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Developing inhibitory peptides against SARS-CoV-2 envelope protein [1]

['Ramsey Bekdash', 'Department Of Rehabilitation', 'Regenerative Medicine', 'Columbia University', 'New York', 'United States Of America', 'Columbia Stem Cell Initiative', 'Department Of Pharmacology', 'Kazushige Yoshida', 'Manoj S. Nair']

Date: 2024-03

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected approximately 800 million people since the start of the Coronavirus Disease 2019 (COVID-19) pandemic. Because of the high rate of mutagenesis in SARS-CoV-2, it is difficult to develop a sustainable approach for prevention and treatment. The Envelope (E) protein is highly conserved among human coronaviruses. Previous studies reported that SARS-CoV-1 E deficiency reduced viral propagation, suggesting that E inhibition might be an effective therapeutic strategy for SARS-CoV-2. Here, we report inhibitory peptides against SARS-CoV-2 E protein named iPep-SARS2-E. Leveraging E-induced alterations in proton homeostasis and NFAT/AP-1 pathway in mammalian cells, we developed screening platforms to design and optimize the peptides that bind and inhibit E protein. Using Vero-E6 cells, human-induced pluripotent stem cell-derived branching lung organoid and mouse models with SARS-CoV-2, we found that iPep-SARS2-E significantly inhibits virus egress and reduces viral cytotoxicity and propagation in vitro and in vivo. Furthermore, the peptide can be customizable for E protein of other human coronaviruses such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The results indicate that E protein can be a potential therapeutic target for human coronaviruses.

Data Availability: All relevant data are within the paper and its Supporting Information files. The data can be also accessible through Icahn School of Medicine at Mount Sinai Data Management and Research Compliance Committee. All materials and plasmid constructs used in this study have been maintained by Dr. Yazawa’s laboratory and are available upon request. In the future, the constructs will be deposited and available to order through Addgene.

( A ) Alignment of Envelope of SARS-CoV (SARS1-E) and SARS-CoV-2 (SARS2-E), showing the difference in amino acids (red), the targeted amino-terminal region named MY18 (red underlined), and putative transmembrane region (black underlined, 18–39). ( B ) Representative confocal fluorescent and bright field images of NIH 3T3 cells loaded with DND-189, a lysosomal pH green fluorescent dye, and transfected with MY18 peptide construct, empty vector (mock) and SARS2-E fused with mKate2 red fluorescent protein (2E-mKate2). Scale bar, 5 μm. ( C ) Relative fluorescent intensity of DND-189 dye in NIH 3T3 cells transfected using mock (n = 40) or 2E-mKate2 plasmid without (-, n = 36) and with MY18 plasmid (+MY18, n = 30). One-way ANOVA with Tukey’s multiple comparisons test (**** P < 0.0001; n.s. not significant). ( D ) The sequences of cell-penetrating peptide candidates, 6-Arg (6Arg), TAT and Penetratin (Pen), for MY18 peptide uptake in mammalian cells. ( E ) Relative fluorescent intensity of DND-189 dye in NIH 3T3 cells incubated with MY18 (10 μM, n = 93), 6Arg-MY18 (0.1 μM, n = 40; 1, n = 41; 10, n = 106), TAT-MY18 (0.1 μM, n = 27; 1, n = 20; 10, n = 92), and Pen-MY18 peptides (0.1 μM, n = 27; 1, n = 32; 10, n = 68) with 2E-mKate2 plasmid transfection. Mock (n = 116) and non-treated 2E-mKate2 (-, n = 139) were also tested as their controls. In 1 μM condition, TAT-MY18 peptide is not different from mock (blue highlighted) while 6Arg and Pen are significantly lower than mock. One-way ANOVA with Dunnett’s multiple comparisons test (**** P < 0.0001; *** P < 0.001; ** P < 0.01; n.s. not significant, compared to mock). The data underlying this figure can be found in S1 Data . All the graphs in the figure are mean ± SD. NIH, National Institute of Health; SARS‑CoV‑2, Severe Acute Respiratory Syndrome Coronavirus 2.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) [ 1 – 3 ] has affected approximately 800 million people and counting in the world. More than 6 million people have passed away due to the viral infection. Because the mutagenesis rate in SARS-CoV-2 genes such as Spike is high [ 4 – 6 ], it is a challenge to develop sustainable approaches for prevention and treatment. While several new vaccines and drug candidates have become available, the number of COVID-19 infections and deaths are still increasing, and new variants are being reported [ 7 – 10 ]. Therefore, it would be ideal if we could target coronavirus genes that are highly conserved in SARS-CoV-1 and SARS-CoV-2 variants, as well as other human coronaviruses. Human coronaviruses such as SARS-CoV-2 and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) express an Envelope (E) protein that forms an ion channel essential for viral function called a viroporin [ 11 – 17 ]. E protein is known to induce cellular toxicity via a number of different molecules and signaling pathways [ 12 , 16 , 18 – 23 ] and E protein is also thought to be involved in the Spike protein protection and maturation in host cells [ 20 , 24 ]. Compared to the other molecules, E protein is highly conserved among coronaviruses: SARS-CoV-2 E (2E) protein’s 75 amino acid residues have high homology with SARS-CoV-1 E protein (approximately 96%), with identical amino (N)-terminus, transmembrane, and pore structures while their carboxyl (C)-terminus is slightly different [ 11 , 25 – 31 ] ( Fig 1A ). Previous studies reported that deficiency of SARS-CoV-1 E gene significantly reduced viral propagation [ 12 ], suggesting that 2E may also play essential roles in viral function and can be a potential therapeutic target for COVID-19 and future variants [ 10 ]. Therefore, in this study we seek to develop screening platforms and applied them to identify drug candidates against 2E. In addition, we examined whether our therapeutic approach targeting 2E can be applicable to the E protein of other human coronaviruses.

