Journal: Cell Reports

Article Title: IgG targeting distinct seasonal coronavirus- conserved SARS-CoV-2 spike subdomains correlates with differential COVID-19 disease outcomes

doi: 10.1016/j.celrep.2022.110904

Figure Lengend Snippet:

Article Snippet: Human SARS coronavirus (SARS-CoV) Spike glycoprotein Gene ORF cDNA clone expression plasmid (Codon Optimized), C-Flag tag , SinoBiological , VG40150-CF.

Techniques: Virus, Recombinant, Cell Culture, Enzyme-linked Immunosorbent Assay, In Vitro, Transfection, Luciferase, Lysis, Expressing, Plasmid Preparation, Software, Sequencing

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: The illustration is based on a multiple sequence alignment using the Clustal W algorithm (see for accession numbers used in the alignment) of MCVSyn and all full length MCPyV sequences deposited in the NCBI Database as of August 2011. Aligned genomes were compared to the consensus sequence (which is identical to MCVSyn as well as the isolates 17b, 18b and 20b). Nucleotide substitutions/mismatches relative to this sequence are shown as vertical black bars, whereas deletions are shown in red. Nucleotide insertions in a given sequence are shown as blue bars, and register as gaps in the backbone of the remaining genomes. Genomes that were isolated from MCC or MCC-derived cell lines are marked by an asterisk; the mutations which lead to the truncation of LT-Ag sequences in these genomes are likewise marked.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Sequencing, Isolation, Derivative Assay

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: Cell lines used to study MCVSyn replication, early and late transcription as well as particle formation.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques:

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: (A) 100 ng of intramolecular religated SV40 viral DNA was transfected in CV-1 cells and cells were lysed 12 h, 24 h, 36 h, 2d and 7d post transfection. Protein lysates were subsequently analyzed for SV40 LT-Ag (Pab419 antibody) and VP1 expression (α-VP1 polyclonal rabbit serum) by SDS-page and Western Blotting. Staining of actin was used to ensure that equal protein amounts were loaded per lane. (B) Low molecular weight DNA was isolated from SV40 DNA transfected CV-1 cells at the indicated time points by HIRT extraction, 1 µg DNA was DpnI and EcoRI digested; DNA was separated on an agarose gel and stained with EtBr (left panel), followed by southern blotting and detection of viral DNA using a 32 PdCTP-labeled SV40 LT-Ag PCR fragment as a probe. The blot was exposed for 30 min. Numbers below the lanes correspond to the quantification of newly replicated DNA using a Fuji phosphoimager FLA7000 and MultiGauge software.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Transfection, Expressing, SDS Page, Western Blot, Staining, Molecular Weight, Isolation, Extraction, Agarose Gel Electrophoresis, Southern Blot, Labeling, Software

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: 5×10 4 H1299, PSFK-1 or 293 cells were transfected with 100 ng re-circularized MCVSyn DNA or equivalent amounts of pUC18 DNA (Mock control). At the indicated time points, cells were lysed and analyzed by immunoblotting for MCPyV LT-Ag expression using the monoclonal LT-Ag antibody Cm2B4. Equal protein loading was confirmed by re-incubating the membrane with an anti-actin antibody. An LT-Ag expression control (pos. control; transient transfection with a CMV-promoter driven LT-Ag expression construct for 48 h) was loaded as an internal control.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Transfection, Control, Western Blot, Expressing, Membrane, Construct

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: RNA was isolated at the indicated time points after transfection, DNAse I digested and used for cDNA synthesis followed by real time PCR using a LT-Ag or VP1 specific primer set. Results were normalized against GAPDH transcript levels.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Isolation, Transfection, cDNA Synthesis, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: (A) Double staining of CV-1 cells transfected with SV40 viral DNA. 4d p.t. the cells were fixed, and VP1 was detected with a polyclonal anti-VP1 antibody. LT-Ag was visualized with the monoclonal anti-LT antibody Pab419. Z-stack pictures were taken using confocal microscopy. Each picture represents an individual Z-stack. VP1 staining was observed primarily in speckles close to or at the nuclear membrane. LT-Ag staining was observed throughout the nucleoplasm with the nucleoli excluded. In some cells granular LT-Ag staining was observed. The panel on the lower right represents a 3× zoomed picture of a CV1 transfected cell with the two channels merged. Double staining of Merkel cell polyomavirus VP1 and LT-Ag in H1299 cells (B) and PFSK-1 cells (C) 4d p.t. reveals inner peripheral nuclear localization of MCVSyn VP1 protein. VP1 was visualized with a polyclonal anti-VP1 serum and anti-rabbit FITC, while LT-Ag was visualized with the monoclonal antibody Cm2B4 specifically recognizing MCPyV LT-Ag. 40 Z-stack pictures were taken scanning through the cells using a 63× magnification and 2fold zoom on a confocal microscope. The picture shown represents an individual image from the center of a Z-stack.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Double Staining, Transfection, Confocal Microscopy, Staining, Membrane, Microscopy

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: Optiprep™ gradient centrifugation was performed with cell lysates from CV1 (A) and H1299 (B) cells 4d after transfection with viral DNA. 15×250 µl fractions were collected (fraction 1 represents the fraction with the highest density and fraction 15 represents the lowest density fraction). (A) Left panel: Real time PCR of micrococcal nuclease treated fractions was performed using SV40 VP1 primer sequences. 20 µl of each gradient fraction was loaded on a 10% SDS-page followed immunoblotting using anti-VP1 serum. Right panel: Negative EM staining of SV40 particles identified in fraction 9. (B) Left panel: Real time PCR results of H1299 MCVSyn gradient fractions after micrococcal nuclease treatment using MCPyV VP1-specific primers. Right panel: Negative EM staining of particles identified in fractions 10 and 6.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Gradient Centrifugation, Transfection, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Staining

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: Images were prepared from PFSK-1 cultures at 8 days post transfection with MCVSyn DNA. (A and B) ∼40 µm electron dense particles were observed in approximately 1 out of 50 cells with the particles localizing in the nucleus close to membrane structures (additional particles in B that are located outside of the enlarged inset are marked by arrows). (C) Membrane-attached MCPyV particles, reminiscent of the structures observed in SV40 infected cells as shown in .

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Transfection, Membrane, Infection

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

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: Expression constructs (plasmids) containing codon-optimized cDNA clones encoding SARS-CoV-2 proteins M, N, S, and E were purchased from Sino Biological (#VG40608-UT, #VG40588-CY, #VG40589-CH, and #VG40609-CF, respectively).

Techniques: Inhibition