Journal: Frontiers in Immunology

Article Title: Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity

doi: 10.3389/fimmu.2021.719077

Figure Lengend Snippet: Sindbis-Spike vaccine prevents infection of SARS-CoV-2 in hACE2 transgenic (hACE2-Tg) mice. Luciferase -encoding SARS-CoV-2 spike pseudotyped lentivirus was incubated with mouse sera collected at (A) 21 and (B) 75 days post vaccination with SV.Spike, SV.Spike in combination with αOX40 and αOX40 antibody alone compared and unvaccinated naïve groups. Area under the curve (AUC) values of serum antibodies were calculated from reciprocal dilution curves in antibody detection assay. The data presented are the mean of 5 biological replicates with two technical replicates. Statistics were performed using a One-way ANOVA with the Bonferroni correction in GraphPad Prism. n.s. > 0.05; ****P<0.0001. (C) Expression of pseudotyped SARS-CoV-2-spike-lacZ lentivirus in whole mouse lung following intranasal delivery. One week following vector nasal administration to the right nostril of four weeks old hACE2 transgenic and control mice (B6(Cg)-Tg(K18-ACE2)2Prlmn/J), expression of LacZ was analyzed in mice airways. X-Gal stained whole lungs from (left) hACE2 non carrier control mouse and (right) hACE2 transgenic mouse, both dosed with SARS-CoV-2-spike-lacZ pseudotyped lentivirus. (D) Schematic of the “challenge” experiment with SARS-CoV-2-spike-lacZ lentivirus. (E) On day 21 (upper panels) and 75 (lower panels) after the initial infection hACE2-Tg were “challenged” with 3.6 x 10 5 PFU of SARS-CoV-2-spike-lacZ pseudotyped lentivirus and then analyzed for X-Gal staining at day 7 “post-challenge.” Three non-vaccinated naïve animals were included as a positive control in the “challenge” experiment. (F–H) hACE2-Tg mice were vaccinated with SV.Spike and/or αOX40 and challenged with 10 4 particles of live SARS-CoV-2 coronavirus at day 21 post immunization. Weight loss and mortality were observed daily for 14 days after live virus infection and compared to the naïve unvaccinated group. (G) Change of body weight during systemic infection with SARS-CoV-2 coronavirus. Percent weight loss (y-axis) is plotted versus time (x-axis). Data points represent mean weight change +/− SEM. (H) Survival curves of SV.Spike with or without αOX40 treated and naïve unvaccinated mice. n = 4 or 5 mice per group. One mouse belonging to the SV.Spike+aOX40 group died for inapparent reasons 7 days after challenge with the authentic SARS-CoV_2. Graph points in (G) were calculated as average of 4 mice instead of 5.

Article Snippet: SARS-CoV-2 spike (BPS Bioscience) and p24 (Abcam) recombinant proteins were used as positive controls ( ).

Techniques: Infection, Transgenic Assay, Luciferase, Incubation, Detection Assay, Expressing, Plasmid Preparation, Staining, Positive Control

Journal: Frontiers in Immunology

Article Title: Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity

doi: 10.3389/fimmu.2021.719077

Figure Lengend Snippet: Challenging immunized mice with spike antigen promotes a fast and coordinated response of the two arms of the adaptive immune system. Humoral and T cell immune responses were assessed in vaccinated mice after “challenge” with Sindbis carrying SARS-CoV-2-spike (SV.Spike). (A) Design steps of the “challenge” experiment in immunized C57BL/6J mice evaluated by (B) T cell cytotoxic assay, (C–F) Flow cytometry indicating cytotoxic CD8 T cell effector response by GrB+ positive CD8 T cells and activation of CXCR5+ICOS+ positive Tfh cells upon “challenge”, (G) binding IgA, IgM, IgG antibody ELISA to SARS-CoV-2-spike recombinant protein (n=5 mice per group, or as otherwise indicated). Each symbol represents one individual mouse. Bars or symbols represent means ± SEM, and statistical significance was determined with one-way ANOVA with the Bonferroni correction (B, G) or with the Kruskal-Wallis test followed by Dunns’ test (C–F) . n.s. > 0.05, **p<0.005, ***p≤ 0.001, ****p ≤ 0.0001.

Article Snippet: SARS-CoV-2 spike (BPS Bioscience) and p24 (Abcam) recombinant proteins were used as positive controls ( ).

