DNA constructs and peptides
The plasmids encoding the SARS-CoV-2 spike protein (S protein) (pTwist-EF1 alpha-SARS-CoV-2-S-2xStrep was a gift from Nevan Krogan  (Addgene plasmid #141,382)), human ACE2 (hACE2 was a gift from Hyeryun Choe  (Addgene plasmid #1786; http://n2t.net/addgene:1786; RRID: Addgene_1786)), and TMPRSS2 (TMPRSS2 was a gift from Roger Reeves  (Addgene plasmid #53,887; http://n2t.net/addgene:53887; RRID: Addgene_53887)) were purchased from Addgene. After the bacteria were amplified, plasmids were purified using GenElute Plasmid Maxiprep Kit (Sigma-Aldrich). Peptides were synthesized by Biomatik, dissolved in DMSO to a stock solution of 10 mM for treating HEK293T cells and 50 mM for the in vitro blocking assay, or were dissolved in ethanol to a stock solution of 50 mM for for in vivo treatments in mice. For each intranasal treatment in mice, 12 µl of peptide stock solution was used.
Cell culture and DNA transfection
HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37 °C. Cells that were grown to 70–80% confluency were transiently transfected with DNA constructs using X-treme gene 9 transfection reagent (Roche) following the manufacturer’s instructions, and. Cells were used for various experiments 24 or 48 h after transfection. Transfected cells were treated with either vehicle, TAT, or peptides 16 h after transfection, and 24 h after treatment, cells were treated again for 1 h before harvesting. For detection of the S-ACE2 interaction, cells were transfected with both S protein and hACE2. For testing of the cleavage of S protein, cells were transfected with S protein, hACE2, and TMPRSS2.
Co-immunoprecipitation and Western blot
Co-immunoprecipitation and Western blot analyses were performed as previously described [18, 19, 22]. After treatment, cells were lysed with lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS) with protease inhibitor cocktail (Sigma-Aldrich). After shaking at 4 °C for 1 h, the mixture was centrifuged at 10,000 g for 10 min at 4 °C, and the supernatant was collected as the total protein extract. Protein concentration was quantified by BCA protein assay (Pierce). For co-immunoprecipitation, 800 µg of cellular protein extract was incubated in the presence of primary antibody against S protein or control IgG (3 µg) as well as 25 µl of protein A/G plus agarose (Santa Cruz Biotechnology) for 12 h at 4 °C. Pellets were washed, boiled for 5 min in SDS sample buffer and subjected to SDS-PAGE. 5–50 µg of cellular protein extract was used as the control in each experiment. The blots were imaged using the ChemiDoc MP Gel Imaging System (Bio-Rad) and the densitometric analysis was conducted using ImageLab (Bio-Rad). Antibodies used for co-immunoprecipitation and Western blot include: anti-SARS-CoV-2 spike antibody (GeneTex, mouse, Cat No. GTX632604), anti-hACE2 (R&D Systems, goat, Cat No. AF933), anti-α-Tubulin (Sigma-Aldrich, mouse, Cat No. T8203), normal mouse IgG (Santa Cruz Biotechnology, Cat No. sc-2025), HRP-conjugated anti-goat secondary antibody (Thermo Fisher Scientific, Cat No. A15999), and HRP-conjugated anti-mouse secondary antibody (Cell Signaling Technology, Cat No. 7076).
In vitro testing of S-ACE2 binding
To assay binding of purified ACE2 and S protein in vitro, we used the RayBio® COVID-19 Spike-ACE2 binding assay kit. Briefly, the RBD domain of SARS-CoV-2 S protein was coated onto a 96-well plate, and the same concentration of purified ACE2 was added together with different concentrations of TAT-ACE2-2 (0, 0.2, 2, and 10 mM) simultaneously to each well. ACE2 binding to S protein was detected using anti-ACE2 antibodies and HRP-conjugated secondary antibodies, and visualized with TMB one-step substrate reagent that reacts with HRP. The reaction was stopped with stop solution, and absorbance at 450 nm was measured with the Synergy H4 plate luminometer (Biotek).
Cell–cell fusion assay
Cell–cell fusion assays were conducted as previously described [9, 29]. Briefly, HEK293T cells transiently transfected with hACE2 and TMPRSS2 were used as target cells, and HEK293T cells transiently transfected with SARS-CoV-2 S protein and GFP were used as effector cells. 40 h after transfection, target cells were treated with vehicle, TAT or peptide (10 µM) 15 min before adding the effector cells. The effector cells were detached from their culture dishes with 0.25% trypsin and overlaid onto a target cell monolayer at an effector:target cells ratio of 1:3. After 2 h incubation, 10–21 images were randomly taken to count the number of fused vs. unfused cells using fluorescence microscopy (Bio-Rad). The cells number were counted using ImageJ (NIH). Effector cells incubated in DMEM supplemented with 10% FBS without target cells were used as a negative control. All experiments were performed in triplicate. This assay was performed in a double-blind fashion.
