Journal: bioRxiv

Article Title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

doi: 10.1101/2021.11.09.467981

Figure Lengend Snippet: (A) Schematic of proposed SARS-CoV-2 Spike binding assay. HEK293T cells with stable integration of ACE2 cDNA for overexpression (HEK293T- ACE2 ) are incubated with Alexa Fluor 488-conjugated SARS-CoV-2 Spike protein (Spike488). Spike488-binding cells are then detected by flow cytometry. (B) Representative flow cytometry plots for WT HEK293T and HEK293T- ACE2 incubated with Spike488. See also for gating strategy. (C) Titration of HEK293T- ACE2 ( ACE2 ) cells with WT HEK293T cells. 1% HEK293T- ACE2 cells showed sufficient difference to baseline non-specific binding, a condition that likely mimics the real screen condition. Histogram summary showing mean fluorescence intensity (MFI) of flowed cells. (D) Schematic of CRISPR activation (CRISPRa) system used. HEK293T cells express synergistic activation machinery (SAM), which includes VP64-dCas9-VP64 protein and helper proteins MS2, p65 and HSF. When transduced with single guide RNA (sgRNA) plasmids, the assembled CRISPRa complex uses MS2 stem loops to recruit the MS2-p65-HSF transcriptional activators. The sgRNA plasmid also encodes the PCP-p65-HSF complex which is recruited to PP7 aptamers in the sgRNA scaffold. (E) Representative plot of flow cytometry analysis for a clonal HEK293T-CRISPRa cell line transduced with NTC sgRNA or ACE2 sgRNA (expression confirmation via RT-qPCR in ).

Article Snippet: LRRC15-TurboGFP fusion constructs (Origene, RG225990 and RG221437) were used for flow cytometry, immunoprecipitation, signaling and immunocytochemistry experiments while LRRC15-myc-DDK fusion constructs (Origene, RC225990 and RC221437) were utilized for SARS-CoV-2 pseudovirus and SARS-CoV-2 live virus inhibition experiments.

Techniques: Binding Assay, Over Expression, Incubation, Flow Cytometry, Titration, Fluorescence, CRISPR, Activation Assay, Transduction, Plasmid Preparation, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

doi: 10.1101/2021.11.09.467981

Figure Lengend Snippet: (A) RT-qPCR of ACE2 expression in 3 SAM clonal cell lines transduced with 3 sgRNAs that increase expression of ACE2, and with HEK293T-ACE2 cells. Results calculated using -□□C T method and normalized to non-targeting control (NTC) sgRNA-transduced HEK293T-CRISPRa cells. (B) FACS gating strategy. Cells were first gated by forward (FSC) and side scatter (SSC) before filtering for singlets. Spike fluorescence was gated by comparison with non-targeting sgRNA transduced cells. Similar strategy was applied to all flow cytometry experiments. (C) FACS results for 3 whole genome CRISPRa screens with NTC as negative controls. For screen 1, cells were incubated with Alexa Fluor 488-conjugated SARS-CoV-2 HexaPro Spike (Addgene #154754) and selected on puromycin for 3 days. For screen 2, cells were incubated with Alexa Fluor 488-conjugated SARS-CoV-2 Spike glycoprotein (residues 1-1208, complete ectodomain; gift from Dr. Florian Krammer) and selected on puromycin for 3 days. For screen 3, cells were incubated with Alexa Fluor 488-conjugated SARS-CoV-2 HexaPro spike (Addgene #15474) and selected on puromycin for 8 days.

Article Snippet: LRRC15-TurboGFP fusion constructs (Origene, RG225990 and RG221437) were used for flow cytometry, immunoprecipitation, signaling and immunocytochemistry experiments while LRRC15-myc-DDK fusion constructs (Origene, RC225990 and RC221437) were utilized for SARS-CoV-2 pseudovirus and SARS-CoV-2 live virus inhibition experiments.