Techniques: Flow Cytometry, Activation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity

doi: 10.3389/fimmu.2021.719077

Figure Lengend Snippet: Characterization of Sindbis vector carrying the SARS-CoV-2 spike. (A) Schema of SARS-CoV-2 spike gene cloned into Sindbis vector system. (B) Western Blot of SARS-CoV-2 spike produced from the Sindbis vector. Lanes shown are titration of the vector, and recombinant spike control produced in HEK cells. (C) Schematic of vaccination. C57BL/6 mice were immunized with 1× 0.5 ml SV.Spike/and or αOX40 antibody (250 μg/dose) on day 0. A boost injection of SV.Spike/and or αOX40 were once given on day 14. On days 7, 14 and 21, 75, and 100, blood was taken to determine Sars-Cov-2 spike specific antibodies by ELISA. Spleens were excised and a single cell suspension was stained and analyzed by flow cytometry. T cells were isolated and were used for ELISPOT assay and Seahorse. As control, naïve C57BL/6J mice were used.

Article Snippet: SARS-CoV-2 spike (BPS Bioscience) and p24 (Abcam) recombinant proteins were used as positive controls ( ).

Techniques: Plasmid Preparation, Clone Assay, Western Blot, Produced, Titration, Recombinant, Injection, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Isolation, Enzyme-linked Immunospot

Journal: Frontiers in Immunology

Article Title: Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity

doi: 10.3389/fimmu.2021.719077

Figure Lengend Snippet: SARS-CoV-2 spike specific antibodies induced by Sindbis vector. Characterization of serum IgA, IgM, and IgG in C57BL/6J mice vaccinated with SV.Spike at days 21, 75, and 100 post-immunization. (A) The levels of Spike-specific IgA, IgM, and IgG isotypes in sera (diluted 1:100) of immunized mice at different time windows. P values were calculated by one-way ANOVA with the Bonferroni correction in Graphpad Prism. n.s. > 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001. (B) The kinetics of Spike-specific IgA, IgM, and IgG isotypes in sera of immunized mice at different time windows. Mean comparison between time points and isotypes and relative P values were determined by two-way ANOVA with the Bonferroni correction in GraphPad Prism. The data presented are the mean of three technical replicates. The median values of (A) OD450 or (B) calculated log2 antibody levels were plotted for each isotype of three antibodies. Log2-scale in (B) was used to better represent the proportional relation between antibody titers and sera reciprocal dilution. The data presented are the mean of five biological replicates with two technical replicates.

Article Snippet: SARS-CoV-2 spike (BPS Bioscience) and p24 (Abcam) recombinant proteins were used as positive controls ( ).

Techniques: Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity

doi: 10.3389/fimmu.2021.719077

Figure Lengend Snippet: Blockade of SARS-CoV-2 spike-hACE2 binding and spike protein-mediated cell–cell fusion by anti-SARS-CoV-2 spike neutralizing antibodies. (A, B) In the assay, anti-SARS-CoV-2 neutralizing antibodies from immunized C57BL/6J mice, block recombinant Spike protein from binding to the hACE2 protein pre-coated on an ELISA plate. Percentage of inhibition distributed along y-axis of SARS-CoV-2 spike–hACE2 interaction for the indicated reciprocal plasma dilutions by mouse sera collected at (A) 21 and (B) 75 days post vaccination with Sindbis expressing SARS-CoV-2 spike (SV.Spike), SV.Spike in combination with αOX40 and αOX40 alone compared to the naive group. Area under the curve (AUC) values of serum antibodies were calculated from reciprocal dilution curves in antibody detection assay. The data presented are the mean of 5 biological replicates with two technical replicates. Statistics were performed using a One-way ANOVA with the Bonferroni correction in Graphpad Prism. n.s. > 0.05; *P < 0.05; **P < 0.01; ***P<0.001; ****P<0.0001. (C) Images of SARS-CoV-2 spike-mediated cell–cell fusion inhibition on 293T/ACE2 cells by sera from C57BL/6J vaccinated mice. SARS-CoV-2 spike-transfected 293T were incubated with mice serum at 1:100 dilution and applied onto 293T/ACE2 cells for 24 h. Scale bar: 100 µm. (D) Quantification of the number aggregates (left panel) and inhibition of cell–cell fusions (right panel) induced by SARS-CoV-2 spike following pre-incubation with naïve, SV.Spike, SV.Spike+αOX40 and αOX40 alone are shown. N = 5 biological replicates with two independent technical replicates. One-way ANOVA with Bonferroni correction *P < 0.05, **P < 0.01, and ***P < 0.001. (E) Representative confocal images of 293T/ACE2 cells treated with serum from Naïve and SV.Spike+αOX40-immunized mice pre-incubated with SARS-CoV-2 spike recombinant protein and stained for hACE2 (green), SARS-CoV-2 spike (red), and DAPI (blue). Scale bar: 20 μm.

