI) [11,12], the semenogelin peptides [13] or peptides derived from HIV-1 envelope glycoproteins, for example EF-C [14], P13 and P16 [15]. However, the formation of fibrils from semen-derived peptides is difficult (strong agitation of the peptide solution for days) and HIV-1 envelope-derived peptides are raising some concerns in terms of immunogenicity.
We recently focused our attention on the LAH4 peptide family, previously designed as antimicrobial agents while at the same time being efficient transfection agents for DNA and siRNA [16][17][18][19][20][21][22]. Some LAH4-derived peptides have been shown to strongly promote the transduction of human primary cells, especially hematopoietic stem/ progenitor cells with various lentiviral pseudotypes, [23][24][25]. Furthermore, the lead peptide LAH4-A4, also called vectofusin-1 (VF-1), can be dosed to result in an infectivity of a single virus per cell, is not toxic on stem cells, and reduces the inoculum of viral vector needed by 2-3-fold, especially for the clinically used VSV-G-LV pseudotypes [24,25]. VF-1 promotes the adhesion and fusion between viral and cellular membranes [7,24]. Although the peptide has empirically shown to have many advantages over the other compounds used so far, the molecular basis of its mechanism of action and the reasons for its more favourable behaviour remain to be elucidated.
Here we investigated the VF-1 structure and its putative fibril formation as a function of environmental conditions by using thioflavin-T fluorescence, circular dichroism and infrared spectroscopies, as well as confocal, electron and atomic force microscopies. In contrast to the β-amyloid-like nanofibrillar arrangements by other peptides tested for viral transduction enhancement [10], VF-1 associates in a reversible manner from α-helical building blocks. The implications for the VF-1 mechanism of action and how these structural and biophysical insights can be used for the rational design of new transduction enhancing peptides are discussed.

Peptide synthesis
Vectofusin-1 (sequence: KKALLHAALAHLLALAHHLLAL-LKKA-NH 2 ) was synthesised with an amidated carboxy-terminus using a Millipore 9050 automated peptide synthesiser and standard Fmoc chemistry. The resulting product was purified by reverse-phase HPLC, and to exchange the trifluoroacetic acid counter ions washed three times with 4% (vol) acetic acid, freeze-dried, and stored at −18°C. Its mass was verified by MALDI-ToF mass spectrometry and its purity confirmed by HPLC. For infrared spectroscopy, acetic acid counter ions were exchanged for chloride by three cycles of dissolving the peptide in a 10 mM HCl solution at a concentration of 1 mg/ml and lyophilisation. For some experiments (viral pull-down assay, AFM and confocal microscopy), VF-1 and LAH2-A4 (sequence: KKALLAAALAALLALAHHLLALLKKA-NH 2 , substituted amino acids underlined) were obtained from Genecust (Dudelange, Luxembourg) and used without further treatment.

.2.2. Viral vector production
Lentiviral vectors pseudotyped with the VSV-G envelope glycoprotein and encoding the modified nerve growth factor receptor marker (ΔNGFR) were labelled with the mCherry-Vpr fusion protein (VSV-G-mCherry-LV) and generated by transient calcium phosphate transfection of 293T cells with a 6-plasmid vector system as described previously [7] but using the pmCherry-Vpr plasmid [26]. Chromatography-purified VSV-G-LV particles encoding the enhanced green fluorescent protein (eGFP) were produced as described previously [27]. Infectious titres are expressed as the number of transduction units per millilitre (TU/ml) and physical titres as the number of p24 antigens per millilitre (ng p24/ml).

