Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: Replication synergy in co-infection of SARS-CoV-2 variants and high antiviral potency of TG against all variants . Confluent cells were primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS, and infected with A variant, B variant, and D variant at 0.1 MOI in single virus infections (A, B and D) and in co-infections (AB, AD, and BD) for 1.5 h, washed twice with PBS and incubated in infection media. Notably, co-infected cells received the same amount of each virus as used in single virus infection. At 24 (panel a) and 72 hpi (panel a and b), total viral RNA from media was subjected to one-step reverse transcription qPCR, using primer set (1, 2) specific to all 3 variants, to quantify viral spike glycoprotein RNA by relative Ct method. Synergy in progeny production was evident in AB and AD co-infections where total virus RNA detected was greater than the sum of corresponding single-virus infection RNA evident at 24 and 72 hpi (panel a). Indicated significance relative to corresponding A/DMSO control based on 2-way ANOVA with Tukey’s multiple comparisons. Replication of all single virus- and co-infections was effectively blocked for at least 3 days by single pre-infection priming of TG. In AD co-infection, the most prolific infection group, combined viral RNA from TG-primed cells fell by 99.6% relative to corresponding DMSO control (panel b). Indicated significance relative to corresponding DMSO control based on 2-way ANOVA with Sidak’s multiple comparisons. Data shown are representative of three independent experiments and performed in quadruplicates

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Infection, Control, Variant Assay, Virus, Incubation, Reverse Transcription

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: Replication comparisons of each variant in single virus- and co-infections . Confluent Calu-3 cells were primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS, and infected with A variant, B variant, and D variant at 0.1 MOI in single variant infections (A, B and D) and in co-infections (AB, AD, and BD) for 1.5 h, washed twice with PBS and incubated in infection media. Co-infected cells received the same amount of each virus as in single variant infection. At 24, 48 and 72 hpi, viral RNA from media was subjected to one-step reverse transcription qPCR with variant-specific primers that can discriminate between variants in co-infected samples to detect relative spike glycoprotein gene expression. All single variant- and co-infections were blocked for at least 3 days by single pre-infection priming of TG (panels a to c). Production of A variant vRNA in AB and AD co-infections at 72 hpi was clearly enhanced (relative to single-virus A variant infection) (panel a). Production of B variant vRNA in BA and BD co-infections at 72 hpi was also enhanced (panel b). However, production of D variant vRNA in DA and DB co-infections at 72 hpi was attenuated relative to single-variant D variant infection; reduction of the D variant vRNA in DA was more than 2 times that in DB co-infection (panel c). Unless otherwise indicated significance relative to corresponding DMSO control based on 2-way ANOVA with Tukey’s multiple comparisons; % reduction is relative to corresponding DMSO group. Data shown are representative of three independent experiments and performed in quadruplicates

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Variant Assay, Virus, Control, Infection, Incubation, Reverse Transcription, Gene Expression

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: Comparative vRNA production of emergent SARS-CoV-2 variants . Confluent Calu-3 cells were primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS, and separately infected with A variant, B variant, and D variant at 0.1 MOI in for 1.5 h, washed twice with PBS and incubated in infection media. At 24, 48 and 72 hpi, viral RNA from media was subjected to one-step reverse transcription qPCR with variant-specific primer sets to detect relative spike glycoprotein RNA of each variant. Increase of virus output of each variant was determined between the periods of 24 to 48 hpi, and 24 to 72 hpi. Relative rates of progeny virus production of variants are such that D > A > B (panel a). Relative increase in virus output under TG inhibition (panel b) followed the same pattern as DMSO controls but was drastically reduced. Indicated significance of D is relative to B and A variants of each corresponding interval period based on 2-way ANOVA with Tukey’s multiple comparisons. Data shown are representative of three independent experiments and performed in quadruplicates

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Control, Infection, Variant Assay, Incubation, Reverse Transcription, Virus, Inhibition

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: Replication rates of A, B and D variants in single variant- and co-infections . Data points, from earlier viral supernatant results of spike glycoprotein gene expression in single virus- and co-infections at 24, 48 and 72 hpi , were used to generate indicated growth curves and equations to determine the rate of viral RNA production (gradient) at a given time point. The D variant in single-variant infection, until saturation at 72 hpi, had the highest rate of viral RNA production relative to A and B variants in DMSO control (panel a) and TG-primed (panel b) Calu-3 cells. In co-infections, the D variant boosted the production rate of A and B variants (panel c and d) but at the expense of its initial performance (panel e). Negative values in production rate are likely due to virus saturation from limited cell number and even virus breakdown in media (panel a), or low initial virus growth (panel e). Data shown are representative of three independent experiments

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Variant Assay, Gene Expression, Virus, Infection, Control

