2.4.6. Adenovirus

HEK-293 cells (4 × 10<sup>5</sup> cells/well) were seeded in 12-well plates in a total volume of 1 mL and incubated overnight at 37 ◦C. Cells were treated with medium containing 0.1% DMSO and the indicated concentrations of CPXV012 peptide or 0.1% DMSO only as vehicle control at 37 ◦C for 1 h. Afterward, cells were infected with an MOI of 10 with AdGOva for 24 h. Photometric analysis was performed with the Infinite 200 PRO Tecan to quantify the percentage of infected cells based on GFP-expression.

### 2.4.7. Rift Valley Fever Virus

MJS cells (2 × 10<sup>4</sup> cells/well) were seeded in a 96-well plate and incubated for two days at 37 ◦C. Cells were treated with medium containing 0.1% DMSO and the indicated concentrations of either CPXV012 peptide or control peptide UL49.5 or 0.1% DMSO only as vehicle control. After 15 min, RVFV-eGFP was added to the cells. Twenty-four hours after infection, cells were harvested and the percentage of eGFP-positive cells was determined using flow cytometry.

### 2.4.8. Coxsackievirus B3

MJS cells (10<sup>4</sup> cells/well) were seeded in a 96-well plate. After overnight incubation at 37 ◦C, cells were infected with RLuc-CVB3 in the presence of medium containing 0.1% DMSO and the indicated concentrations of CPXV012 peptide or 0.1% DMSO only as vehicle control. After 7 h of infection, cells were lysed and renilla luciferase expression levels were quantified using the Renilla Luciferase Assay System kit (Promega).

### *2.5. Preparation of Large Unilamellar Vesicles (LUVs)*

Calcein-encapsulated LUVs were prepared using 1,2-dioleoyl-*sn*-glycero-3-phosphocholine (DOPC) or a mixture of DOPC and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) (Avanti Polar Lipids) in a 7:3 molar ratio. A stock solution of DOPC and DOPS in chloroform (10 mM) were mixed in a glass tube. The solvent was evaporated with dry nitrogen gas yielding a lipid film that was subsequently kept in a vacuum desiccator for 20 min. Lipid films were hydrated for 30 min in bu ffer containing 10 mM Tris, 50 mM NaCl at pH 7.4 resulting in a total lipid concentration of 10 mM. For calcein-encapsulated LUVs, 50 mM of calcein was added during hydration. The lipid suspension was freeze-thawed ten cycles, at temperatures of −80 and +40 ◦C, respectively, and eventually extruded 10 times through 0.2 μM-pore size filters (Anotop 10, Whatman, UK). For the preparation of calcein-encapsulated LUVs, free calcein was separated from calcein-filled LUVs using size exclusion column chromatography (Sephadex G-50 fine) and eluted with 10 mM Tris-HCl, 150 mM NaCl bu ffer at pH 7.4. Finally, the phospholipid content of lipid stock solutions and vesicle preparations was determined as inorganic phosphate according to Rouser [29].

### *2.6. Circular Dichroism*

Circular dichroism (CD) experiments were performed as described previously (15). Briefly, the CD spectra of 625 μM LUVs and/or 100 μg/mL peptides diluted in 10 mM MES bu ffer (pH 6.2) were recorded on a Jasco 810 spectropolarimeter (Jasco, Easton, MD) over a wavelength range of 200 to 250 nm. Each reported spectrum is the average of five independent scans recorded every 1 nm at a scan rate of 20 nm/min at room temperature in cuvettes with a path length of 1.0 mm.

### *2.7. Langmuir Monolayers*

Peptide-induced changes in the surface pressure of a monomolecular layer (monolayer) of phospholipids at a constant surface area were measured using a Langmuir Microtrough XL device (Kibron, Helsinki, Finland). A Teflon trough was filled with 16 mL PBS (pH 7.4) and lipid monolayers of DOPC or DOPC/DOPS (ratio 7:3) were spread from a 0.5 mM stock solution in chloroform at the air–bu ffer interface. The bu ffer below the lipid monolayer (subphase) was continuously stirred using a magnetic stirrer. Upon stabilization of the initial surface pressure to 25 mN/m, a freshly prepared stock of peptide in DMSO was injected into the subphase, resulting in a final peptide concentration of 0.25 μM.

### *2.8. Membrane Permeability Assay*

Membrane permeability was measured in standard 96-well transparent microtiter plates in a plate reader (Spectrafluor, Tecan, Salzburg, Austria). Peptides (5 μL of 0.2 mM in DMSO) were added to calcein-containing LUVs (195 μL of lipid vesicles (50 μM) in 10 mM Tris–HCl, 100 mM NaCl bu ffer (pH 7.4)). As positive control human islet amyloid polypeptide (hIAPP; Bachem) 5 μL of a 0.2 mM in DMSO) was added to calcein-containing LUVs. As a negative control, murine IAPP ((mIAPP; Bachem) 5 μL of 0.2 mM in DMSO) was added to calcein-containing LUVs. For blank only, 5 μL DMSO was added to calcein-containing LUVs. Directly after the addition of all components, the microtiter plate was shaken for 10 s. Fluorescence was measured from the top, every 5 min, using a 485 nm excitation filter and a 535 nm emission filter at 25 ◦C. The maximum leakage at the end of each measurement was

determined by adding 1 μL of 10% Triton X-100 to a final concentration of 0.05% (*v*/*v*). The release of fluorescent dye was calculated according to Equation (1):

$$\mathbf{L(t)} = (\mathbf{Ft} - \mathbf{F0}) \langle \mathbf{F100} - \mathbf{F0} \rangle \tag{1}$$

L(t) is the fraction of dye released (normalized to membrane leakage), Ft is the measured fluorescence intensity, and F0 and F100 are the fluorescence intensities at times t = 0 and after addition of Triton X-100, respectively. All membrane leakage assays were performed two times, each in duplicate, on di fferent days, using di fferent IAPP stock solutions.

### *2.9. Statistical Analysis*

Statistical significance was analyzed by one-way ANOVA testing, followed by Dunnett's multiple comparisons test (the mean of each column was compared to that of the DMSO control), using GraphPad Prism 8.0.1. In Figure 1D, statistical analysis was performed on the plotted data transformed as follows: Y = Log(Y). *p*-values of <0.05 were considered significant (\* *p* < 0.05; \*\* *p* < 0.01, \*\*\* *p* < 0.001).