Article Snippet: SARS-CoV-2 spike (BPS Bioscience) and p24 (Abcam) recombinant proteins were used as positive controls ( ).

Techniques: Binding Assay, Blocking Assay, Recombinant, Protein Binding, Enzyme-linked Immunosorbent Assay, Inhibition, Expressing, Detection Assay, Transfection, Incubation, Staining

Journal: Frontiers in Immunology

Article Title: Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity

doi: 10.3389/fimmu.2021.719077

Figure Lengend Snippet: Sindbis expressing SARS-CoV-2 spike+αOX40 C57BL/6J vaccinated mice are characterized by a unique transcriptional signature of T cells. Combination therapy markedly changes the transcriptome signature of T cells favoring T-cell differentiation towards effector T-cells with a Th1 type phenotype 7 days after prime vaccination. (A) Principal component analysis (PCA) of RNA seq data from naïve, SV.Spike, and/or αOX40 groups. (B) Venn diagrams summarizing the overlap between differentially expressed genes (DEGs) from SV.Spike (blue), αOX40 (pink), and SV.Spike+αOX40 (purple). Up-regulated DEGs (left) and down-regulated (right). (C) MA plots of differentially expressed genes in T-cells of naive versus SV.Spike (top graph), αOX40 (middle graph), and combination (bottom graph). Significantly (p<0.05) upregulated and downregulated DEGs are depicted in red or blue, respectively. (D) Pathway and network analysis based on GSEA in T-cells isolated from mice treated with combination therapy. Downregulated (blue circle) and upregulated (red circles) pathways are shown, respectively. (E) Pathway and network analysis based on GSEA in T-cells isolated from mice treated with single dose of SV.Spike. Top 10 hub biological process gene ontology (GO) terms ranked by the Cytoscape plugin cytoHubba (red, highest ranks; yellow, lowest ranks) in the SV.Spike only (F) versus combination immunized group (G) . Heatmap analysis of selected genes based on normalized read counts linked to T-cell differentiation in the SV.Spike and/or αOX40 immunized mice compared to naïve (H) . Highlighted selected gene set enrichment analysis (GSEA) pathways based on DEG in naive versus SV.Spike (I) and combination treated group (J) .

Article Snippet: SARS-CoV-2 spike (BPS Bioscience) and p24 (Abcam) recombinant proteins were used as positive controls ( ).

Techniques: Expressing, Cell Differentiation, RNA Sequencing Assay, Isolation

Journal: Frontiers in Immunology

Article Title: Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity

doi: 10.3389/fimmu.2021.719077

Figure Lengend Snippet: Reprogrammed T cells in SV.Spike+αOX40 vaccinated mice display enhanced Th-1 T-cell phenotype mediated cytokine production and cytotoxic T-cell activity. Spleens of C57BL/6J naïve and vaccinated mice were excised on day 7 after prime vaccine doses for flow cytometry analysis (A–J) . T cells were further isolated for (K) Interferon-g (IFN γ ) enzyme-linked immunospot analysis (ELISpot) and (L, M) cytotoxicity analysis. Percentage of (A) CXCR3 and (B) CX3CR1 expressing CD4+ T cells indicating Th1-like T-cell effector phenotype. (C) Percentage of Tbet+ICOS+ positive Tfh-like effector CD4+ T-cell polarization. (D) Representative blots. (E) Percentage of granzyme B (GrB) positive CD4+ T cells from indicated groups using flow cytometry. (F) Representative blots. (G) Percentage of GrB positive CD8+ T cells from indicated groups using flow cytometry. (H) Representative blots. (I) Percentage of Perforin positive CD8+ T cells. (J) Representative blots. Bars represent means ± SEM (A–J) and each symbol represent an individual mouse (n=5 per group). Statistical significance was determined with the Kruskal-Wallis test followed by Dunns’ test. Results are representatives of at least two independent experiments. (K) Amount of IFN γ spots per 10 5 T cells determined by ELISpot. (L, M) Cytotoxic activity of T cells harvested on day 7 from control and treated mice (n = 5 mice per group). T-cells were isolated from splenocytes and were co-cultured with 293T/ACE2 cells for 2 days. Effector-to-target (E/T) cell ratio (T cells/ACE2 cells) was 30:1. Cytotoxicity was determined for each group of mice by measuring the infectivity of luciferase-encoding pseudotyped particles with (L) Spike protein of SARS-CoV-2 or (M) VSV-G and is shown relative to naive T cells. Bars or symbols represent means ± SEM, and statistical significance was determined with one-way ANOVA with the Bonferroni correction. n.s. > 0.05, *p<0.05, **p<0.01, ***p≤ 0.001, ****p ≤ 0.0001.