Techniques: Quantitative RT-PCR, Expressing, Transduction, Fluorescence, Flow Cytometry, Incubation

Journal: bioRxiv

Article Title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

doi: 10.1101/2021.11.09.467981

Figure Lengend Snippet: (A) Schematic of CRISPRa screen for identification of novel SARS-CoV-2 Spike-binding proteins. HEK293T-CRISPRa cells were transduced with a whole genome activation library at MOI = 0.5 and selected on puromycin. HEK293T-CRISPRa cells incubated with Spike488 were analyzed by FACS and genomic DNA extracted from both sorted cells and unsorted diversity control HEK293-CRISPRa cells. Genomic DNA underwent next generation sequencing for gene enrichment analysis. (B) Ranking of all genes in screen 1 by log 2 fold change calculated using MAGeCK and plotted using MAGeCKFlute. See also Supplementary Table S1 (C) Gene enrichment analysis of Screen 1 performed using MAGeCK. Horizontal dotted line indicates p-value = 0.05. Vertical dotted lines indicate log 2 fold changes (LFCs) of -2 and 2. P-values and LFCs for all genes are reported in Supplementary Table S1 . Plot generated using EnhancedVolcano (v1.10.0) R package. (D) sgRNA Z-scores for screen 1 unsorted and sorted samples. Density curve for all sgRNA Z-scores insample (i.e. sorted or unsorted) is shown in grey. Z-scores for ACE2 sgRNA are indicated by vertical blue lines. Z-scores for LRRC15 sgRNAs are indicated by vertical red lines. (E) Flow cytometry analysis of HEK293T-CRISPRa cells transduced with three independent LRRC15 sgRNAs. HEK293T- ACE2 ( ACE2 sgRNA3) cells were used as a positive control and NTC sgRNA-transduced HEK293T-CRISPRa cells were used as a negative control. (F) Quantification of Spike647 binding in ACE2 sgRNA3 and LRRC15 sgRNA1 cells via flow cytometry. Dissociation constant (Kd) was calculated by fitting with non-linear regression (one site --specific binding).

Article Snippet: LRRC15-TurboGFP fusion constructs (Origene, RG225990 and RG221437) were used for flow cytometry, immunoprecipitation, signaling and immunocytochemistry experiments while LRRC15-myc-DDK fusion constructs (Origene, RC225990 and RC221437) were utilized for SARS-CoV-2 pseudovirus and SARS-CoV-2 live virus inhibition experiments.

Techniques: Binding Assay, Transduction, Activation Assay, Incubation, Next-Generation Sequencing, Generated, Flow Cytometry, Positive Control, Negative Control

Journal: bioRxiv

Article Title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

doi: 10.1101/2021.11.09.467981

Figure Lengend Snippet: (A) LRRC15 contains 15 leucine-rich repeats, a short cytoplasmic C-terminus, and 2 glycosylation sites. (B) Predicted protein structure of LRRC15. (C) LRRC15 is part of the LRR - Tollkin family of proteins. (D) Flow cytometry analysis of Alexa Fluor-647 (Spike647) binding in WT HEK293T cells, (E) HEK293T- ACE2 and (F) HEK293T cells with stable expression of both ACE2 cDNA and TMPRSS2 cDNA (HEK293T- ACE2 - TMPRSS2 ). Each cell line was transfected with plasmids encoding cDNA for GFP -tagged LRRC15 (transcript 1 or 2) or with empty GFP vector as negative control plasmid. (E) Histogram summary shows mean fluorescence intensity (MFI) of (D-F). (F) Representative images of interaction between LRRC15-GFP and Alexa Fluor 647-conjugated SARS-CoV-2 HexaPro Spike protein in HEK293T cells. Images were taken at 40x magnification. Green = LRRC15-GFP, Red = Spike647, Blue = Hoechst-stained nuclei. Scale bar = 25 μ m. (G) Immunoprecipitation of LRRC15 with Spike protein. Lysates of HEK293T cells transfected with GFP-tagged LRRC15 (transcript 1 or 2, LRRC15_1 and LRRC15_2, respectively) incubated with SARS-CoV-2 HexaPro Spike protein were immunoprecipitated using anti-LRRC15 primary antibody. Immunoblots were performed for LRRC15 and for SARS-CoV-2 HexaPro spike. I = input, FT = flow-through, E = elute.

Article Snippet: LRRC15-TurboGFP fusion constructs (Origene, RG225990 and RG221437) were used for flow cytometry, immunoprecipitation, signaling and immunocytochemistry experiments while LRRC15-myc-DDK fusion constructs (Origene, RC225990 and RC221437) were utilized for SARS-CoV-2 pseudovirus and SARS-CoV-2 live virus inhibition experiments.