Results

Previous studies have reported oligomeric structures and molecular interactions in SARS-CoV-1 E and 2E [25,26,32]. Following the results, we hypothesized that the highly conserved N-terminal region is crucial for oligomerization and that the N-terminal fragment might be able to disrupt 2E protein oligomerization and function because the N-termini might interact with each other in the protein oligomers. Because our previous study demonstrates that the overexpression of 2E affects proton homeostasis in intracellular organelles such as Golgi apparatus and lysosomes in mammalian cells [19], we examined the effect of the N-terminal fragment, which is named MY18 (18 amino acids, MYSFVSEETGTLIVNSVL), on 2E function using DND-189 pH fluorescent dye and MY18 plasmid transfection in mammalian cells. DND-189-based pH fluorescent imaging shows that MY18 co-overexpression in 2E-expressing mammalian cells significantly restores DND-189 fluorescence to the normal level (Fig 1B and 1C). These results encouraged further investigation in using MY18 as a peptide that inhibits 2E. Next, to apply MY18 as an exogenous synthetic peptide, we tested 3 cell-penetrating amino acid motifs: an arginine repeat, TAT and Penetratin [33] (Fig 1D). The DND-189-based pH imaging demonstrates that the TAT version of MY18 is the most promising cell-penetrating peptide among the cell-penetrating peptide candidates (Fig 1E).

While DND-189-based pH fluorescent imaging is useful as a drug screening platform of live mammalian cells, the dynamic range of the dye is somewhat limited, the standard deviation of fluorescent readout is relatively large, and its throughput is not as high as that of an assay for screening. These limitations may give rise to difficulties in further optimizing the MY18 peptide using molecular biological approach with mutagenesis. To develop a higher-throughput screening platform, we overexpressed 2E in mammalian cells and explored other reliable and quantitative readouts. Interestingly, global proteomics results showed increases of various key signaling molecules such as JUN/AP-1 (Fig 2A). A follow-up experiment using quantitative RT-PCR (qPCR) confirmed that the expression of most of the genes including JUN transcript are significantly increased in 2E-expressing cells compared to mock (Figs 2B and S1). The transcript expression of a JUN-related molecule, NFATC4/NFAT3, is also up-regulated significantly (Fig 2C), though the increase of NFATC4/NFAT3 protein (approximately 120%) did not reach to significance in statistical analysis of the global proteomics results with the standard false discovery rate (FDR, 0.05, Fig 2A). Following these results, we decided to apply the NFAT response element of the human IL-2 gene where NFAT and JUN/AP-1 interact [34]. To obtain precise readouts, we used a dual luciferase reporter system containing NFAT Firefly luciferase reporter and pRL-TK-Renilla luciferase reporter as the transfection control using HSV TK, herpes simplex virus thymidine kinase, promoter [35] (Fig 2D). The luciferase assay result obtained using plasmid DNA co-transfection shows that 2E overexpression significantly increases the Firefly luciferase activity in mammalian cells and that MY18 co-expression significantly suppresses the effect of 2E on the NFAT/AP-1 pathway, though it does not fully restore it to mock levels (Fig 2E). These results suggest that MY18 is not sufficient to prevent 2E from altering the NFAT/AP-1 pathway completely, though we observed that MY18 restores proton homeostasis in DND-189-based pH fluorescent imaging (Fig 1). Looking to optimize MY18, we next examined whether deletion or extension of MY18 might improve the effect on interrupting 2E function using the luciferase reporter. We found that none of the constructs significantly improved the effect, as the majority reduced efficacy (Fig 2F). Next, we tested a variety of MY18 mutant constructs using mutagenesis, co-transfection and luciferase assay, and found that the substitution of glutamate to aspartate at seventh and eighth residues (EE7-8DD or 2ED) significantly improved MY18 (Fig 2G). We combined the mutant 2ED and TAT cell-penetrating motif (Fig 1D and 1E) and called TAT-MY18-2ED “iPep-SARS2-E” (inhibitory Peptide against SARS-CoV-2 Envelope). In addition, we confirmed that iPep-SARS2-E had the same rescuing effect on lysosomal pH phenotype in 2E-transfected mammalian cells as the plasmid construct did (S2A Fig).