Article Snippet: SARS-CoV-2 spike (BPS Bioscience) and p24 (Abcam) recombinant proteins were used as positive controls ( ).

Techniques: Activity Assay, Flow Cytometry, Isolation, Enzyme-linked Immunospot, Expressing, Cell Culture, Infection, Luciferase

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: Identification of SARS-CoV-2 RNA elements with structural similarity to the canonical Cp GAIT element. (a) Bioinformatics-predicted secondary structures of the Cp GAIT element and SARS-CoV-2 S and ORF1a VAIT elements. VAIT sequences were identified using the Foldalign program and secondary structure and free energy predictions were made using RNA Folding server RNAstructure version 6.3 (see Materials and Methods). For the Cp GAIT element, the numbers in parentheses indicate nucleotide position in the 3′ UTR (counting from the first base after the stop codon); for the SARS-CoV-2 VAIT elements, the nucleotide position in the virus genome is given. (b) 1D- 1H-NMR spectroscopic analysis of Cp GAIT and SARS-CoV-2 S and ORF1a VAIT elements. The imino region of 1D- 1H-NMR spectra obtained for chemically synthesized and HPLC-purified RNA elements at four different temperatures are shown. Sharp resonances between 10 and 14.5 ppm (typical for canonical Watson–Crick or G-U wobble base pairing) indicate that all three RNA elements adopt structures consistent with in silico predictions shown in (a).

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: Synthesized, Purification, In Silico

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: In vitro translation of reporter mRNAs containing S and ORF1a VAIT elements is suppressed by extracts from SARS-CoV-2 spike protein-treated lung cells. (a) Chimeric luciferase reporter mRNAs harboring either the S VAIT element or the Cp GAIT element in their 3′UTRS were translated in vitro in rabbit reticulocyte lysates in the presence of [ 35 S]-Methionine. Cell extracts prepared from SARS-CoV-2 spike protein (S1 subunit) or IFN-γ -treated (for 24 h) human bronchial epithelial cells (HBTEC), A549 cells or U937 cells were added to the translation reactions. [ 35 S]-Methionine-labeled translation products from the luciferase reporter and phage T7 gene 10 cRNA (included in the reaction as an internal control, no GAIT or VAIT element present) were resolved by SDS-PAGE (indicated by arrows). The experiment shown in the right panel includes a “luciferase-only” reporter (no GAIT or VAIT element, first two lanes) to illustrate that repression of luciferase translation by extracts of S protein-treated lung cells requires the VAIT element. (b) Translational control assays were performed as described in (a) with luciferase reporters containing either the SARS-CoV-2 ORF1a VAIT element (left panel) or the Cp GAIT element (right panel) in their 3’UTRs.

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: In Vitro, Luciferase, Labeling, SDS Page

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: SARS-CoV-2 spike protein pseudotyped lentivirus or virus like particles (VLPs) containing S protein can induce VAIT element-mediated translational control in human bronchial epithelial cells. (a) Translational control assay performed as in with a luciferase reporter mRNA containing the SARS-CoV-2 S VAIT element in the 3’UTR. Cell extracts added to in vitro translation reactions were prepared from HBTEC cells left untreated or treated for 72 h with bald (“blank”) lentivirus, SARS-CoV-2 spike protein pseudotyped lentivirus, or VLPs (containing structural proteins M, N, and E only or M, N, E, and S). (b) Transduction of SARS-CoV-2 spike protein pseudotyped lentivirus in human bronchial epithelial cells was confirmed by luciferase expression. (c) Presence of SARS-CoV-2 structural proteins in VLPs purified from the culture medium of A549 cells 48 h after transfection with plasmids directing expression of SARS-CoV-2 M, N, and E, or M, N, E, and S proteins. Viral structural proteins were detected in VLPs by Western blotting using antibodies against either the native protein (M) or tags (HA, FLAG, and His) attached to the viral N, E, and S proteins.

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: Control Assay, Luciferase, In Vitro, Transduction, Expressing, Purification, Transfection, Western Blot

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: Ribosomal protein L13a is required for SARS-CoV-2 S protein-induced suppression of translation of VAIT element-containing mRNAs. Extracts were prepared from human bronchial epithelial cells (HBTEC) (a) and human alveolar epithelial cells (A549) (b) left untreated or treated with recombinant spike protein (S1 subunit). The immunodepletion of L13a was confirmed by immunoblot (a, right panel). Extracts were added to in vitro translation reactions performed as described for using the same luciferase reporter cRNAs harboring S or ORF1a VAIT elements (as shown below the panels). To test for involvement of L13a, L13a protein was immunodepleted from cell extracts by preincubation with anti-L13a antibody. IgG antibody was used as a negative control for immunodepletion.

