# Structurally Modified Bioactive Peptide Inhibits SARS-CoV-2 Lentiviral Particles Expression
## 3 Results
### SARS-CoV-2 Spike-Pseudotyped Lentiviral Particles Assay  
The spike lentivirusirus entry assay as conducted as shown in Figure 7A using 293T-ACE2.TMPRSS2 (mCherry) cells, with abundant ACE2 and TMPRSS2, the receptor for viral entry and the serine protease for spike protein priming, respectively (Figure 7B) [22]. Our microscopy results showed that induction of SARS-CoV-2 spike-pseudotyped lentiviral particles significantly increased the overexpression of green fluorescent protein (GFP) in cells (Figure 7C). The increased GFP confirmed the infection of 293T-ACE2 cells with lentivirusirus particles. In line with our protein expression findings (Figure 6A), the co-treatment of cells with A9 and A14 (50 \(\upmu\)M), strongly allaged the expression of GFP in 293T-ACE2 cells (Figure 7C and Figure S4). These findings were then confirmed using flow cytometry analysis (FITC screening). Similar to microscopy results, both IRW analogs, A9 and A14 decreased the levels of GFP expression compared to the spike infection (1.102% and 0.474% vs. 9.834%) (Figure 7D). These findings showed that both the compounds could inhibit lentivirus growth in vitro. However, the direct measurement of lentivirus particles is key for validation of both microscopy and flowcytometry findings on GFP expression in 293T-ACE2 cells (Figure 7C,D). Our flowcytometry analysis, using SARS-CoV-2 Spike glycoprotein S1 subunit for detection of lentivirus showed a strong increase in spike lentivirus in spike group compared to the control group (Figure 7E). Similar to results from florescence microscopy and flowcytometry analysis for GFP detection), the co-treatment of cells with A9 and A14 (50 \(\upmu\)M), inhibited the production of spike lentivirus in 293T-ACE2 cells (Figure 7E). These results confirmed the ability of two novel peptides A9 (Acetyl-Ile-Arg-Trp-Amide) and A14 (Formyl-Ile-Arg-Trp-Amide) to inhibit SARS-CoV-2 Spike-pseudotyped lentiviral particles in cells.  
Figure 6: Impact of peptides A9 and A14 on over expressing SARS-CoV-2 S (spike) protein in HEK293T cells. Immunohistochemistry showing the changes in (**A**) SARS-CoV-2 S (spike) and (**B**) OXPHOS levels of HEK293T cells after treatment with A9 and A14 at the tested concentration. HEK293T cells were grown in DMEM complete media containing 10% FBS with antibiotics. The SARS-CoV-2 S (spike) protein was overexpressed in cells. These cells were treated with vehicle (nuclease free water) or A9 or A14 at 50 \(\upmu\)M for 24 h. Thereafter, protein was extracted using RIPA buffer and was stored at \(-20\)\({}^{\circ}\)C till further analysis. These experiments were repeated thrice independently. \(\upmu\)M: micromolar.  
Figure 7: Impact of peptides A9 and A14 on SARS-CoV-2 Spike-pseudotyped Lentiviral Particles. Lentiviral pseudotyping schematic showing the use of **(A)** 293T cells as packaging cells for producing a 293T cell.  
lentivirus particles using Viral Entry Protein (S D614G Glycoprotein), Lentiviral Backbone (Luc2; ZsGreen), and helper plasmids expressing the other HIV proteins needed for virion formation (Tat, Gag-Pol, and Rev). The transfected 293T cells produce lentiviral particles with surface Spike and can infect (**B**) 293T-ACE2.TMPRSSS2 (mCherry) cells (**C**) Bright-field and fluorescence microscopy images; and (**D**) flowcytometry analysis showing changes in ZsGreen expression in 293T-ACE2.TMPRSSS2 cells at 24 h after incubation with Spike-pseudotyped lentiviral particles with the ZsGreen backbone in presence of A9 and A14 at 50 uM and (**E**) the spike lentivirus cum in the 293T-ACE2.TMPRSSS2 cells transfected with the Spike lentiviral particles with the ZsGreen backbone in presence of A9 and A14 at 50 uM. Flow cytometry was used to measure Spike by staining with Recombinant Anti-SARS-CoV-2 Spike Glycoprotein S1 antibody followed by staining (1:500 dilution) with an APC AffiniPure Goat Anti-Human IgG, Fcy fragment specific antibody (Jackson Labs, 109-135-098) (1:100 dilution).