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: TG reduced vRNA synthesis and progeny production of Beta variant of SARS-CoV-2 in Calu-3 cells . Confluent cells were primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS and infected with the B variant at 0.05 MOI in infection media for 3 h followed by three washes with PBS and incubated in fresh infection media. At indicated hpi, total cellular RNA was extracted (panel a); progeny virus in media was subjected to focus forming assay (FFA) (panel b) and viral RNA extraction (panel c). Total RNA was converted to cDNA for qPCR of SARS-CoV-2, normalized to 18s rRNA (panel a). FFA was based on the quantification of virus-positive Vero E6 cells at 18 hpi by immunochemical detection of viral spike glycoprotein (error bars = SEM; ns = not significant between time points) (panel b). Viral RNA from media was subjected to one-step reverse transcription qPCR to detect relative copy number of SARS-CoV-2, based on relative Ct method (panel c). Notably, despite increasing viral RNA accumulation in media of infected cells with time (panel c), viral gene expression (panel a) and infectious progeny virus (panel b) were in relative decline by 72 hpi. Indicated significance relative to corresponding DMSO control based on 2-way ANOVA with Sidak’s multiple comparisons. Indicated % refers to reduction in viral detection relative to corresponding DMSO control

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Variant Assay, Control, Infection, Incubation, Virus, Focus Forming Assay, RNA Extraction, Reverse Transcription, Gene Expression

Journal: Virulence

Article Title: Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin

doi: 10.1080/21505594.2021.2006960

Figure Lengend Snippet: TG was effective in inhibiting emergent SARS-CoV-2 variants in pre-infected Calu-3 cells . Confluent cells were separately infected with SARS-CoV-2 variants at about 0.2 MOI for 2 h followed by three washes with PBS and incubated in fresh infection media. At 24 hpi, cultured media were removed and kept for re-seeding back to the same wells. Infected cells were then primed with 0.5 µM TG or DMSO control for 30 min, washed twice with PBS and the retained media were put back to the corresponding wells. At 48 and 72 hpi (i.e. at 24 h and 48 h post-priming with TG/DMSO respectively), viral RNA was extracted from spun supernatants for one-step reverse transcription qPCR to detect relative abundance of viral RNA that codes for spike glycoprotein (using primers 1 and 2). Notably, TG was able to inhibit preexisting active infection to great effect with a single 30 min exposure dose. Indicated significance relative to corresponding DMSO control based on 2-way ANOVA with Tukey’s multiple comparisons. Indicated % refers to reduction in viral detection relative to corresponding DMSO control

Article Snippet: Briefly, Vero E6 cells infected with a fixed volume of supernatant for 18 h were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed three times with PBS, followed by peroxidase treatment for 10 min and incubation with a 1:1000 dilution of primary mouse monoclonal antibody, specific to the viral spike glycoprotein (1A9 clone, GeneTex GTX632604), at room temperature for 1 h. The cells were then washed with Tris-buffered saline (TBS), incubated with horse radish peroxidase-labeled polymer for 1 h. After further washing with TBS, the cells were incubated with DAB substrate-chromogen solution for a few min (Envision+ system-HRP kit, Dako).

Techniques: Infection, Incubation, Cell Culture, Control, Reverse Transcription

Journal: Nucleic Acids Research

Article Title: In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins

doi: 10.1093/nar/gkab390

Figure Lengend Snippet: Chloroplast genome-wide analysis of RNAs associated with dPPR rbcL in vivo . ( A ) Immunoblot analysis of an immunoprecipitation of dPPR rbcL performed with HA antibodies on stroma extracts from the complemented mrl1:dPPR rbcL or Col-0 Arabidopsis plants (negative control). Pel : immunoprecipitation pellet, Sup : supernatant; IgG : Immunoglobuline G. IgGs in the experimental pellet are detected by the secondary antibody used to probe the immunoblot. A portion of the blot stained with Coomassie blue is shown to display equal loading. ( B ) RIP-seq analysis of chloroplast RNAs that associates with dPPR rbcL in vivo . The mean coverage in kiloreads per nucleotide of two replicates for the experimental dPPR rbcL immunoprecipitation and negative Col-0 control are plotted along the chloroplast genome on the same graph. Since the read coverage was too high for the chloroplast rRNA loci, these were split and displayed in a separate graph to the right. The main peaks are labeled with the names of the locus they belong to. Data for replicate experiments are shown in and the read counts are provided in . ( C ) Local enrichment (Ratio dPPR rbcL /Col-0) of rbcL RNA. The highest enrichment (more than 700-fold) is found in the 5′ UTR region where the binding site of dPPR rbcL is located. The positions of the rbcL initiation (ATG) and stop (TAG) codons are indicated with their genomic position. ( D ) Slot blot hybridization analysis of RNAs that coimmunoprecipitate with dPPR rbcL in vivo . The experimental and control immunoprecipitations were both performed with mrl1:dPPR rbcL stroma but used different antibodies: α-HA that detects dPPR rbcL or α-Myc as a negative control. Four replicate blots were hybridized with strand and gene specific oligonucleotide probes. The same blot was used for hybridization with psbT and psbE probes, psbC and psaB probes and psbA and atpF probes after stripping. The hybridization signals in the pellet and supernatant of dPPR rbcL immunoprecipitation were quantified for each gene and their ratio is displayed to the right.

Article Snippet: PsbE and PsbA (D1) antibodies were purchased from Agrisera.

Techniques: Genome Wide, In Vivo, Western Blot, Immunoprecipitation, Negative Control, Staining, Control, Labeling, Binding Assay, Dot Blot, Hybridization, Stripping Membranes