Techniques: Flow Cytometry, Binding Assay, Expressing, Transfection, Plasmid Preparation, Negative Control, Fluorescence, Staining, Immunoprecipitation, Incubation, Western Blot

Journal: bioRxiv

Article Title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

doi: 10.1101/2021.11.09.467981

Figure Lengend Snippet: (A) Luciferase assay for quantification of SARS-CoV-2 pseudovirus infection in (B) WT HEK293T (n=4) and (C) HEK293T- ACE2 - TMPRSS2 (n=3). Cells were transfected with plasmid encoding LRRC15 transcript 1 or empty vector as a control. Luminescence for LRRC15 cells were normalized to Control cells. Significance was determined by two-way ANOVA, Sidak multiple comparison test; ****p<0.0001,***p<0.001,**p<0.01,*p<0.05. (D) Cell death assay for quantification of D614G SARS-CoV-2 live virus infection in HEK293T- ACE2 - TMPRSS2 cells. Cell death was determined via nuclei counts 48 hours after addition of virus. (E,F) Quantification of cell survival after incubation with (E) D614G (n=4) and (F) Delta (n=3) SARS-CoV-2 live virus. Significance was determined by two-way ANOVA, *p<0.05.

Article Snippet: LRRC15-TurboGFP fusion constructs (Origene, RG225990 and RG221437) were used for flow cytometry, immunoprecipitation, signaling and immunocytochemistry experiments while LRRC15-myc-DDK fusion constructs (Origene, RC225990 and RC221437) were utilized for SARS-CoV-2 pseudovirus and SARS-CoV-2 live virus inhibition experiments.

Techniques: Luciferase, Infection, Transfection, Plasmid Preparation, Incubation

Journal: bioRxiv

Article Title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

doi: 10.1101/2021.11.09.467981

Figure Lengend Snippet: (A) SARS-CoV-2 pseudovirus carrying a firefly luciferase cassette was applied to HEK293T, HEK293T- ACE2 and HEK293T- ACE2 - TMPRSS2 cells for 24 hours before luminescence quantification. HEK293T cells were relatively resistant to infection while HEK293T- ACE2 and HEK293T- ACE2 - TMPRSS2 expressing cells were infectable. (B) Pseudovirus added to ACE2-expressing cells in the context of LRRC15. Titration of 15×10 6 , 62.5×10 6 , 250×10 6 and 1000×10 6 lentiviral particles in HEK293T-ACE2 cells transfected with 0, 156.25, 312.5, 625, 1250 and 2500 ng of LRRC15 plasmid DNA. (C) Normalized LRRC15 -mediated inhibition in HEK293T- ACE2 cells.

Article Snippet: LRRC15-TurboGFP fusion constructs (Origene, RG225990 and RG221437) were used for flow cytometry, immunoprecipitation, signaling and immunocytochemistry experiments while LRRC15-myc-DDK fusion constructs (Origene, RC225990 and RC221437) were utilized for SARS-CoV-2 pseudovirus and SARS-CoV-2 live virus inhibition experiments.

Techniques: Luciferase, Infection, Expressing, Titration, Transfection, Plasmid Preparation, Inhibition

Journal: bioRxiv

Article Title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

doi: 10.1101/2021.11.09.467981

Figure Lengend Snippet: (A) Overview of cell types expressing LRRC15 from existing single cell RNA-sequencing datasets. (B) UMAP plot of lung single nucleus RNAseq dataset (Melms et al). (C) Feature plot and (D) Dotplot shows LRRC15 is expressed in fibroblasts and neuronal cells. Expression of LRRC15 in fibroblasts is also observed in fibroblasts of separate studies (See ). (E) Fibroblasts have intrinsic spike binding ability that can be further enhanced by LRRC15 overexpression. Fibroblasts were transfected with empty vector control or LRRC15 cDNA, and spike binding capacity was quantified via flow cytometry. MFI = Mean Fluorescence Intensity. (F) Fibroblasts do not have innate tropism for SARS-CoV-2 and overexpression of LRRC15 does not mediate infection. Untransfected, GFP and LRRC15-GFP transfected fibroblasts were transduced with 5×10 8 SARS-CoV-2 pseudovirus particles for 24 hours before quantification via luciferase assay. Transduction efficiency (luciferase luminescence) was compared to permissive cell line HEK293T- ACE2 - TMPRSS2 . (G) LRRC15 expressing fibroblasts reduced SARS-CoV-2 pseudovirus transduction in HEK293T -ACE2 - TMPRSS2 . Luminescence of LRRC15 + co-culture was normalized to control GFP co-culture, and significance was determined by Mann-Whitney One-tailed test, *p<0.05.