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: Recombinant, Western Blot, In Vitro, Luciferase, Negative Control

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: Formation of L13a-dependent, SARS-CoV-2 VAIT element-specific RNA-protein complexes in spike protein-treated human bronchial epithelial cells (HBTEC) and A549 cells. Complexes were detected by RNA-EMSA in which biotin-labeled VAIT or GAIT element probes were incubated with extracts prepared from cells treated with spike protein or IFN-γ and then subjected to native gel electrophoresis. (a) RNA-EMSA analysis of the S VAIT element with cell extracts from S protein-treated HBTEC and of the Cp GAIT element with extracts from IFN-γ-treated U937 cells as indicated below (RNA element/probe) and above (cell extract) each lane. (b) RNA-EMSA analysis of the S VAIT element with extracts prepared from S protein-treated A549 cells. (c, d) RNA-EMSA analysis of the ORF1a VAIT element using extracts prepared from S protein-treated HBTEC (c) and A549 cells (d). L13a dependency was shown by immunodepleting L13a protein from cell extracts with anti-L13a antibody as in specificity of the complexes formed on S (a, b) and ORF1a (c, d) VAIT elements was determined by competition EMSA using 10- and 100-fold molar excess of unlabeled (“cold”) RNA oligos corresponding to the S VAIT, ORF1a VAIT, and Cp GAIT elements.

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: Labeling, Incubation, Nucleic Acid Electrophoresis

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: While exogenous addition of S protein induces VAIT element-mediated translation suppression, intracellular S protein production does not. (a) Expression of SARS-CoV-2 S protein in A549 cells stably transfected with either an empty vector (left panel) or a plasmid directing expression of S protein (pCDH-S, right panel) was detected by immunofluorescence. Cells were co-stained with antibodies against SARS-CoV-2 S protein (green) ( antibodies-online.com #ABIN1030641), the membrane protein ezrin, (red) (Thermofisher #MA5-13862), and DAPI (blue; to visualize DNA). (b) Western blot analysis of extracts made from the same cells as in (a) using an antibody specific for SARS-CoV-2 S protein (antibodies-online.com #ABIN1030641). (c) Translational control assay performed as in using the luciferase reporter mRNA harboring the S VAIT element and cell extracts made from A549 cells left untreated, treated exogenously treated with recombinant S protein, or stably transfected with pCDH-S (directing intracellular synthesis of S protein) or empty vector (as a negative control). For comparison, two lanes corresponding to the exogenous treatment and intracellular production (by stable expression) are marked with a star.

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Western Blot, Control Assay, Luciferase, Recombinant, Negative Control

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: The ACE2 receptor is required for SARS-CoV-2 S protein-induced translation control in lung cells. (a) RNAi-mediated depletion of ACE2 protein from A549 cells abrogates VAIT element-mediated translational control. In vitro translation reactions were performed as described for with luciferase reporter cRNAs harboring S or ORF1a VAIT elements as indicated below lanes. Cell extracts added to in vitro translation reactions were prepared from A549 cells that were transfected with a mock (nontargeting) siRNA pool or a specific ACE2 siRNA pool and then transduced with SARS-CoV-2 spike protein pseudotyped lentivirus as indicated above lanes. (b) Reduced expression of ACE2 protein in A549 cells transfected with ACE2-specific siRNA (but not in A549 cells transfected with a nontargeting “mock” siRNA pool) was confirmed by Western blotting with anti-ACE2 antibody. Actin was used as a specificity/loading control. (c) RNA-EMSA performed with biotin-labeled S and OFR1a VAIT element RNA probes and extracts prepared from A549 cells treated as in (a) (transfected with ACE2-targeting or nontargeting siRNA pools and transduced with S pseudotyped lentivirus).

