Membrane Fusion

To monitor the fusion of OMV with airway epithelial cells, OMV were fluorescently labeled with a probe that fluoresces upon membrane fusion. OMV purified with the method described above were resuspended in labeling buffer (50 mM Na₂CO₃, 100 mM NaCl, pH 9.2). Rhodamine isothiocyanate B-R18 (Molecular Probes), which integrates in the membrane of the OMV, was added at a concentration of 1 mg/ml for 1 hour at 25°C, followed by ultracentrifugation at 52,000×g for 30 min at 4°C. Rhodamine isothiocyanate B-R18 fluorescence is quenched at high concentrations in bilayer membranes, and fluorescence is dequenched when the probe is diluted upon vesicle fusion. Subsequently, rhodamine labeled-OMV were resuspended in PBS (0.2 M NaCl) and pelleted at 52,000×g for 30 min a 4°C. After a final centrifugation step, the labeled-OMV were resuspended in 1 ml PBS (0.2 M NaCl) containing a protease inhibitor cocktail tablet (Complete Protease Inhibitor Tablet, Roche). Labeled-OMV were applied to the apical side of airway epithelial cells at 1∶4 dilution of labeled-OMV to Earle's Minimal Medium (MEM, Invitrogen) and fluorescence was detected over time as indicated on a fluorescent plate reader (Ex 570 nm; Em 595 nm). Fluorescence intensity was normalized for fluorescence detected by labeled-OMV in the absence of airway epithelial cells at the indicated time points.

The instructor provided to the students a brief introduction
to the most important features of the structure of SARS-CoV-2. The
four major structural proteins are displayed: the envelope (E), membrane
(M), nucleocapsid (N), and spike (S) proteins (Figure 1).^{7 (link)}It is highlighted that spike protein (approximately 180–200
kDa) is the surface glycoprotein anchored to the viral membrane that
plays an essential role when the infection process of SARS-CoV-2 takes
place. This protein is a trimer of three identical protomers (Figure 2). Each protomer
contains three segments: a short intracellular tail (IC), a transmembrane
anchor (TM), and a large ectodomain that extends outward from the
virus which is coated with sugar chains to hide the virus from the
immune system^{8 (link)} and comprises S1 and S2
subunits.
Next, the students are invited to study the ectodomain by analyzing
the requested structural features that they must observe manipulating
PyMOL.
Although hundreds of structures of this spike protein
are already
available in the Protein Data Bank, the one with the code 7DWY(9 (link)) has been selected and must be loaded in a PyMOL session.
They are encouraged to distinguish the four different levels of the
protein structures: primary, secondary, tertiary, and quaternary,
changing the representation of the molecule from lines or wireframe
to cartoon.
They must learn how to select individual residues
or different
chains, how to change their colors, how to generate objects, how to
show and hide different parts of the protein, how to measure distances
and angles for bonds, and how to generate surfaces.
They have
to realize that the spike protein is a complex of three
identical chains. A schematic illustration of the spike protein (Figure 3) is given to the
students, and they must recognize every single domain in the ectodomain,
extracting them as different objects and coloring them in the suggested
color.
The S1 subunit has an N-terminal
domain (NTD) and a receptor-binding
domain (RBD) located in the C-terminal domain, which is implied in
recognition and binding to the host cell receptor. S2 consists of
the fusion peptide (FP), two heptad repeats 1 (HR1 and HR2) which
operate the fusion of viral and host membranes, a transmembrane domain
(TM), and a cytoplasmic tail (CT).
When different species of
coronavirus are compared, the S2 subunit
is highly conserved, but the sequence of the S1 subunit varies greatly.
S1 and S2 are connected to the S1/S2 cleavage site in which specific
proteases act. The cleavage transforms the spike protein into a fusion
competent form that suffers several conformational changes and allows
it to anchor to the host membrane leading to the membrane fusion.^{10 (link)}

