Journal: Molecular Cell

Article Title: The SARS-CoV-2 subgenome landscape and its novel regulatory features

doi: 10.1016/j.molcel.2021.02.036

Figure Lengend Snippet: Experimental strategy and analysis for global mapping of template switches (A) Experimental design for decoding SARS-CoV-2 subgenome dynamics at different time points after infection of Vero E6 and Caco-2 cells. (B) Distribution of poly(A) tail length in Nanopore reads in different samples. (C) SARS-CoV-2 RNA genome life cycle and the analysis strategy. The template switches are represented by curved dashed lines and identified by junctions in NGS and Nanopore reads. (D) Reproducibility of two replicates of NGS data. Each dot represents the read counts of one junction in replicates 1 (x axis) and 2 (y axis). Red points represent the significant junctions identified by statistical analysis. (E) Global view of NGS-consistent and Nanopore-consistent JSs in Vero E6 cells 48 h after infection. Each dot represents a junction linking the start (x axis) and end genomic position (y axis). NGS-only, Nanopore-only, and both consistent JSs are represented in green, blue, and red, respectively. (F) Comparison of the signal coverage for each type of sgRNA between Nanopore and NGS platforms in Vero E6 cells 48 h after infection. (G) Global view of NGS-derived JSs in VeroE6 cells infected with SARS-CoV-2 at 6 h, 12 h, 24 h, and 48 h. Red points represent statistically significant JSs. (H) Same as (G) for Caco-2 data at 12 h and 24 h. (I) Statistics of sgRNA composition in different samples based on Nanopore (top) and NGS (bottom) reads. See also and .

Article Snippet: Using poly(A) RNAs enriched from total RNAs extracted from monkey Vero E6 cells (ATCC number CRL-1586) and human Caco-2 cells infected with SARS-CoV-2 (WIV04, IVCAS 6.7512), we constructed RNA-seq libraries with duplicates for the NGS Illumina and Nanopore MinION platforms, respectively.

Techniques: Infection, Comparison, Derivative Assay

Journal: Molecular Cell

Article Title: The SARS-CoV-2 subgenome landscape and its novel regulatory features

doi: 10.1016/j.molcel.2021.02.036

Figure Lengend Snippet: Global landscape of SARS-CoV-2 subgenomes (A) Global view of consistent template switches in NGS and Nanopore data. Each template switch is represented as a point by the genomic positions of its upstream and downstream JSs in the genome. Three types of template switches are shown in different colors (leader/S-N, red; ORF1ab/S-N, blue; S-N/S-N, green). The densities of upstream and downstream JSs are shown in the top right and top left bar graphs, respectively. (B) Distribution of the downstream JSs for leader-group sgRNAs (48 h, Vero E6 cells). The sgRNA names were assigned based on the first annotated gene downstream of the junction. Strong sites (with more than 100 NGS read support) are marked as red lines, of which the major site (with the largest number) in each sgRNA group is marked with an asterisk. (C) Subgenome clusters reconstructed from Nanopore long reads. Representative examples of five different types of subgenomes (colored legend) are shown by row in global (left) and magnified (center) views with the number of supporting reads (right). Boxes and lines represent transcribed and skipped regions, respectively, because of template switches. The top 10 leader-type and top 5 other types of subgenomes are shown. The label of the subgenome was assigned by the first ORF after the template switch. (D) Statistics for 10 subgenome types classified by the first complete ORF in the subgenome (Vero E6 cells, 48 h). For each type of sgRNA, the number of clusters, sgRNAs, Nanopore reads, and cumulative count of Nanopore reads containing the ORF are shown. Because S sgRNAs are the longest canonical sgRNAs, they might be sequenced less efficiently by Nanopore. (E) The number of subgenome clusters, subgenomes, and subgenome reads for five types of subgenomes. (F) Examples of multi-switch sgRNAs with common junctions (28,525–28,576). There are 7 bi-switch and 4 tri-switch sgRNAs (numbers of supporting Nanopore reads are shown on the right). (G) Comparison of the number of multi-switch reads versus single-switch reads with a specific junction for leader-type sgRNAs. The Spearman correlation coefficient is labeled. See also and .