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TIFF original image Download: Fig 2. Mutagenesis of MY18 peptide to develop iPep-SARS2-E. (A) Volcano plots of global proteomics of HEK 293S cells transfected with SARS2-E fused to mKate2 (2E-mKate2). Red plots demonstrate significant increases in 2E-mKate2 compared to empty vector (mock) (FDR: 0.05). (B and C) The expressions of JUN/AP-1 (B) and NFATC4 transcripts (C) significantly increased in HEK 293S cells transfected to 2E-mKate2 compared to mock. Unpaired Student’s t test was used (** P < 0.01; * P < 0.05, n = 3–4). (D) Schematic representation of dual luminescence reporter system using 4-repeated NFAT response element (RE) of human IL-2 gene and Firefly luciferase gene (NFAT-FLuc) and herpes simplex virus thymidine kinase (HSV TK) promoter-driven Renilla luciferase gene (TK-RLuc) as transfection control. (E) Relative NFAT-FLuc/TK-RLuc activity in HEK 293T cells transfected using empty vector (mock, n = 9) or 2E-mKate2 plasmid without (-, n = 9) and with MY18 plasmid (+MY18, n = 12). One-way ANOVA with Tukey’s multiple comparisons test was used (**** P < 0.0001). (F) Relative NFAT-FLuc/TK-RLuc activity in HEK 293T cells transfected using empty vector (mock, n = 9) or 2E-mKate2 plasmid without (-, n = 6) and with various sizes of SARS2-E amino-terminal constructs including MY18 (each, n = 6, inset). One-way ANOVA with Dunnett’s multiple comparisons test was used (**** P < 0.0001; ** P < 0.01; * P < 0.05; n.s., not significant, compared to MY18). (G) Relative NFAT-FLuc/TK-RLuc activity in HEK 293T cells transfected using empty vector (mock, n = 3) or 2E-mKate2 plasmid without (-, n = 3) and with MY18 mutants (each, n = 3). One-way ANOVA with Dunnett’s multiple comparisons test was used (**** P < 0.0001; ** P < 0.01; * P < 0.05; n.s., not significant, compared to MY18 wild-type, WT, n = 9). S-T switch, Ser and Thr replaced each other. The inset demonstrates the amino acid sequence of MY18-2ED (EE7-8DD, underlined) mutant, which significantly improves the inhibitory effect on SARS2-E-mediated NFAT-JUN/AP-1 activation. The data underlying this figure can be found in S1 Data. All the graphs in the figure are mean ± SD. FDR, false discovery rate; HEK, human embryonic kidney. https://doi.org/10.1371/journal.pbio.3002522.g002

To characterize iPep-SARS2-E, we first produced a monoclonal antibody against the N-terminal region of 2E. Following previous studies of viral envelope topology, we believed that the N-terminus may be in the extracellular region of SARS-CoV-2 [25,36]. To produce the monoclonal antibody, we used a synthetic peptide composed of the first 18 amino acids of E protein (i.e., MY18) with keyhole limpet haemocyanin (KLH) as the antigen. Western blotting result reveals that a hybridoma produces a 2E monoclonal antibody (2E-N; clone, N2A5E8) that can recognize 2E proteins expressed in a mammalian heterologous expression system (S2B Fig). Next, we conducted an ELISA using the antibody to compare the affinity of our 2E-N antibody to our 2ED and wild-type peptides. We confirmed that the 2ED mutation reduces the binding affinity of the 2E antibody, which was produced using the wild-type MY18 peptide as the antigen (S2C Fig). Next, we used ELISA to examine how stable iPep-SARS2-E is in phosphate-buffered saline (PBS) at 37°C. We did not observe obvious peptide degradation in 24 h, though the peptide might become unstable after 48 h because the standard deviation became larger compared to earlier time points (S2D Fig). Using an apoptosis/necrosis assay with flow cytometry, we confirmed that iPep-SARS2-E does not cause cellular toxicity in mammalian cells in vitro (S2E–S2G Fig).

To investigate the interaction of MY18-2ED and 2E protein biochemically, we first conducted immunoprecipitation using 6xHis-tagged MY18-2ED (His-MY18) and 2E-YFP with Ni column. The western blotting using anti-GFP antibody, which can recognize 2E-YFP protein band, demonstrates that Ni column with His-MY18 can pull down 2E-YFP proteins (Fig 3A), suggesting an interaction between MY18-2ED and 2E protein, though there was no obvious difference in MY18-2E protein interaction between the wild-type and 2ED peptide constructs, according to the immunoprecipitation result (S2H Fig). To confirm this in situ, we co-expressed 2E-mKate2 with His-MY18 in NIH 3T3 cells using lipofection and anti-His tag antibody conjugated to Alexa Fluor 488 to examine whether MY18 peptide interacts 2E protein directly. The fluorescent imaging result reveals that His-MY18 are co-localized with 2E proteins in the mammalian cells (Fig 3B). To further investigate the molecular mechanism underlying the inhibitory effect of MY18-2ED, we next used our established electrophysiological recording [19] to examine whether MY18-2ED co-transfected with 2E has a direct effect on 2E channel activity. The result demonstrates that MY18-2ED significantly reduced 2E channel current in HEK cells (Fig 3C and 3D), suggesting that MY18-2ED may inhibit 2E function directly. Together, the results suggest that MY18-2ED might be integrated into 2E protein oligomers and inhibit 2E function.