To examine the capacity of OligoBinders
to prevent the infection of cells by SARS-CoV-2, we employed a cell-based
screening assay using HiBiT-tagged PsVLP from Promega Biotech. The
genome-free nature of SC2-VLPs eliminates the need for biosafety level
3 (BSL3) facilities during handling. HiBiT technology provides the
benefits of high sensitivity and convenience of a single-reagent-addition
step, all while overcoming the disadvantage of difficulty quantifying
PsVLP cell entry and membrane fusion.
Serial dilutions of OligoBinder-1
and OligoBinder-3 were prepared in the range 0.2–40000 pM.
Next, 4× OligoBinder-1 and OligoBinder-3 were incubated with
SARS-CoV-2 S(G614) HiBiT-SC2-VLPs dissolved in assay buffer for 30
min at 37 °C. Next, SARS-CoV-2 HEK293T (LgBiT) target cells were
thawed and transferred to a 96-well plate containing HiBiT-SC2-VLPs
and OligoBinders. After 3 h of incubation at 37 °C, 25 μL
of 5× Nano-Glo live cell reagent was added to each well, and
luminescence was measured after 15 min of incubation at 37 °C.
In the presence of inhibitory particles, SC2-VLP entry and fusion
with target cells were blocked; therefore, this prevented HiBiT release,
and no luminescent signal was produced. Assay buffer was used to calculate
the baseline signal. Next, maximum entry values of SC2-VLPs into cells
were calculated by dividing baseline-corrected values by values from
control samples containing no oligomeric particle. The obtained luminescence
values were normalized to neutralization%, where the highest luminescence
value was defined as 0%, and the lowest luminescence values were defined
as 100%. GraphPad Prism V6.01 (GraphPad Software, Inc.) was used to
fit the obtained values to a nonlinear regression curve (least-squares
fitting method) to determine the IC₅₀.

Liposomes were characterized in terms of their size, zeta potential, concentration, membrane fluidity, morphology and IR-780 content. For HELs and HMLs, the total protein content was also determined. The liposome size and concentration were evaluated using a nano-tracking analysis device (NanoSight NS500, NanoSight, Amesbury, UK) equipped with a 532 nm laser and an EMCCD 215S camera. The vesicle suspensions were diluted in a ratio of 1:100,000 before analysis and automatically injected into the sample compartment. Final sample concentrations and size distributions were obtained using the NTA 3.4 software (NanoSight, Amesbury, UK). Subsequently, liposomes’ zeta potentials were determined using a NanoBrook Zetaplus (Brookhaven, GA, USA). The membrane fluidity of spin-labeled vesicle suspensions was evaluated by EPR measurements using a Bruker EMX Plus spectrometer (Rheinstetten, Germany) operating in the X-band (approximately 9.4 GHz) with a 4119-HS resonant cavity and the following instrumental parameters: microwave power, 2 mW; modulation frequency, 100 kHz; amplitude of modulation, 1 G; magnetic field scan, 100 G; scan time, 168 s; and detection time constant, 41 ms. The maximum hyperfine splitting parameter (2A_||) values were obtained from the experimental spectra and were used as a measure of membrane rigidity, as previously described [25 (link)]. Liposome morphologies were assessed by transmission electron microscopy (TEM). The vesicle suspensions were fixed using a buffered formaldehyde solution (25%, pH = 7.0) and post-fixed with an osmium tetroxide 4% solution and subsequently dehydrated in ethanol. Finally, the samples were deposited on carbon films of a TEM copper grid and colored by 0.5% aqueous uranyl acetate. The images were acquired using a JEOL JEM-2100 microscope (Tokyo, Japan). The IR-780 content was determined using a Cary 50 UV-Vis spectrophotometer (Varian Inc., Palo Alto, CA, USA) using a calibration curve of known IR-780 concentrations. HEL and HML total protein content was determined using a commercial kit (Sigma-Aldrich, Burlington, MA, USA) based on the reaction of bicinchoninic acid (BCA) to confirm membrane fusion. Briefly, HEL/HML samples were added to a solution of the BCA reagent, and after incubation for 30 min at 37 °C, the absorbance was measured at 562 nm. The protein concentration was determined using a calibration curve prepared with known concentrations of bovine serum albumin.