Article Snippet: Using poly(A) RNAs enriched from total RNAs extracted from monkey Vero E6 cells (ATCC number CRL-1586) and human Caco-2 cells infected with SARS-CoV-2 (WIV04, IVCAS 6.7512), we constructed RNA-seq libraries with duplicates for the NGS Illumina and Nanopore MinION platforms, respectively.

Techniques: Comparison, Labeling

Journal: Molecular Cell

Article Title: The SARS-CoV-2 subgenome landscape and its novel regulatory features

doi: 10.1016/j.molcel.2021.02.036

Figure Lengend Snippet: RNA-RNA pairing determinants in template switching efficacy (A) The RNA-RNA base-pairing patterns for the 9 canonical SARS-CoV-2 sgRNAs. The presence/absence of sgRNAs in 7 Nanopore samples (by column) are shown on the left with filled circles or an empty circle (NS7b sgRNA). Base pairings between the TRS-L and anti-TRS-B segments are represented by blue dots, and the TRS motifs are highlighted in gray. The heatmaps on the right represent base pairings between the UR-DR pair (UR and anti-DR) and UL-DL pair (UL and anti-DL). The red or orange squares represent paired states, whereas white squares represent an unpaired state for base pairings between two specific segments flanking the upstream and downstream JSs for template-switching events by row, as illustrated by the arcs linking the predicted base pairs for the first row of template switches (S sgRNA). Red indicates a consecutive paired state in a 6-nt segment with at least 5 nt. (B) Illustrations and examples of the positive-to-positive (top, UR-DR pair, canonical) and negative-to-negative (bottom, UL-DL pair) template switch modes. Known TRS motifs are highlighted in a gray box. The number of NGS reads in 48-h Vero E6 cell data and the MFEs between different pairing segments are shown. (C) Heatmaps as in (A), representing RNA-RNA base pairings in two modes (UR-DR pair and UL-DL pair) for consistent and core template switch junctions from Vero E6 cell 48-h data. The junctions shown by row are detected in NGS and Nanopore reads, with the largest numbers of read support in 5-nt windows from the leader-type sgRNAs. The rows are sorted by the number of supporting NGS reads. (D) Global view of negative-to-negative (top) and positive-to-positive (bottom) template switches for consistent junctions in NGS and Nanopore data 48 h after infection. The numbers of supporting NGS reads are shown by color-scaled lines. (E) Consistent UL-DL junctions observed in Nanopore reads from different samples. The junctions are ordered by column according to their total number of reads in all 7 samples (top). The presence of junctions in each sample (by row) is represented by black rectangles. The total numbers of complete Nanopore reads for all samples are shown on the right. (F) The relationship between the MFE and the number of NGS reads for 9 major leader-group sgRNA junctions (48 h, Vero E6 cells). The Spearman correlation coefficient is labeled. (G) Boxplots of MFEs for leader-group sgRNA junctions sub-grouped by the number of NGS reads (48 h, Vero E6 cells). The number of junctions in each group and the p values from one-sided t tests are shown at the top. (H) Representative examples showing that RRI features affect template switching efficacy. The RNA base-pairing pattern, MFE, terminal paired/unpaired status, and number of observed reads are shown for each example. (I) RNA-RNA base-pairing visualization as in (A) between UR and anti-DR segments flanking template switch sites. The columns indicating the pairing states of the two terminal bases are marked by red arrows. Neighboring junctions with similar pairing patterns were grouped together, and the terminal pairing states for the major junctions in each group was marked by color (red for paired and blue for unpaired). The corresponding sgRNA, terminal pairing state, and read numbers (48 h, Vero E6 cells) for each junction are shown by row on the right. See also and .

Article Snippet: Using poly(A) RNAs enriched from total RNAs extracted from monkey Vero E6 cells (ATCC number CRL-1586) and human Caco-2 cells infected with SARS-CoV-2 (WIV04, IVCAS 6.7512), we constructed RNA-seq libraries with duplicates for the NGS Illumina and Nanopore MinION platforms, respectively.