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TIFF original image Download: Fig 3. Characterization of iPep-SARS2-E. (A) Immunoprecipitation of 2E protein using Ni column and HEK 293T cells transfected using 2E-YFP with or without 6xHis-MY18-2ED (His-MY18). Anti-GFP antibody was used to blot 2E-YFP protein bands. (B) Representative epi-fluorescence image of NIH 3T3 cells co-transfected with 2E-mKate2 (red) and His-MY18 plasmids. Anti-His tag antibody conjugated to Alexa Fluor 488 (green) and Hoechst 33258 dye (blue, nucleus) were used after cell fixation. White arrowheads, co-localization of red and green fluorescence. Scale bar, 5 μm. (C) Representative SARS2-E currents in 2E-PM-expressing HEK 293S cells mock-transfect and co-transfected with MY18-2ED peptide construct. (D) MY18-2ED significantly blocked 2E currents. Student’s t test was used (*** P < 0.001). (E) Representative immunoblot images of HEK 293T cells transfected with SARS2-E fused with YFP (2E-YFP) and 48 h treated with 10 μM TAT-MY18-2ED or MY18-WT peptides (negative control). Anti-GFP (for 2E-YFP, top) and GAPDH antibodies (as loading control, bottom) were used. Putative aggregates of 2E-YFP proteins (&) were observed even though Urea-based lysis buffer was used for the sample preparation. #, nonspecific bands around 40 and 50 kDa according to the YFP blotting (S3B Fig). The whole blotting image of GAPDH is shown in S3A Fig. (F and G) Quantification of 2E-YFP monomeric form (F) and aggregates (G) of HEK 293T cells transfected with 2E-YFP and treated using TAT-MY18-2ED (n = 4) or MY18-WT peptides (n = 4). Student’s t test was used (*** P < 0.001). (H) Representative immunoblot images of HEK 293T cells transfected with YFP plasmid and 48 h treated with TAT-MY18-2ED (10 μM, n = 3) and MY18-WT peptides (10 μM, n = 3, a negative control) and non-treated cells (n = 3, another negative control). Anti-GFP (for YFP, top) and GAPDH antibodies (as loading control, bottom) were used. The whole blotting images and quantification are shown in S3B and S3C Fig, respectively. (I) Schematic representation of the molecular mechanism underlying the effect of MY18 peptide on 2E protein and lysosomal function. The images are prepared using BioRender: Top, 2E induces deacidification in lysosome (Fig 1B and 1C); bottom, MY18 peptides binds 2E proteins (Fig 3A and 3B), resulting in 2E inhibition (Fig 3C and 3D) and restored lysosomal activity (Figs 1B, 1C and S2A) and 2E protein reduction (Fig 3E–3G). The data underlying this figure can be found in S1 Data. All the graphs in the figure are mean ± SD. HEK, human embryonic kidney; NIH, National Institute of Health. https://doi.org/10.1371/journal.pbio.3002522.g003

To examine the effect of MY18-2ED on 2E protein, we incubated mammalian cells with iPep-SARS2-E and transfected them with 2E-YFP construct. Interestingly, iPep-SARS2-E significantly reduced 2E-YFP protein expression in mammalian cells. While we observed both monomeric and aggregate bands of 2E proteins, both forms were significantly decreased in the treated cells (Figs 3E–3G and S3A). As a control experiment, we used YFP-transfected cells with peptide treatment and did not observe any effect of iPep-SARS2-E on YFP protein expression (Figs 3H, S3B and S3C). The results indicate that iPep-SARS2-E does not affect lipofection but reduces 2E expression in mammalian cells. Based on the results obtained in the series of experiments, the suggested molecular mechanism underlying the inhibitory effect of iPep-SARS2-E is as follows (Fig 3I): the peptides might interact with 2E proteins (Fig 3A and 3B) and inhibit 2E channel function (Fig 3C and 3D), resulting in lysosomal pH restoration (Figs 1B–1E and S2A) and 2E protein degradation (Fig 3E–3G).