Viral pull-down assay
Chromatography-purified VSV-G-LV particles were diluted in culture medium to a final concentration of 100 ng p24/ml, and 500 µl of viral vectors were mixed with the indicated culture additive, homogenised, and centrifuged at 15,870 g for 10 min at room temperature. Supernatants were discarded and pellets suspended in 100 µl Dulbecco's modified Eagle's medium (DMEM, GIBCO Life Tech., St-Aubin, France). Viral pull-down efficiencies were evaluated by quantifying the p24 content in the pellets using a commercial HIV-1 p24 ELISA kit (Perkin Elmer, Courtaboeuf, France).
Images of VF-1 fibrils (12 µg/ml) in contact with VSV-G-mCherry-LVs (6 × 10 5 TU/ml) were obtained with a scanning confocal microscope LEICA SP8 (Leica Microsystems, Germany). VF-1 (excitation: 488 nm, emission: 500-540 nm) and VSV-G-mCherry-LV emission (excitation: 552 nm, emission: 590-700 nm) were imaged with a 63x PL APO CS2 oil immersion objective 1.40 NA (Leica Microsystems, Germany). A z-stack was created by acquiring images every 0.4 µm. All images were deconvolved using Huygens Professional software (Scientific Volume Imaging, The Netherlands) before three-dimensional sample reconstruction. Finally, images were treated with a contrast enhancement algorithm (histogram equalization) and a 3-pixel radius median filter for background subtraction. For confocal images in presence of cells, HC-T116 cells were cultured on Lab-Tek II Chamber Slide with cover (Lab-Tek, USA). LVs/VF-1 mix was added to cell culture for 1 h at 37°C. Cells were then fixed with 1% PFA. The slide was mounted with Prolong Diamond with DAPI (Thermofisher, France) and using grade #1.5 24 mm × 50 mm coverslip (Dominique Dutscher, France). Images were then acquired. The DAPI channel (excitation: 405 nm, emission: 415-470 nm) was added to the optic configuration described previously and images were treated with the same contrast enhancement algorithm.
Emission spectra (ex. 488 nm) of VF-1 fibrils were acquired using a multimodal plate reader spectrofluorometer (2300Enspire) and the turbidity of the VF-1 solutions was evaluated by measuring the absorbance at 600 nm with a KC4 spectrophotometer (BioTek Instruments, St Quentin-en-Yvelines, France).

Atomic force microscopy (AFM)
Vectofusin-1 (1 mg/ml) was incubated at room temperature in DMEM culture medium. After incubation times ranging from 3 min to 1 day, the solution was diluted in DMEM culture medium to obtain a final VF-1 or LAH2-A4 peptide concentration of 10 µg/mL. A 10 µL droplet was deposited on a freshly cleaved mica surface and left to interact for 30 s. The mica surface was then rinsed with 0.02% uranyl acetate solution and dried with filter paper. AFM images recorded in air were acquired with a Nanoscope V Multimode 8 microscope (Bruker, Santa Barbara, CA, USA) in PeakForce Tapping mode using Scanasyst-Air-HR probes (Bruker). Images were recorded at 1024 × 1024 pixels at a line rate of 3 Hz for Fig. 3A, B, C; and at 3072 × 3072 pixels at 1.5 Hz for Fig. 3D.

AFM image analysis and statistical methods
The "particle analysis" tool on the Nanoscope Analysis software (version 1.50) was used to determine the diameter of 50 annular structures (Fig. 3B), for each incubation time and from at least three independent samples [28]. Filamentous structures, aggregates or structures of which the contours could not be determined were excluded from this analysis. The "section" tool on the same software was used to determine the height and periodicity along the fibril axis.

Transmission electron microscopy
For transmission electron microscopy, a solution of 5 mg/ml (1.6 mM) vectofusin-1 in 10 mM sodium phosphate at pH 4 or pH 8 was deposited on a carbon grid. After 2 min the excess sample was removed with filter paper and a solution of filtered (0.22 µm) ammonium molybdate (1% w/v) at pH 4 or pH 8 was added and left to interact for 2 min prior to the removal of excess solution with filter paper. TEM images were recorded with an AMT Hamamatsu digital camera (Advanced Microscopy Techniques, Woburn, MA, USA) on a Hitachi (Krefeld, Germany) H-7500 electron microscope at 80 kV.