VSV-ΔG-SARS-CoV-2 pseudovirus packaging and concentration
The VSV-ΔG-GFP Plasmid Expression Vector was purchased from Kerafast INC (EH1004 and EH1019). The pTwist-EF1 alpha-SARS-CoV-2-S-2xStrep vector was purchased from Addgene (141,382). To generate high titer stocks of VSV-ΔG-GFP, HEK 293 T cells were transfected with VSV-G helper vector (EH1012, Kerafast INC) using X-treme Gene HP transfection reagent (Roche) for 24 h, then infected with Pseudotyped ΔG-GFP (G*ΔG-GFP) rVSV virus at an MOI of 0.1. The supernatants were collected after 48 h and clarified by centrifugation at 1000×g for 7.5 min and stored at − 80℃. HEK 293 T cells were plated on a T75 flask in DMEM + 10% FBS at 100,000 cells/cm2. After 6–8 h, 13 μg of pTwist-EF1alpha-SARS-CoV-2-S-2xStrep were transfected into these HEK 293 T cells using X-treme Gene HP transfection reagent according to the procedure recommended by the manufacturer. Cells were then infected with Pseudotyped ΔG-GFP (G*ΔG-GFP) rVSV virus at an MOI of 2. The transfected cells were incubated at 37℃ and 5% CO2 overnight. The supernatant containing pseudovirus was collected after 24 h and passed through a 0.45 μm filter and stored at -80℃ for later use or at 4℃ for immediate use .
To concentrate the VSV-ΔG-SARS-CoV-2 pseudovirus, Beckman Ultra-Clear centrifuge tubes (Cat # 344,058) were first sterilized for 15 min with UV light in a biological safety cabinet. Supernatant was added to these tubes and centrifuged at 100,000 rpm for 90 min at 4 °C in a Beckman SW32Ti rotor. Following centrifugation, the supernatant was carefully decanted and discarded, leaving the final 1 ml, which was kept and pooled with other tubes for the final round of centrifugation with the same parameters. Pellets were then resuspended and kept overnight at 4℃ or stored at − 80℃.
Assays of VSV-ΔG-SARS-CoV-2 pseudoviral entry
White opaque 96-well plates (Sarstedt) were coated with 0.2 mg/ml poly-D-lysine (Sigma-Aldrich, P0899) overnight at 37℃. HEK293T cells were plated into the wells at the density of 2 × 104 cells per well in 200 µl DMEM supplemented with 10% FBS. The cells were transiently transfected with hACE2 and TMPRSS2. 24 h after transfection, cells were treated with vehicle, TAT, or peptides at 5 µM 1 h before adding pseudovirus. After 1 h, the cells were treated for the second time with vehicle, TAT, or peptides at 5 µM, and the VSV-ΔG-SARS-CoV-2 pseudovirus was added immediately. 24 h later, fluorescent intensity in each well was quantified using the Synergy H4 plate luminometer (Biotek) and photographed through the microscope (Bio-Rad).
All animal experimental procedures were conducted in accordance with the protocol (#849) approved by the Animal Care Committee in Centre for Addiction and Mental Health.
Eight to ten week-old B6.Cg-Tg (K18-ACE2)2Prlmn/J mice were purchased from Jackson laboratories (Stock #034860). Mice were housed in ventilated cages in a controlled environment that included standard enrichment. Animals were closely monitored for health and well-being daily by the investigator and supervised by a certified veterinarian in accordance with standard animal care guidelines by CACC (Canadian Council on Animal Care). Animals were acclimatized to the environment for at least 7 days before experiments. Pseudoviral work, including intranasal inoculation, were performed in a biosafety risk level 2 + laboratory. Mice were anesthetized with isoflurane using a vaporizer (Benson Medical Industries INC, 5% at induction). TAT-ACE2-2 peptide, vehicle or TAT peptide were delivered intranasally 15 min before or 1 h after intranasal pseudovirus inoculation (1 × 107 VSV-ΔG-GFP-SARS-CoV-2 pseudovirus in10 µl). Pseudovirus was delivered with a micropipette with gel-loading tips in both nostrils. After 72 h, animals were sacrificed and perfused with PBS followed by 4% paraformaldehyde (PFA) solution. Lung and olfactory bulb were dissected and fixed in 4% PFA for 48 h followed by 20% and 30% sucrose for another 48 h before cryostat sectioning. Tissues were cut into 25 µm slices and examined under confocal microscopy (FV1200, Olympus INC). The fluorescent intensity in lung tissue and GFP-positive cell numbers in olfactory bulb tissue were analyzed using ImageJ (NIH).
Frozen coronal sections of olfactory bulb were initially permeabilized with PBS and 0.3% Triton X-100 (Sigma-Aldrich) for 30 min, and incubated with blocking solution (0.3% Triton X-100 and 3% BSA (Sigma-Aldrich) in PBS) for 2 h at room temperature to reduce nonspecific background. The sections were then incubated with primary antibodies and diluted in PBS containing 0.3% Triton and 1% BSA overnight at 4℃. After this, the sections were washed with PBS for 3 times, and incubated with secondary antibodies for 2 h at room temperature. Finally, the sections were mounted using Prolong Gold Antifade Mountant (Thermo Fisher Scientific). The primary antibodies used were: anti-calbindin (1:30, rabbit, Cat No. ab108404, Abcam), and anti-hACE2 (1:150, goat, Cat No. AF933, R&D Systems). Fluorescent secondary antibodies conjugated to either Alexa 594 (1:200, Invitrogen) were used to detect the primary antibodies. DAPI (Invitrogen) was used to stain the nuclei.
Data were analyzed by one-way ANOVA test followed by Dunnett’s or Tukey’s post hoc test using GraphPad Prism (GraphPad Software). For EC50 analysis, data were analyzed by the nonlinear curve fit module using GraphPad Prism (GraphPad Software).