To examine the kinetics of peptide penetration into mammalian cells, we conjugated a fluorescent probe, Alexa Fluor 594, to the N- or C-terminus of iPep-SARS2-E. Fluorescent imaging in situ reveals that the C-terminal fused version exhibited faster cell-penetration than the N-terminal version in NIH 3T3 cells. We found that most cells can uptake the peptides in 2 h (S4A and S4B Fig). This result suggests that the N-terminal conjugation of Alexa Fluor 594 might slightly affect the function of the TAT motif in the peptide. To examine the off kinetics, we next treated cells with iPep-SARS2-E fluorescent peptide (C-terminal version, TAT-MY18-2ED-Alexa Fluor 594) for 24 h, washed out the culture medium, and then monitored the red fluorescence (S4C Fig). The in situ imaging suggests that the peptide is not reduced or degraded for 96 h; however, the peptide might become unstable and aggregated after 72 h at 37°C in the live cells, since larger fluorescent puncta were observed compared to earlier time points (S4D Fig). In addition, we tested the C-terminal version in another mammalian cell line, Vero-E6, which has been commonly used in virological studies using SARS-CoV-2. We confirmed that the peptides can penetrate into these cells as well (S5A and S5B Fig).

To examine the effect of iPep-SARS2-E on SARS-CoV-2 infection, as a proof-of-concept experiment in vitro, we conducted a cytopathic assay using a mammalian cell line, Vero-E6, and SARS-CoV-2 (WA1 strain, Fig 4A). We used the wild-type MY18 peptide (non-TAT version, MY18-WT) as a negative control in the assay because MY18-WT does not have any cell-penetrating motifs or effect on 2E in the pH imaging in NIH 3T3 cells (Fig 1E). The cytopathic assay result demonstrates that iPep-SARS2-E significantly inhibits viral toxicity in vitro (Fig 4B: IC 50 of iPep-SARS2-E, approximately 400 nM). Following these results, we next conducted time-course experiments to elucidate the mechanism underlying the inhibitory effect of iPep-SARS2-E on viral function (Fig 4C). The qPCR result demonstrates that there is no difference in SARS-CoV-2 nucleocapsid (N) gene expression, suggesting no effect of iPep-SARS2-E on virus transcription and entry (Fig 4D). On the other hand, there is a significant difference in SARS-CoV-2 N gene detection between the culture supernatants of PBS-treated control and iPep-SARS2-E-treated cells sampled at 24 h post-infection (Fig 4E), demonstrating a significant reduction of virus release from iPep-SARS2-E-treated cells. Importantly, we found that iPep-SARS2-E could significantly restore the expression of JUN/AP-1 (Fig 4F), which we had used as a reporter of SARS2-E cellular toxicity in this study to optimize the MY18 peptide series (Fig 2). Electron microscopy reveals that viral particles can be observed in large vacuoles of PBS-treated cells (Fig 4G and 4H), which could be deacidified and disrupted lysosomes, according to a previous study [37]. However, no vacuoles containing multiple viral particles were found in iPep-SARS2-E-treated cells, while small particles were found in the endoplasmic reticulum and nuclear envelope (Figs 4I and S6A). This suggests that the particles might be virions, though it is not clear whether the virions are mature in iPep-SARS2-E-treated cells. Therefore, we next examined infectivity of these intracellular particles from iPep-SARS2-E-treated cells treated via an endpoint titration assay used to quantitate intracellular virus particles (S6B Fig) [38–40]. We found that there was a modest but significant (P < 0.0001) reduction in virus titers of iPep-SARS2-E-treated cell samples compared to PBS-treated control cells, but that the intracellular particles of iPep-SARS2-E-treated cell samples are still infectious (S6C Fig).

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TIFF original image Download: Fig 4. iPep-SARS2-E in vitro test. (A) Experimental design for the iPep-SARS2-E (TAT-MY18-2ED) test using SARS-CoV-2 WA1 virus (MOI, 0.10) and Vero-E6 cells in vitro. (B) Inhibition of iPep-SARS2-E in the cytopathic effect of SARS-CoV-2 WA1 virus on Vero-E6 cells. MY18-WT (non-TAT) was used as a control. (C) Design of time-course experiment using SARS-CoV-2 WA1 virus (MOI, 0.10) and Vero-E6 cells in vitro. (D) Time-course qPCR of SARS2-CoV-2 nucleocapsid (SARS2 N) expression of PBS- and iPep-SARS2-E (10 μM)-treated Vero-E6 cells. The expression of N gene was normalized to a house-keeping gene, GAPDH. Student’s t test was used at each time point (n.s., not significant). (E) qPCR of SARS2 N expression of PBS- and iPep-SARS2-E (10 μM)-treated Vero-E6 cell culture supernatant (sup) at 24-h post-infection. Student’s t test was used (**** P < 0.0001). (F) qPCR of JUN/AP-1 expression of PBS- and iPep-SARS2-E (10 μM)-treated Vero-E6 cells comparing to non-infected cells. The expression of JUN was normalized to GAPDH. One-way ANOVA with Tukey’s multiple comparisons test was used (* P < 0.05; n.s., not significant). (G) Representative electron microscopic (EM) image of PBS-treated Vero-E6 cells at 24 h post-infection. Scale bar, 500 nm. (H) Higher magnification of the EM image of PBS-treated Vero-E6 cells at 24 h post-infection (a box shown in Fig 4G). Scale bar, 100 nm. (I) Representative EM image of iPep-SARS2-E-treated Vero-E6 cells at 24 h post-infection. Arrowheads, virus-like particles accumulated in the endoplasmic reticulum. The other EM image is also shown in S6A Fig. Scale bar, 500 nm. (J) Representative confocal fluorescent images of Vero-E6 cells treated with PBS or iPep-SARS2-E at 24 h post-infection. SARS2 N antibody (red) and Hoechst 33258 dye (blue, for nucleus) were used with antibodies of subcellular organelle markers (green): BiP for endoplasmic reticulum (ER), ERGIC-53 for ER Golgi inter compartment (ERGIC), and LAMP1 for lysosome. Scale bar, 5 μm. The data underlying this figure can be found in S1 Data. All the graphs in the figure are mean ± SD. EM, electron microscopic; ER, endoplasmic reticulum; ERGIC, ER-Golgi inter-compartment; PBS, phosphate-buffered saline; qPCR, quantitative polymerase chain reaction; SARS‑CoV‑2, Severe Acute Respiratory Syndrome Coronavirus 2. https://doi.org/10.1371/journal.pbio.3002522.g004