Circular dichroism (CD) spectroscopy
A stock solution of 1 mg/ml (0.3 mM) in 10 mM sodium phosphate at pH 4.3 was prepared and diluted 10 times with 10 mM sodium phosphate. Throughout the manuscript we use the term "phosphate" to cover both the H 2 PO 4 − and the HPO 4 2− ions. With a phosphate pKa of 7.2, the ionisation state can be deduced from the context. From a stock solution of 2-3 µl, 0.5 M NaOH was added to increase the pH in a stepwise manner and a CD spectrum was recorded using a quartz cuvette with a path length of 1 mm on a Jasco J-810 spectrometer (Jasco, Tokyo, Japan) at 25°C, with wavelengths between 260 and 190 nm in steps of 1 nm with a stepping speed of 50 nm/min. Baselines of samples without peptide were subtracted and except for summing the 10 scans acquired for each spectrum and conversion from machine units to mean residual ellipticity (deg cm 2 dmol −1 ), no spectral processing was carried out.

Attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectroscopy
ATR-FTIR analyses were performed on a Bruker Vertex 70 FTIR spectrometer (Karlsruhe Germany) equipped with a Harrick-Diamond ATR. 3 µL of a solution of 20 mg/ml (6.6 mM) vectofusin-1 in D 2 O (Sigma-Aldrich, 99,9% purity) or 20 mM sodium phosphate in D 2 O at pH 4 or pH 8 were deposited on the diamond plate and dried under an argon flow. Infrared spectra were recorded in the 4000-700 cm −1 spectral range with a resolution of 4 cm −1 and 256 scans co-added.

Statistical considerations associated with CD-and ATR-FTIR spectra
To analyse the structural changes as a function of lipid concentration or pH only spectral changes that are significantly more pronounced than the noise level were taken into consideration. Notably, whereas considerable errors can occur during the deconvolution of CD-and FTIR spectra [29] here we performed a semi-quantitative analysis and/or followed the relative changes in spectral intensities during titration experiments.

Vectofusin-1 forms peptide aggregates that sediment viral particles
VF-1 stock solutions are soluble in water (pH < 6) at concentrations around 1-5 mg/ml, but the solution becomes turbid when suspended in phosphate buffered saline (PBS). In X-vivo20 culture medium, used in clinical settings for viral transduction of hematopoietic stem cells, macroscopic aggregates are formed (Fig. 1A-B). Interestingly, these VF-1 aggregates can be observed because of their photoluminescence (Fig. 1C), with an emission peak around 518 nm (Fig. 1D). A dense network of small aggregates is observed in PBS and large aggregates in X-vivo20 medium ( Fig. 1C and Supplemental videos). To characterise the network of VF-1 fibrils formed in stock solution (5 mg/ml), electron microscopy images were acquired in phosphate at pH 4 and pH 8 (below and above the pKa of histidine). At pH 4, no supramolecular aggregate formation was observed (not shown), while at pH 8 a dense network of fibrils is visible (Fig. 1E).
Numerous known viral transduction enhancers self-assemble in supramolecular structures capable of trapping viral particles [10]. This feature can be evaluated using a viral pull-down assay, as described previously for the EF-C or SEVI fibrils [14,30]. Whereas the biological efficiency of VF-1 has been compared to retronectin and SEVI in previous publications [23,24] here we included EF-C another highly efficient enhancer peptide for viral transduction. Notably by centrifugation alone viruses remain in solution and cannot be analysed. As shown in Fig. 2A, VF-1 promotes the sedimentation of 46% to nearly 100% of viral particles at concentrations typically used in biological settings (12-24 µg/ml corresponding to 4.3-8.6 µM) being as efficient as EF-C. To observe the complex between VF-1 peptide aggregates and lentiviral particles, the latter were labelled with mCherry fluorescent proteins (VSV-G-mCherry-LVs), incubated with VF-1 aggregates, and sedimented by centrifugation. Images of the resulting fibril-virion complexes obtained by deconvoluted confocal microscopy are shown in Fig. 2B, and confirm the association of viral particles with a network of VF-1 aggregates. When mixtures of VF-1 and LVs were added to HCT116 cells, some VF-1/LV complexes seem to co-localize with the cell surfaces but the intense autofluorescence at the same frequency of both cells and VF-1 elongated aggregates make the exact delineation of these structures difficult (Fig. S1).