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: In Vitro, Luciferase, Transfection, Transduction, Expressing, Western Blot, Labeling

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: Treatment of A549 lung cells with SARS-CoV-2 S pseudotyped lentivirus triggers phosphorylation and release of L13a from the ribosome. (a) Detection of ribosome-associated and free L13a in A549 cells exposed to S protein. Extracts from A549 cells treated with “bald” (negative control lacking S protein) or S protein pseudotyped lentivirus for the indicated amounts of time were separated into polysome (bottom) and ribosome-free cytosolic (top) fractions, which were then immunoblotted with anti-L13a or anti-L19 antibodies (Thermofisher #14701-1-AP). (b) (left panel), Confirmation of ribosomal and non-ribosomal fractions used in (a). RNA was extracted from the separated fractions using TRIzol, resolved on an agarose gel, and visualized by staining with ethidium bromide. 8b (right panel), Treatment with SARS-CoV-2 S pseudotyped lentivirus induces DAPK-dependent serine phosphorylation of L13a in A549 cells. A549 cells were pretreated with DAPK inhibitor KN62 (or DMSO solvent as a negative control) for 1 h before incubation with the lentivirus. After the indicated amounts of time, cell extracts were prepared and subjected to immunoprecipitation (IP) with anti-L13a antibody, followed by immunoblotting with anti-phosphoserine or anti-L13a antibodies. (c) Requirement of DAPK1 in SARS-CoV-2 S protein-induced and VAIT element-mediated translation control. A549 cells were either untreated or treated with a DAPK1 specific or a non-targeting (used as negative control) siRNA and the steady state level of DAPK1 and beta actin were monitored by immunoblot analysis with anti-DAPK1 and anti-beta actin antibodies. 48 h after siRNA transfection, cells were incubated with SARS-CoV-2 S protein pseudotyped lentivirus for 24 h. Cell lysates were then prepared and used in translation control assay as described earlier. Results showed DAPK1 knocked down cells failed to inhibit translation from chimeric RNA of luciferase and VAIT element (lanes 3 and 4 vs lane 5).

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: Negative Control, Agarose Gel Electrophoresis, Staining, Incubation, Immunoprecipitation, Western Blot, Transfection, Control Assay, Luciferase

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: The SARS-CoV-2 S VAIT element controls translation of the full-length S mRNA in A549 cells. (a) A mutant S VAIT element with altered secondary structure fails to support S protein-induced translation control. Right panel: Structures of the wild type and U22741C mutant S VAIT elements predicted using m-fold software. Left panel: In vitro translation of chimeric luciferase reporter cRNAs containing wild type or U22741C mutant S VAIT elements (as indicated below lanes) in the presence of extracts prepared from A549 lung cells treated with bald or S pseudotyped lentivirus (as indicated above lanes). Arrows show luciferase and T7 gene 10 (internal control) translation products. (b) VAIT element structure determines the polysomal association of full-length S mRNA in A549 cells in response to S protein treatment. Polyribosomal and free ribosomal fractions were prepared from A549 cells transfected with plasmids expressing full-length native S cDNA harboring either the wild type (upper panels) or U22741C mutant (lower panels) S VAIT element (and treated with either bald (left panels) or S pseudotyped (right panels) lentivirus). S mRNA and GAPDH mRNA (control) in each fraction was determined by RT-PCR. For each panel, a plot of the A 254 values of the fractions (1 through 12) is provided above agarose gels showing ethidium bromide-stained RT-PCR products in the corresponding fractions.

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: Mutagenesis, Software, In Vitro, Luciferase, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: Sequence conservation of SARS-CoV-2 VAIT elements. The colored bar provides a schematic representation of the SARS-CoV-2 genome structure spanning nucleotides 0 to ∼30,000. The black histogram above the colored bar shows the degree of diversity among currently available SARS-CoV-2 sequences at each position of the genome (data from https://nextstrain.org/ncov/ ), thus illustrating mutation host spots. The positions and sequence diversity of VAIT elements in ORF1a and ORF S are shown in expanded form below the colored bar.

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: Sequencing, Mutagenesis

Journal: Journal of Virology

Article Title: A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells

doi: 10.1128/JVI.01678-21

Figure Lengend Snippet: Model for VAIT element-driven regulation of SARS-CoV-2 S and ORF1a protein synthesis in host cells. S protein-mediated interaction of SARS-CoV-2 virus with ACE2 at the cell surface leads to phosphorylation of L13a and its release from ribosomes through a novel signaling pathway that involves DAPK but is otherwise not yet defined. The released extra-ribosomal phosphorylated L13a joins with other (currently unknown) proteins to form complexes on VAIT elements in viral RNAs, which suppresses their translation. Inhibition of viral protein synthesis via this mechanism might benefit the host by reducing lung damage and/or promoting host-virus homeostasis through the reduction of ER stress. Whether any host mRNAs are regulated by this translational control mechanism is currently unknown.