E protein is thought to be involved in Spike protein protection and maturation [20,24]. Therefore, we examined the effect of iPep-SARS2-E on Spike protein expression. The western blotting result demonstrates significant reductions of Spike protein expression (S6D Fig). Immunocytochemistry confirms that, in iPep-SARS2-E-treated cells, N proteins are co-localized to BiP/GRP78 (a marker of endoplasmic reticulum, or ER), and to ERGIC-53 (a marker of ER-Golgi inter-compartment, or ERGIC); in PBS-treated cells, N proteins were more highly and broadly expressed, and co-localized to LAMP1 (a lysosomal marker) (Fig 4J).

Following these results, we conducted in vitro experiments using iPep-SARS2-E at a later time-point to examine its effect further. qPCR shows that there is a significant decrease in SARS-CoV-2 N and E gene expression in iPep-SARS2-E-treated Vero-E6 cells at 48 h post-infection compared to the PBS-treated control cells (S6E and S6F Fig). In addition, iPep-SARS2-E significantly restores the expression of JUN/AP-1 (S6G Fig). Importantly, detection of SARS-CoV-2 genes in the culture supernatant harvested at 48 h post-infection is significantly reduced in iPep-SARS2-E-treated cells compared to PBS-treated control cells (S6H Fig), demonstrating that iPep-SARS2-E suppresses virus release. Immunocytochemistry shows that the majority of infected Vero-E6 cells became round and apoptotic-like in the PBS-treated group, while iPep-SARS2-E-treated cells exhibited moderate expression of viral N and normal cellular morphology (S6I Fig). This is consistent with the results of the cytopathic assay (Fig 4B).

Next, to validate the inhibitory effect of iPep-SARS2-E further, we conducted a preclinical experiment in vivo using iPep-SARS2-E intravenous (i.v.) injection to Balb/c mice infected with a mouse-adapted strain of SARS-CoV-2 (MA10, [41–43]). First, we conducted i.v. injection of the C-terminal fluorescent version of iPep-SARS2-E (TAT-MY18-2ED-Alexa Fluor 594) to mice and confirmed that the peptide can permeate and is detectable in mouse lung tissues 2 h after administration (Fig 5A). Following this result, we next injected iPep-SARS2-E post-infection. The mice were sacrificed 4 days after MA10 viral infection, and their lungs were harvested for viral titer, qPCR, and histology (Fig 5B). The results show no difference in body weight between the groups, but a significant reduction of viral propagation in iPep-SARS2-E-treated mouse lungs compared to the control (Fig 5C and 5D). qPCR also confirms the inhibitory effect of iPep-SARS2-E on the viral propagation in vivo (Fig 5E). Lung histology reveals no immune infiltration or alveolar damage in iPep-SARS2-E-treated mouse lungs, while minimal interstitial infiltrates with patchy lymphoid aggregates and protein accumulation were observed in the non-treated control group (Fig 5F).