Vectofusin-1 peptides self-assemble as nanofibrils
To study the nature of the VF-1 aggregates in further detail, they were visualized using atomic force microscopy at the concentration optimal for viral transduction (10 µg/ml, i.e. 500-fold less concentrated than the EM image shown in Fig. 1E). Images show isolated as well as clustered nanostructures with a spherical appearance (Fig. 3). Interestingly, those structures associate over time in annular and linear arrangements (Fig. 3A-C) with a periodicity of 10 nm (Fig. 3D). The diameters of the annular structures increase upon the first minutes of incubation (Fig. 3B). Their height profiles are dominated by a 3 nm population with a smaller fraction measuring 4.5 nm (Fig. 3C) and some scatter at 5-12 nm. At later time points, extended linear structures are observed. A variant of VF-1 that lacks two critical histidine residues (LAH2-A4) (Fig. 4A), does not promote lentiviral transduction [25]. No supramolecular structures were observed for this inactive peptide (Fig. 4C), suggesting that larger molecular peptide assemblies are required for transduction enhancement. This is in line with the lack of viral pull-down efficiency by LAH2-A4 (Fig. 4D).

The vectofusin-1 nanofibrils have α-helical structure
To further investigate the structure of these nanofibrils and their pH dependence, a pH titration by circular dichroism (CD) spectroscopy was carried out. Fig. 5A shows that VF-1 is unstructured at low pH and adopts an α-helical structure above pH 6.1. An isodichroic point at 205 nm is observed, confirming a two-state transition from unstructured to α-helical. So far, the observed behaviour is similar to many other cationic amphipathic peptides such as LAH [31], LAK, LAO [32], and LADap peptides [33] that are used for nucleic acid delivery or as antimicrobials. Notably, for VF-1, the ratio of molar ellipticities at 222 nm and 208 nm (θ 222 /θ 208 ) is >1.0, indicative of helix association [34]. The helix formation precedes the formation of larger self-associated complexes: above pH 6.1 the spectra no longer pass through the isodichroic point at 205 nm, but a new isodichroic point at 200 nm appears. Fig. 5B shows the ellipticity per residue as function of pH at each of the isodichroic points. Curve fitting estimates the pKc values of these conformational transitions at 5.5 and 6.7. Although the titration shown here was started at low pH, the reverse titration gives the same results and thus indicates that the peptide self-association is reversible.
Interactions of bivalent ions have been found important for the assembly of some β-amyloids [35], cationic polymers [36] or the packing of peptide oligomers [37]. In order to define the role of phosphates for the VF-1 supramolecular structures, the peptide in water was titrated with increasing phosphate concentrations at pH 4.6 and 7.4 ( Supplementary Figs. S2 and S3). At pH 4.6, VF-1 remains unstructured after addition of 5.3 mM phosphate (160 phosphate ions per peptide), even after 10 days at room temperature. At pH 7.4, no change in the α-helical structure was detected up to phosphate concentrations of 2.4 mM (66 phosphates per peptide), but at 4 mM (111 per peptide) a phosphate-induced increase in the ratio of molar ellipticities at 222 nm and 208 nm (θ 222 /θ 208 ) was observed (Fig. S3), which has been taken as an indicator for oligomeric coiled-coil structures [34].
To the best of our knowledge, all the short peptides with viral transduction enhancing activities form amyloid β-sheets [10]. In contrast, our structural investigations by CD spectroscopy show that VF-1 occurs either as an unstructured peptide at low pH or as an α-helix under biological conditions, while the CD spectra of EF-C β-sheet nanofibrils exhibit different features (SI of [14]). Since circular dichroism is known to be more sensitive to α-helical structures, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used as a complementary technique, known to be more prone to reveal β-sheets. Spectra were recorded in D 2 O in absence or presence of 20 mM phosphate. At pH 4.0, a non-viscous solution was obtained, but at pH 8.0 a transparent, viscous gel formed in the tube. This gel was deposited on the crystal. The amide I region of the processed spectrum is shown in Fig. 6A. In all conditions tested, the spectral profile of VF-1 is clearly different from the designed amyloid peptides [38,39] and transduction enhancing EF-C β-sheet nanofibrils [14], that exhibit typical and predominant features at 1695-1672 cm −1 and 1638-1615 cm −1 [40]. A comparison of IR spectra recorded from β-sheet and VF-1 nanofibrils is shown in Fig.  S4. These spectroscopic data are further confirmed by the absence of thioflavin T fluorescence in presence of VF-1 (Fig. 6B), an assay thought to specifically label β-amyloids [41] such as EF-C. Although CD spectroscopy suggests the presence of unstructured peptide at low pH, no random coil signal at 1670 cm −1 is detected: the higher concentration and the drying of the peptide on the crystal induce the self-association even at low pH. Taken together, the IR peak position near 1650 cm −1 , the lineshape of the circular dichroism spectra and the lack of thioflavin-T fluorescence indicate that VF-1 forms α-helical coiled-coil fibrils at physiological pH.