Article Snippet: Lentiviral particles pseudotyped with full-length SARS-CoV-2 spike glycoprotein were purchased from Genecopoeia (Rockville, MD; #SP101-100; 6.62 × 10 5 TU [(transduction unit)/mL]) and bald lentiviral pseudovirion particles with no VSV-G or SARS-CoV-2 spike protein were purchased from BPS Bioscience (San Diego, CA; #79943).

Techniques: Inhibition

Journal: Journal of Neuroimmune Pharmacology

Article Title: ACE-2-interacting Domain of SARS-CoV-2 (AIDS) Peptide Suppresses Inflammation to Reduce Fever and Protect Lungs and Heart in Mice: Implications for COVID-19 Therapy

doi: 10.1007/s11481-020-09979-8

Figure Lengend Snippet: Designing a peptide for disruption of ACE2 and SARS-CoV-2 interaction. a A rigid-body in silico docked pose of human ACE2 (green) and SARS-CoV-2 spike S1 (magenta). b Sequence of wild type and mutated ACE2-interacting domain of SARS-CoV-2 (AIDS) peptides. Positions of mutations are underlined. c Inhibition of ACE2 to SARS-CoV-2 spike S1 binding by wtAIDS, but not mAIDS, peptide. *** p < 0.001 vs. spike S1 . Human A549 lung cells pretreated with different concentrations of wtAIDS and mAIDS peptides for 15 min were stimulated with 1 ng/ml recombinant SARS-CoV-2 spike S1 under serum-free condition for 4 h followed by monitoring the mRNA expression of IL-6 ( d ) and IL-1β ( e ) by real-time PCR. Similarly, the effect of wtAIDS and mAIDS peptides on the mRNA expression of IL-6 ( f, h & j ) and IL-1β ( g, i & k ) was examined in polyIC- ( f & g ), HIV-1 Tat- (H & I) and flagellin- ( j & k ) stimulated A549 cells by real-time PCR. *** p < 0.001 vs. spike S1. Similarly, the effect of wtAIDS and mAIDS peptides on the activation of NF-κB was examined in spike S1- ( l ), polyIC- ( m ), HIV-1 Tat- ( n ), and flagellin- ( o ) stimulated A549 cells by EMSA. In this case, cells were stimulated with spike S1, polyIC, HIV-1 Tat, and flagellin for 1 h. Results represent three independent experiments

Article Snippet: The effect of wtAIDS and mAIDS peptides on the binding of ACE2 and SARS-CoV-2 spike was examined using the ACE2:SARS-CoV-2 Spike inhibitor screening assay kit (BPS Bioscience, San Diego, CA) according to manufacturer’s instructions.

Techniques: In Silico, Sequencing, Inhibition, Binding Assay, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Journal of Neuroimmune Pharmacology

Article Title: ACE-2-interacting Domain of SARS-CoV-2 (AIDS) Peptide Suppresses Inflammation to Reduce Fever and Protect Lungs and Heart in Mice: Implications for COVID-19 Therapy

doi: 10.1007/s11481-020-09979-8

Figure Lengend Snippet: Intranasal delivery of wtAIDS peptide decreases lung infiltration and inflammation and reduces fever in a mouse model of COVID-19. Six-eight week old C57/BL6 mice (n = 9) of both sexes were treated intranasally with wtAIDS or mAIDS peptides (100 ng/mouse/d). After 10 min, mice were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) via intranasal route. a Schematic presentation of experiments. After 7d of treatment, the activation of NF-κB was checked in lung tissues by EMSA ( b ) followed by monitoring the mRNA expression of IL-6 ( c ) and IL-1β ( d ) in lung by real-time PCR. IL-6 protein was measured in lung tissue homogenates by ELISA ( e ). Levels of IL-6 ( f ) and CRP ( g ) were also quantified in serum by ELISA. Lung sections were analyzed by H&E ( h , images of different magnification; i , epithelial cell count; j , neutrophil cell count; k , infiltrated cells as percent of epithelial cells; l , lung injury score). Cells were counted from two sections of each of five mice (n = 5) per group. Body temperature ( m ) was monitored by Cardinal Health Dual Scale digital rectal thermometer. Results are mean ± SEM of nine mice per group. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The effect of wtAIDS and mAIDS peptides on the binding of ACE2 and SARS-CoV-2 spike was examined using the ACE2:SARS-CoV-2 Spike inhibitor screening assay kit (BPS Bioscience, San Diego, CA) according to manufacturer’s instructions.