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TIFF original image Download: Fig 5. iPep-SARS2-E in vivo preclinical study. (A) Representative fluorescent and bright field images of lung tissues isolated from mice administrated i.v. with PBS or Alexa594-conjugated iPep-SARS2-E peptide (TAT-MY18-2ED-A594, 300 μM, 2 h and 24 h). After isolating the tissues, the samples were washing using PBS 3 times, and the fluorescent and bright field images were taken by a fluorescent stereoscope. Scale bar, 2 mm. (B) Experimental design using the iPep-SARS2-E i.v. injection in vivo. (C) There is no difference in body weight between PBS control (n = 11) and iPep-SARS2-E-treated mice (n = 7). (D) There is a significant reduction of lung viral titer in iPep-SARS2-E-treated mice compared to the control. Student’s t test was used (* P < 0.05; n.s., not significant). Median TCID is normalized to lung wet weight (g) measured before tissue homogenization to isolate the virus. (E) iPep-SARS2-E significantly reduced the transcript expression of SARS2 N in MA10-infected Balb/c mouse lung tissues (PBS, n = 11; iPep-SARS2-E, n = 7, normalized to mouse Gapdh expression). Student’s t test was used (** P < 0.01). (F) Representative images of hematoxylin and eosin (HE) staining of mouse lung tissues of PBS control and iPep-SARS2-E-treated mice at 4 days post-infection. Protein accumulation (x) and immune cells (arrowheads) are indicated. Scale bar, 50 μm. The data underlying this figure can be found in S1 Data. All the graphs in the figure are mean ± SD. HE, hematoxylin and eosin; PBS, phosphate-buffered saline; TCID, tissue culture infection dose. https://doi.org/10.1371/journal.pbio.3002522.g005

To examine whether iPep-SARS2-E could be applied for prevention of SARS-CoV-2 infection, we conducted another experimental series in vivo using iPep-SARS2-E intranasal administration to Balb/c mice infected with MA10 virus. First, we administrated the C-terminal fluorescent version of iPep-SARS2-E to mice intranasally and confirmed that the peptide can permeate and is detectable in mouse nasal tissues 2 h after administration (S7A and S7B Fig). Next, we conducted a safety study in vivo (S7C Fig) using intranasal administration to Balb/c mice to examine the effect of iPep-SARS2-E on body weight and inflammation markers, comparing to non-treated and PBS-administrated groups. We did not find any significant differences in body weight, Cxcl12 and C5a among iPep-SARS2-E-, PBS-, and non-treated groups (S7D–S7F Fig). Following the results using intranasal administration, we applied iPep-SARS2-E intranasal administration to Balb/c mice infected with MA10 virus (S7G Fig). We found that iPep-SARS2-E significantly prevents the body weight loss and suppresses 2E protein expression in infected mouse lungs in vivo (S7H–S7K Fig). These results using in vivo experiments demonstrate that SARS2-E inhibition can be a novel strategy to prevent SARS-CoV-2 toxicity and propagation in vivo.

To validate iPep-SARS2-E further, we continued to conduct control experiments, preparing an additional negative control and experimental conditions. When we examined the effect of deletion on MY18 constructs using the luciferase reporter, we had found that none of the constructs significantly improved the effect: the majority did not reduce its efficacy significantly, but deletion at the 5th and 17th/18th residues significantly reduced the inhibitory effect of MY18 on 2E-mediated NFAT/AP-1 pathway alteration (Fig 2F). Following the results, we introduced Ala-substitution, targeting these amino acids of MY18 (S8A Fig). NFAT/AP-1 Luc assay and DND-189 imaging results demonstrate that the mutagenesis significantly reduced the inhibitory effect of MY18, although NFAT/AP-1 Luc assay result suggests that it was not sufficient to fully counteract the effect of MY18 (S8B and S8C Fig). After further mutagenesis, we found that the addition of an L12A substitution was able to negate the inhibitory effect of MY18 against 2E. We confirm using qPCR, that, like PBS, the mutant peptide cannot reduce SARS-CoV-2 N expression in Vero-E6 cell culture supernatant (S8D Fig). In the following experiments, we used this mutant peptide as a new negative control.

The qPCR results using Vero-E6 cell culture supernatant and lysate samples at 24 h post-infection with SARS-CoV-2 (Fig 4C–4E) suggest that iPep-SARS2-E may not have any effect on the virus entry. However, this time-course transcription profiling may not be sufficient as a readout of the virus entry. To address this concern, we conducted an experiment using pseudo virus containing SARS-CoV-2 Spike, Membrane, E proteins, and YFP reporter with the negative control peptide and iPep-SARS2-E because pseudo virus is useful to examine the effect of drug candidates on the virus entry [44]. The pseudo virus infection resulted in no difference in YFP-positive cell number between the negative control peptide- and iPep-SARS2-E-treated cells, revealing no effect of iPep-SARS2-E on the virus entry (S8E and S8F Fig).

To validate the inhibitory effect of iPep-SARS2-E peptide further, we conducted in vitro experiments using human pluripotent stem cell-derived branching lung organoids and WA1 virus (Fig 6A and 6B). We found that there is a significant reduction in SARS-CoV-2 N transcript of the culture supernatant at 24 h post-infection between the negative-control peptide- and iPep-SARS2-E-treated organoids but not in the organoid lysate (Fig 6C and 6D), suggesting that virus egress is blocked by iPep-SARS2-E. Immunocytochemistry allows us to observe higher expression of SARS-CoV-2 N in the negative control peptide-treated organoids compared to iPep-SARS2-E-treated organoids (Fig 6E). The results using the lung organoids are consistent with the results using Vero-E6 cells at 24 h post-infection (Fig 4). Our results in monolayer cells and in human 3D lung organoids reveal a new strategy to prevent SARS-CoV-2 propagation using iPep-SARS2-E, which inhibits 2E activity and virus egress.