Discussion
VF-1 has been described as a strong enhancer of viral transduction, acting on the adhesion and fusion steps of lentiviruses developed for gene therapy [7,24,25]. In this manuscript, we show that this peptide forms supramolecular assemblies that co-localize with lentiviral particles (Fig. 2), leading to sedimentation and thus increasing local virus concentrations along the cellular surface of target cells. It may also be expected that virus-cell adhesion is increased because the positively charged peptides reduce the electrostatic repulsion between negatively charged membranes without compromising the viral envelope and cellular membrane integrity [3]. Notably, fibre formation and viral pull-down activity are absent for LAH2-A4, a closely related VF-1 derivative lacking transduction enhancement activities, suggesting that fibril formation and the resulting co-localization of lentiviruses with such VF-1 supramolecular assemblies are important for this activity [10]. In this regard, VF-1 is related to other known viral transduction enhancers that self-assemble in supramolecular structures capable of trapping viral particles [10] such as semen-derived peptides and HIV-1 derived peptides, which enhance transduction by promoting the co-localization of viruses and cells [7]. But in contrast to these peptides, VF-1 is not requiring a strong agitation during hours to form fibrils since VF-1 supramolecular assembly occurs spontaneously in culture medium [24,25].
Using atomic force microscopy we have demonstrated at the nanoscale level that VF-1 self-associates into supramolecular com plexes with spherical appearance in AFM images, that further assemble into fibrils with an annular or a spiral turn appearance (Fig.  3). Within hours these convert into longer fibrils. The formation of annular protofibrils and fibril elongations has been extensively described in preparations of different amyloidogenic proteins and peptides [42][43][44][45]. Interestingly, most of the short peptides with viral transduction enhancing activities form amyloid β-sheets [10], including SEVI [11,12], the semenogelin peptides [13], EF-C [14], P13 and P16 [15].
To better understand the molecular interactions that lead to such supramolecular assemblies, comparison with previously described helical coiled-coil fibrils from natural sources or by design is helpful. Helical coiled-coils are abundant in proteins [46,47] and some fibrils derived from α-helical coiled-coil motifs have been described in native biological systems [48]. Whereas the transition from random coil to helical has been observed for cationic amphipathic peptides with nucleic acid delivery or antimicrobial activities, including LAH [31], LAK, LAO [32], LADap peptides [33] or magainins [49], the design of α-helical peptides for creating synthetic biomaterials, a relatively recent topic, has initially proven to be complex but has taken up speed [50,51]. In a first step, much effort went into the design of coiled-coil building blocks of defined oligomeric size where hy Viral pull-down assay. VSV-G-LVs were mixed with VF-1 or LAH2-A4 peptides, respectively, at the indicated concentrations. After a low speed centrifugation, the HIV-1 p24 antigen content in the pellet was evaluated by an ELISA assay and represented as percentage of viral pull-down normalized to the viral input ± SD (n = 2). drophobic interactions, electrostatic repulsion and attraction are carefully tuned [52]. In a next step, order along the long axis of the fibre was introduced which usually involved much longer peptides. For example, contacts in the longitudinal direction were based on a staggered 5-heptat repeat where pH dependence arises from arginine and glutamate residues [53,54]. Alternatively, 'sticky ends' were created by overhanging coiled-coil dimers from two different peptides with complementary charge [55], as well as blunt end [39] or lock-washer interfaces which associate by charge complementarity [52]. Furthermore, repulsive electrostatic forces at the polar faces of the coiled-coil ensured that lateral aggregation does not interfere with longitudinal order [52]. Based on such features, α-helical coiled-coil sequences were designed de novo that, in a pH-dependent manner, form either fibrils or spherical particles [54]. In contrast, VF-1 does not contain negative charges: an amidated C-terminus and the presence of four lysine residues give it a net charge of +5 at neutral pH, and four histidine residues increase this to +9 at a pH below 6. Considerable ingenuity was required to design α-helical peptide sequences that self-assemble into fibrous structures [50,51]. Therefore, the question arises how VF-1 self-associates into extended fibrils and meshes. The amino acid sequence and comparison to previously published principles, some discussed above, provide some clues for speculation.