Techniques: Infection, Labeling

Journal: Molecular Cell

Article Title: The SARS-CoV-2 subgenome landscape and its novel regulatory features

doi: 10.1016/j.molcel.2021.02.036

Figure Lengend Snippet: TRS motif-independent RRI in template switch (A) Heatmap showing use of TRS motifs in donor and acceptor sites of template switches. The motif sequences, strands, and genomic positions are annotated on the right side. (B) Proportion of TRS-mediated template switches in different types of subgenomes. (C) Representative template switch examples without TRS motifs. The TRS motif is marked in gray, and the number of junction reads detected for each class of NS8 is shown. (D) RT-PCR validation for NS8 #2 sgRNA by clone sequencing. The locations of primers, genome sequences, and cloned sequences are shown on the right. (E) An example of upstream, non-TRS mediated, leader-type sgRNA. The number of NGS reads for Vero E6 48 h cells is shown. The presence of this sgRNA in Nanopore data from different samples is marked. (F) Illustration of the non-TRS sequence (pink) in a conserved loop for non-TRS mediated leader-type sgRNA in (E). (G) Illustration of three types of pairing models mediating template switches for leader-type sgRNAs.

Article Snippet: Using poly(A) RNAs enriched from total RNAs extracted from monkey Vero E6 cells (ATCC number CRL-1586) and human Caco-2 cells infected with SARS-CoV-2 (WIV04, IVCAS 6.7512), we constructed RNA-seq libraries with duplicates for the NGS Illumina and Nanopore MinION platforms, respectively.

Techniques: Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Sequencing, Clone Assay

Journal: Molecular Cell

Article Title: The SARS-CoV-2 subgenome landscape and its novel regulatory features

doi: 10.1016/j.molcel.2021.02.036

Figure Lengend Snippet: Extensive fusion subgenomes between ORF1ab and the N RNA region (A) Nanopore read coverage profiles across the SARS-CoV-2 genome for different time points after infection of Vero E6 cells. The arrows in the ORF1ab region mark two obvious loci with abrupt changes indicating template switch junctions. (B) RT-PCR validation for ORF1ab sgRNA by clone sequencing. The diffusing band indicates diverse types of junctions between the two primers. The locations of primers, genome sequences, and cloned sequences are shown on the right. (C) Global view of Nanopore reads associated with ORF1ab-mediated long-range template switches (Vero E6 cells, 48 h). (D) Distribution of the upstream JS for ORF1ab sgRNAs (Vero E6 cells, 48 h). The sgRNA types were assigned according to the last protein upstream of the junction in ORF1ab. Strong sites are shown as a red line, whereas major sites are marked by asterisks. (E) Counts (left) and cumulative counts (right) of Nanopore reads assigned to the 16 types of ORF1ab sgRNAs (Vero E6 cells, 48 h). (F) Illustration of ORF1ab-type JSs covered by the SARS-CoV-2 Ribo-seq reads (blue curves) and MS peptides (orange curves). (G) Examples of ORF1ab-type sgRNA-derived peptides spanning the sgRNA junctions from MS data. See also Figure S7 .

Article Snippet: Using poly(A) RNAs enriched from total RNAs extracted from monkey Vero E6 cells (ATCC number CRL-1586) and human Caco-2 cells infected with SARS-CoV-2 (WIV04, IVCAS 6.7512), we constructed RNA-seq libraries with duplicates for the NGS Illumina and Nanopore MinION platforms, respectively.

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Sequencing, Clone Assay, Derivative Assay

Journal: Molecular Cell

Article Title: The SARS-CoV-2 subgenome landscape and its novel regulatory features

doi: 10.1016/j.molcel.2021.02.036

Figure Lengend Snippet:

Article Snippet: Using poly(A) RNAs enriched from total RNAs extracted from monkey Vero E6 cells (ATCC number CRL-1586) and human Caco-2 cells infected with SARS-CoV-2 (WIV04, IVCAS 6.7512), we constructed RNA-seq libraries with duplicates for the NGS Illumina and Nanopore MinION platforms, respectively.