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TIFF original image Download: Fig 6. iPep-SARS2-E and its negative control peptide test using in vitro human lung organoid model. (A) Experimental design for the iPep-SARS2-E test using SARS-CoV-2 WA1 virus (MOI, 0.10) and human pluripotent stem cell-derived branching lung organoids in vitro. The image is prepared using BioRender. (B) Representative phase contrast images of human pluripotent stem cell-derived branching lung organoids. Scale bar, 50 μm. (C and D) qPCR of SARS2 N expression of the negative control mutant peptide (neg. Ctrl, S8 Fig)- and iPep-SARS2-E (10 μM)-treated organoids culture supernatant (sup, C) and cells (D) at 24 h post-infection. Student’s t test was used (* P < 0.05; n.s., not significant). (E) Representative merged section images of confocal fluorescence and bright field of human lung organoids treated with neg. Ctrl or iPep-SARS2-E at 24 h post-infection. SARS2 N antibody (red, arrowheads), EpCAM antibody (green), and Hoechst dye (blue, for nucleus) were used. Scale bar, 20 μm. The data underlying this figure can be found in S1 Data. All the graphs in the figure are mean ± SD. qPCR, quantitative polymerase chain reaction; SARS‑CoV‑2, Severe Acute Respiratory Syndrome Coronavirus 2. https://doi.org/10.1371/journal.pbio.3002522.g006

Next, we conducted another in vivo mouse study (S8G Fig) using a single intranasal administration to Balb/c mice to examine the effect of iPep-SARS2-E and the negative control peptide on body weight, SARS-CoV-2 titer, and N transcript in the mouse lungs. We found that iPep-SARS2-E could significantly prevent their body weight loss while the negative control did not (S8H Fig). The viral titer and qPCR results confirm the inhibitory effect of iPep-SARS2-E in vivo (S8I and S8J Fig). These in vivo results demonstrate that SARS2-E inhibition using a single intranasal dose of iPep-SARS2-E can be useful to prevent SARS-CoV-2 toxicity and propagation in vivo.

Next, we hypothesized that this peptide design and strategy could be customized and applied to other human coronaviruses because coronavirus E proteins are highly conserved (Figs 7A and S9). Therefore, following iPep-SARS2-E development, we designed inhibitory peptide constructs against E proteins from each of the other human coronaviruses: MERS-CoV, HCoV-NL63, -OC43, -HKU1, and -229E (Fig 7B). First, we found that the overexpression of MERS-CoV and HCoV-NL63 E proteins significantly increased NFAT/AP-1 luciferase reporter activity in mammalian cells while the E proteins of HCoV-OC43, -HKU1, and -229E do not have an effect on NFAT/AP-1 pathway (Fig 7C). Following these results, we focused our testing to MER-CoV and HCoV-NL63 MY18 WT peptide constructs using the NFAT/AP-1 luciferase reporter assay. The reporter assay results demonstrate that MERS-CoV and HCoV-NL63 MY18 WT could significantly reduce the effect of each E protein on NFAT/AP-1 reporter while substitution of Glu/E to Asp/D or Asp/D to Glu/E does not improve the MY18 constructs for MERS-CoV and HCoV-NL63, respectively (Fig 7D and 7E). To improve each MY18 further, we conducted mutagenesis of MERS-CoV MY18 and HCoV-NL63 MY18 constructs. We found that HCoV-NL63 MY18 2DE and N9D and MERS-CoV MY18 R8H are the best to inhibit the effect of HCoV-NL63 and MERS E proteins on NFAT/AP-1 pathway in mammalian cells, respectively (Fig 7D and 7E). Next, using DND-189 pH imaging, we found that the overexpression of MERS-CoV, HCoV-NL63, and -HKU1 E proteins significantly reduced DND-189 fluorescence in mammalian cells while the E proteins of HCoV-OC43 and -229E do not have a significant effect on lysosomal proton homeostasis (Fig 7F). Following these results, we focused on testing MY18 constructs on MERS-CoV, HCoV-NL63, and -HKU1 and found that MERS-CoV MY18 R8H, HCoV-NL63 MY18 2DE and N9D, HCoV-HKU1 MY18 D8E peptide constructs could significantly rescue the phenotypes in proton homeostasis in mammalian cells caused by the respective E proteins (Fig 7F). The results of this experiment reveal that the MY18 peptides can be applicable for not only SARS-CoV-2 but also some of the other human coronaviruses such as MERS-CoV, HCoV-NL63, and HCoV-HKU1, demonstrating that E protein can more broadly be a potential therapeutic target for human coronaviruses.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002522

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