While the supramolecular arrangement within VF-1 fibrils remains unknown, a height of about 3.5-4.5 nm and a periodicity along the fibril axis of ∼10 nm (Fig. 3) suggest the involvement of multiple peptides (Fig. S5). Dimensions of a coiled-coiled dimer are 2 nm [56] and increase only slowly when more helices bundle up [52][53][54]. The CD ratio of molar ellipticities at 222 nm and 208 nm (θ 222 /θ 208 ) > 1.0 has been shown to be an indicator of interfacial helix association and the formation of coiled-coil structures [34,57]. Indeed, the presence of multiple leucine residues in the hydrophobic core and their localization on one face of the α-helix (Fig. 4) agrees with the formation of a building block based on leucine-zipper-like interactions [25], where the knobs-into-holes complementarity allows for lateral packing [46,47]. Hydrophobic residues not involved in the zipper could promote longitudinal self-association. The opposite polar face of VF-1 consists of alanine and histidine residues, where the inactive LAH2-A4 [25] has its first two histidine residues replaced with alanine (Fig. 4). The fact that the latter does not form fibrils indicates that those histidine residues are essential for the fibril formation and thus play an important role in intermolecular interactions. Our structural investigations show that VF-1 is highly soluble and unstructured at acidic pH while it rapidly adopts an α-helical conformation above pH 6 and assembles further into coiled-coil structures at the neutral pH used during viral transduction (Figs. 5 and 6). This strengthens the hypothesis of the involvement of histidine (pKa ∼6), and further suggests that the imidazole side chains need to be in their deprotonated, uncharged state for fibril formation to occur. Replacement of histidine with alanine prevents the association into large supramolecular structures (Fig. 4C), indicating that the removal of repulsive Coulombic interactions alone is not sufficient for fibril formation at neutral pH. A deprotonated histidine sidechain is a hydrogen bond acceptor and can participate in π-π stacking or cation-π interactions. Previous studies have indeed shown that His-His interactions are stronger when histidine is deprotonated [58], and that such interactions are quite common in proteins [59]. However, based on the current data, we cannot exclude that interactions between histidine and either lysine, the terminal carboxyamide or the amino-terminus are involved in the self-association.
To make matters even more complex, phosphate buffer was found to promote self-association of α-helical VF-1 even at low ionic strength (Supplementary Figs. S2 and S3). The pH-dependent structural transition of VF-1 shows pKc values of 5.5 and 6.7 (Fig. 5). With the typical histidine pKa being 6.2 in solution and phosphate having a theoretical pKa of 7.2, the pKc of 5.5 seems linked to the (de)protonation of the histidine side chains and the pKc of 6.7 is suggestive that the equilibrium between H 2 PO 4 − and H 2 PO 4 2− from the buffer is involved in the structural transition of VF-1, similar to the involvement of bivalent ions in the formation of some β-amyloids [35], cationic polymers [36] or the packing of peptide oligomers [37]. It is well possible that the histidine pKa is reduced, favouring the uncharged state, by conformational changes that increase Coulombic interactions with nearby positive charges [60] and/or insertion in a more hydrophobic environment [61], for example due to the observed peptide self-association. An important role of phosphates has been described for β-amyloids of β-endorphin [35], a contraction of polylysine polymers has been reported due to the electrostatic interactions of bivalent phosphate and the cationic lysine side chains [36] and phosphate ions have been found important in the packing of a homodimer antimicrobial peptide [37], suggesting that phosphate or  , at pH 4 (grey) or pH 8 (black). The sample was applied to the diamond plate after a 6.6 mM VF-1 solution has been incubated for a few hours of incubation on the bench. Depending on the pH, the term "phosphate" in the legend refers to either predominantly H 2 PO 4 − or HPO 4 2− . (B) A thioflavin T stock solution was diluted either in water (black) or in X-vivo20 medium (white) to a final concentration of 16 μg/ml and incubated with 12 μg/ml VF-1 (circles) or 50 μg/ml EF-C peptide (squares) for the indicated amount of time. The fluorescence intensity (ex. 440 nm, em. 482 nm) was recorded and background without peptide subtracted. Data are representative of three different experiments performed in triplicate ± SD. other anions have a structural role during the VF-1 association process. Additional high-resolution structural investigations are in progress to better define the molecular interactions involved in the self-assembly of VF-1.