Article Snippet: LRRC15-TurboGFP fusion constructs (Origene, RG225990 and RG221437) were used for flow cytometry, immunoprecipitation, signaling and immunocytochemistry experiments while LRRC15-myc-DDK fusion constructs (Origene, RC225990 and RC221437) were utilized for SARS-CoV-2 pseudovirus and SARS-CoV-2 live virus inhibition experiments.

Techniques: Expressing, RNA Sequencing Assay, Binding Assay, Over Expression, Transfection, Plasmid Preparation, Flow Cytometry, Fluorescence, Infection, Transduction, Luciferase, Co-Culture Assay, MANN-WHITNEY, One-tailed Test

Journal: bioRxiv

Article Title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

doi: 10.1101/2021.11.09.467981

Figure Lengend Snippet: (A) UMAP plot of lung single nucleus RNAseq dataset (Delorey et al). (B) Feature plot and (C) Dotplot shows LRRC15 is expressed in Delorey et al. fibroblasts. (D) UMAP plot of lung single nucleus RNAseq dataset (Bharat et al). (E) Feature plot and (F) Dotplot shows LRRC15 is expressed in Bharat et al. Lymphatic Endothelial cells and various populations of fibroblasts. (G) IMR90 fibroblasts express LRRC15 , quantified via RT-qPCR. (H) These fibroblasts have intrinsic spike binding activity. (I) Fibroblasts do not have intrinsic tropism and LRRC15 is not an entry receptor for SARS-CoV-2 pseudovirus.

Article Snippet: LRRC15-TurboGFP fusion constructs (Origene, RG225990 and RG221437) were used for flow cytometry, immunoprecipitation, signaling and immunocytochemistry experiments while LRRC15-myc-DDK fusion constructs (Origene, RC225990 and RC221437) were utilized for SARS-CoV-2 pseudovirus and SARS-CoV-2 live virus inhibition experiments.

Techniques: Quantitative RT-PCR, Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

doi: 10.1101/2021.11.09.467981

Figure Lengend Snippet: (A) Pooled analysis of 3 independent studies show increase in fibroblasts proportion in COVID lungs (7.9% in control (n=19) and 22.9% in COVID (n=47); Unpaired t test, p<0.0001). (B) LRRC15 + fibroblasts have an enhanced collagen gene signature. Dotplots generated from 3 separate studies. Pos = LRRC15 + , Neg = LRRC15 - . (C,D) TGFβ increased LRRC15 and COL1A1 in fibroblasts. Quantification of LRRC15 (C) and COL1A1 (D) expression via RT-qPCR. Significance was determined by Mann-Whitney One-Tailed test, **p<0.01. (E,F) LRRC15 regulates COL1A1 expression. Quantification of LRRC15 (E) and COL1A1 (F) expression in GFP control, Lo LRRC15-GFP and Hi LRRC-GFP fibroblasts via RT-qPCR. Lo LRRC15 increased COL1A1 expression. Significance was determined by Kruskal-Wallis with Dunn’s Multiple Comparisons test, compared to control GFP, *p<0.05. (G-I) Bimodal regulation was also confirmed at the protein level. Representative images of Western blots of LRRC15 and COL1A1 (F, full blots in ). Intensity of LRRC15 and COL1A1 bands were normalized to their respective /3-ACTIN bands. Significance was determined by One-way ANOVA with Dunnett’s Multiple Comparisons test, compared to control GFP, **p<0.01. (J) Summary schematic of LRRC15’s role in COVID. Inflammation increase LRRC15 to immobilize SARS-CoV-2 and regulate collagen production.

Article Snippet: LRRC15-TurboGFP fusion constructs (Origene, RG225990 and RG221437) were used for flow cytometry, immunoprecipitation, signaling and immunocytochemistry experiments while LRRC15-myc-DDK fusion constructs (Origene, RC225990 and RC221437) were utilized for SARS-CoV-2 pseudovirus and SARS-CoV-2 live virus inhibition experiments.

Techniques: Generated, Expressing, Quantitative RT-PCR, MANN-WHITNEY, One-tailed Test, 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: 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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

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: SARS-CoV-2 S gene with the native sequence (Wuhan MN988668) was purchased from Origene (Rockville, MD; #VC102566).

Techniques: Inhibition