Techniques: Virus, RNA Sequencing, Reverse Transcription, Sequencing, Mass Spectrometry, Northern Blot, Software

Journal: Pharmaceutics

Article Title: The Synergistic Inhibition of Coronavirus Replication and Induced Cytokine Production by Ciclesonide and the Tylophorine-Based Compound Dbq33b

doi: 10.3390/pharmaceutics14071511

Figure Lengend Snippet: Combined treatments of ciclesonide and tylophorine-based dbq33b synergistically inhibited coronaviral replication. ( A ) Combined treatment of ciclesonide and tylophorine-based dbq33b synergistically inhibited HCoV-OC43 infection/replication in human fetal lung fibroblast MRC-5 cells assayed by IFA. ( B ) Combined treatment of ciclesonide and tylophorine-based dbq33b synergistically inhibited SARS-CoV-2 infection/replication in Vero E6 cells assayed by IFA. ( C ) Combined treatment of ciclesonide and tylophorine-based dbq33b synergistically reduced infectious SARS-CoV-2 viral loads as determined by plaque formation assays. IFAs were performed with antibody against viral N protein (green) in HCoV-OC43-infected MRC-5 (0.05 MOI) at 30 h.p.i. treated with vehicle (0.5% DMSO) or compounds as indicated. Nuclei (blue) were counterstained with Hoechst dye and used to determine the relative cell viability by using the number of nuclei in vehicle control as 100%. The Methods used for IFA and plaque assays for SARS-CoV-2 infectivity in Vero E6 cells were performed and analyzed as described . The effects of single treatments within indicated concentrations were shown as AVE ± S.D. from three independent experiments ( Aa , Ba , C ). IFA or plaque formation images ( Ab , Bb , C ) shown are representative of three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001). Shown synergy scores are AVE ± S.D. from three independent experiments ( Ac , Bc ) analyzed via the online SynergyFinder ( https://synergyfinder.fimm.fi/ , accessed on 23 March 2021).

Article Snippet: HCT-8 colon adenocarcinoma cells (ATCC ® CCL-244TM), MRC-5 lung fibroblasts cells (ATCC ® CCL-171TM), and the OC43 strain of human coronavirus (HCoV-OC43, ATCC ® VR1558TM) were purchased from American Type Culture Collection (ATCC), USA, and Vero E6 cells (BCRC number: 60476; derived from ATCC CRL-1586) from Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

Techniques: Infection, Control

Journal: Pharmaceutics

Article Title: The Synergistic Inhibition of Coronavirus Replication and Induced Cytokine Production by Ciclesonide and the Tylophorine-Based Compound Dbq33b

doi: 10.3390/pharmaceutics14071511

Figure Lengend Snippet: The synergistic inhibition of coronaviral replication in HCoV-OC43-infected MRC-5 cells and SARS-CoV-2-infected Vero E6 cells after treatment with the combination of ciclesonide and dbq33b.

Article Snippet: HCT-8 colon adenocarcinoma cells (ATCC ® CCL-244TM), MRC-5 lung fibroblasts cells (ATCC ® CCL-171TM), and the OC43 strain of human coronavirus (HCoV-OC43, ATCC ® VR1558TM) were purchased from American Type Culture Collection (ATCC), USA, and Vero E6 cells (BCRC number: 60476; derived from ATCC CRL-1586) from Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

Techniques: Inhibition

Journal:

Article Title: Interferon-Independent, Human Immunodeficiency Virus Type 1 gp120-Mediated Induction of CXCL10/IP-10 Gene Expression by Astrocytes In Vivo and In Vitro †

doi: 10.1128/JVI.75.15.7067-7077.2001

Figure Lengend Snippet: Examination of role of STAT1 in induction of CXCL10/IP-10 RNA expression by soluble recombinant HIV gp120 protein. Astrocyte cultures derived from wild-type or STAT1-null mice were incubated in medium alone or with gp120 IIIB or murine recombinant IFN-γ proteins for 6 h. Each treatment was done in triplicate. Following treatment, the astrocyte cultures were washed twice in PBS, and RNA was extracted with Trizol reagent according to the manufacturer's instructions. For RPA, 5 μg of total RNA was analyzed as described in Materials and Methods. Quantitative analysis of RPA autoradiographs was performed as described for Fig. ​Fig.11.

Article Snippet: To examine the role of STAT1 wild-type and STAT1-null astrocyte cultures were treated with soluble recombinant HIV-1 IIIB gp120 protein or murine recombinant IFN-γ (R&D Systems, Minneapolis, Minn.) for 6 h at 37°C prior to RNA isolation.

Techniques: RNA Expression, Recombinant, Derivative Assay, Incubation