Conclusion
The viral transduction enhancing peptide VF-1 belongs to a novel class of α-helical fibril-forming transduction enhancers and has not shown any propensity for amyloid formation in our experiments. To the best of our knowledge, that makes VF-1 the first transduction enhancing peptide that forms α-helical coiled-coil fibrils instead of amyloid β-sheets [10]. It is of interest that some β-sheet nanofibers or their precursors are associated with pathologies [62], whereas the use of coiled-coil α-helical materials has so far not been a concern. The VF-1 assembles into long fibrils composed of multiple spherical particles with a diameter around 10 nm. They form fibrils that appear to close back on themselves giving them an annular appearance, which is lost during the elongation that takes place on a timescale of hours under the conditions used for gene therapy transduction protocols. The pH dependence and reversibility of this fibril formation bears considerable advantages in handling the peptide under conditions well-adapted to Good Manufacturing Practices and scalable gene therapy protocols using lentiviral vectors in which, at present, the cumbersome retronectin additive is typically used. VF-1 peptides can be stored and dosed precisely from a stock solution at low pH and/or the absence of phosphate and subsequently diluted in culture medium at physiological pH to ensure fibril formation in presence of lentiviral vectors and before addition to the cell suspension. In conclusion, the family of histidine-rich LAH4 peptides bears considerable promise for LV transduction, in particular in clinical settings where the rational design of pH-sensitive self-associating α-helical peptides can contribute to further improvements of gene therapy treatments.