Techniques: Recombinant, Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Counting

Journal: Journal of Neuroimmune Pharmacology

Article Title: ACE-2-interacting Domain of SARS-CoV-2 (AIDS) Peptide Suppresses Inflammation to Reduce Fever and Protect Lungs and Heart in Mice: Implications for COVID-19 Therapy

doi: 10.1007/s11481-020-09979-8

Figure Lengend Snippet: SARS-CoV-2 spike S1 intoxication induces the recruitment of NF-κB to the IL-6 gene promoter in vivo in the lungs: Suppression by wtAIDS treatment. a The map of mouse IL-6 promoter region that harbors one consensus NF-κB-binding site (position − 124 to − 110). Six-eight week old C57/BL6 mice of both sexes were treated intranasally with wtAIDS or mAIDS peptides (100 ng/mouse/d). After 10 min, mice were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) via intranasal route. After 7d of treatment, in situ ChIP for p65 and p50 followed by semi-quantitative ( b ) and quantitative PCR ( c , p65; d , p50; e , p300; f , RNA polymerase; g , control IgG) analyses of IL-6 promoter were performed. Results are mean + SEM of four mice per group. *** p < 0.001. h A schema depicting spike S1-induced transcriptional activation of the IL-6 gene

Article Snippet: The effect of wtAIDS and mAIDS peptides on the binding of ACE2 and SARS-CoV-2 spike was examined using the ACE2:SARS-CoV-2 Spike inhibitor screening assay kit (BPS Bioscience, San Diego, CA) according to manufacturer’s instructions.

Techniques: In Vivo, Binding Assay, Recombinant, In Situ, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Journal of Neuroimmune Pharmacology

Article Title: ACE-2-interacting Domain of SARS-CoV-2 (AIDS) Peptide Suppresses Inflammation to Reduce Fever and Protect Lungs and Heart in Mice: Implications for COVID-19 Therapy

doi: 10.1007/s11481-020-09979-8

Figure Lengend Snippet: Intranasal delivery of wtAIDS peptide protects heart functions in a mouse model of COVID-19. Six-eight week old C57/BL6 mice (n = 9) of both sexes were treated intranasally with wtAIDS or mAIDS peptides (100 ng/mouse/d). After 10 min, mice were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) via intranasal route. After 7d of treatment, the activation of NF-κB was checked in the heart by EMSA ( a ) followed by monitoring heart functions by non-invasive electrocardiography (ECG) using the PowerLab (ADInstruments) [ b , chromatogram of control mice; c , chromatogram of spike S1-intoxicated mice; d , chromatogram of (spike S1 + wtAIDS)-treated mice; e , chromatogram of (spike S1 + mAIDS)-treated mice; f , heart rate; g , RR interval; h , JT interval; i , R amplitude; j , heart rate variability; k , QRS interval; l , QT interval]. m ) Serum LDH was quantified using an assay kit from Sigma. Results are mean + SEM of nine mice per group. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant

Article Snippet: The effect of wtAIDS and mAIDS peptides on the binding of ACE2 and SARS-CoV-2 spike was examined using the ACE2:SARS-CoV-2 Spike inhibitor screening assay kit (BPS Bioscience, San Diego, CA) according to manufacturer’s instructions.

Techniques: Recombinant, Activation Assay

Journal: Journal of Neuroimmune Pharmacology

Article Title: ACE-2-interacting Domain of SARS-CoV-2 (AIDS) Peptide Suppresses Inflammation to Reduce Fever and Protect Lungs and Heart in Mice: Implications for COVID-19 Therapy

doi: 10.1007/s11481-020-09979-8

Figure Lengend Snippet: Intranasal delivery of wtAIDS peptide suppresses disease progression in a mouse model of COVID-19. Six-eight week old C57/BL6 mice (n = 5) of both sexes were treated intranasally with wtAIDS peptide (100 ng/mouse/d) from 1 d after intoxication of SARS-CoV-2 spike S1 (50 ng/mouse/d). After 7d of wtAIDS treatment, body temperature was measured followed by monitoring heart functions by non-invasive electrocardiography (ECG) using the PowerLab (ADInstruments) [ a , schematic presentation of experiments; b , body temperature; c , chromatogram of control mice; d , chromatogram of spike S1-intoxicated mice; e , chromatogram of (spike S1 + wtAIDS)-treated mice; f , heart rate; g , RR interval; H, heart rate variability; I, QRS interval]. *** p < 0.001

Article Snippet: The effect of wtAIDS and mAIDS peptides on the binding of ACE2 and SARS-CoV-2 spike was examined using the ACE2:SARS-CoV-2 Spike inhibitor screening assay kit (BPS Bioscience, San Diego, CA) according to manufacturer’s instructions.

Techniques: