Journal: bioRxiv

Article Title: Rapid decay of host basal mRNAs during SARS-CoV-2 infection perturbs host antiviral mRNA biogenesis and export

doi: 10.1101/2021.04.19.440452

Figure Lengend Snippet: (A) Schematic to show the location of smFISH probe sets targeting the different regions of SARS-CoV-2 mRNA. The ORF1a and ORF1b target the full-length genome, whereas the S and N probe sets target both the full-length genome and various sub-genomic RNAs. (B) smFISH for SARS-CoV-2 full length genome (ORF1a probes) and sub-genomic RNAs (N probes) at indicated times post-infection with SARS-CoV-2 (MOI=5).

Article Snippet: SARS-CoV-2/WA/20/01 (GenBank MT020880 ) was acquired from BEI Resources (NR-52881) and used for all infections.

Techniques: Infection

Journal: bioRxiv

Article Title: Rapid decay of host basal mRNAs during SARS-CoV-2 infection perturbs host antiviral mRNA biogenesis and export

doi: 10.1101/2021.04.19.440452

Figure Lengend Snippet: (A) smFISH for host GAPDH and ACTB mRNAs and SARS-CoV-2 full-length genome (ORF1b) at indicated times post-infection with SARS-CoV-2 (MOI=5) in WT ACE2 A549 cells. (B) Graphs show quantification of GAPDH and ACTB mRNAs as represented in above images. (C and D) Similar to (A and B) but in RL-KO ACE2 A549 cells.

Article Snippet: SARS-CoV-2/WA/20/01 (GenBank MT020880 ) was acquired from BEI Resources (NR-52881) and used for all infections.

Techniques: Infection

Journal: bioRxiv

Article Title: Rapid decay of host basal mRNAs during SARS-CoV-2 infection perturbs host antiviral mRNA biogenesis and export

doi: 10.1101/2021.04.19.440452

Figure Lengend Snippet: (A) Schematic of flag-tagged SARS-CoV-2 protein expression vector transfected into U2-OS cells. (B) Immunoblot for flag confirmed expression of flag-tagged Nsp1 and Nsp15 expression at expected size (Nsp1 ~20 kDa; Nsp15 ~40kDa) in cells transfected with respective expression vectors but not empty vector (EV). (C) Immunofluorescence assay for flag and smFISH for ACTB and GAPDH mRNAs in U2-OS twenty-four hours post transfection with either pcDNA3.1+ (empty vector; EV), flag-Nsp1, or flag-Nsp15 expression vectors. (D) Quantification of ACTB and GAPDH mRNAs as represented in (C). Statistical significance (*p<0.05; **p<0.005; ***p<0.0005) was determined by t-test.

Article Snippet: SARS-CoV-2/WA/20/01 (GenBank MT020880 ) was acquired from BEI Resources (NR-52881) and used for all infections.

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Rapid decay of host basal mRNAs during SARS-CoV-2 infection perturbs host antiviral mRNA biogenesis and export

doi: 10.1101/2021.04.19.440452

Figure Lengend Snippet: (A) smFISH for IFNB1 mRNA and SARS-CoV-2 ORF1a forty-eight hours post-infection. Two fields of view are shown. In the top image, 45% of SARS-CoV-2-positive cells stain for IFNB1 (cell boundary marked by red line), whereas 55% do not (cell boundary marked by green line). Eighty-two percent of cells that contain IFNB1 transcriptional foci (TF) lack abundant disseminated IFNB1 mRNA (dashed red line). The lower image shows a SARS-CoV-2-infected cell that contains abundant and diffuse IFNB1 mRNA in the nucleus and cytoplasm (solid red line), which constitute less than 18% of cells that induce IFNB1 (contain IFNB1 transcriptional foci or mRNA). Cells that do not stain for SARS-CoV-2 are labeled SARS2-negative (SARS2-). (B) smFISH for IFNB1 mRNA and GAPDH mRNA sixteen hours post-poly(I:C) transfection in WT and RL-KO A549 cells (images of individual staining shown in Fig. S4B). In WT cells, 12% do not activate RNase L (RL-). Of the 88% of cells that activate RNase L, 63% (55% of total cells) also induce abundant and disseminated IFNB1 mRNA (cell boundary marked by red line), whereas 37% of RL+ cells do not induce IFNB1 (cell boundary marked by green line). Fifty-nine percent of RL-KO cells induce abundant disseminated IFNB1 mRNA (cell boundary marked by red line), whereas 41% do not (cell boundary marked by green line). (C) Histograms quantifying the percent of SARS-CoV-2 infected cells, poly(I:C)-transfected WT cells that activate RNase L (GAPDH mRNA-negative cells), and poly(I:C)-transfected RL-KO cells that induce IFNB1 , as represented in (A and B). (D) Histograms quantifying the percent of IFNB1 -positive cells in which IFNB1 smFISH staining is predominantly localized to IFNB1 transcriptional foci (TF) or diffuse.

Article Snippet: SARS-CoV-2/WA/20/01 (GenBank MT020880 ) was acquired from BEI Resources (NR-52881) and used for all infections.

Techniques: Infection, Staining, Labeling, Transfection

Journal: bioRxiv

Article Title: Rapid decay of host basal mRNAs during SARS-CoV-2 infection perturbs host antiviral mRNA biogenesis and export

doi: 10.1101/2021.04.19.440452

Figure Lengend Snippet: (A) smFISH for IFNB1 mRNA, GAPDH mRNA, and SARS-CoV-2 ORF1b mRNA in WT ACE2 and RL-KO ACE2 cells forty-eight hours post-infection with SARS-CoV-2 (MOI=5). Spectral crossover from the SARS-CoV-2 ORF1b RF into the IFNB mRNA channel is indicated be white arrows. The green arrows indicate cells in which IFNB mRNA is retained in the nucleus. The blue arrows indicate cells in which IFNB mRNA is localized to the cytoplasm. (B) Scatter plot quantifying IFNB1 mRNA in the nucleus (y-axis) and in the cytoplasm (x-axis) in individual WT ACE2 or RL-KO ACE2 cells infected with SARS-CoV-2, or RL-KO cells 48 hrs. post-infection with DENV or 8 hours post-transfection with poly(I:C). (C) Representative smFISH for IFNB1 and GAPDH mRNAs in RL-KO A549 cells forty-eight hours post infection with DENV (MOI=0.1) or 16 hours post-transfection with poly(I:C). (D) Quantification of IFNB1 mRNA via smFISH in the nucleus (N) or cytoplasm (C) of either WT ACE2 or RL-KO ACE2 cells infected with SARS-CoV-2, and WT or RL-KO cells transfected with poly(I:C) or infected with DENV2 as represented in (A and C). Poly(I:C) and DENV2 data was obtained from . Statistical significance (*p<0.05; **p<0.005; ***p<0.0005) was determined by t-test.

Article Snippet: SARS-CoV-2/WA/20/01 (GenBank MT020880 ) was acquired from BEI Resources (NR-52881) and used for all infections.

Techniques: Infection, Transfection

Journal: bioRxiv

Article Title: Rapid decay of host basal mRNAs during SARS-CoV-2 infection perturbs host antiviral mRNA biogenesis and export

doi: 10.1101/2021.04.19.440452

Figure Lengend Snippet: Schematic modeling how antiviral mRNA biogenesis is inhibited during SARS-CoV-2 infection. SARS-CoV-2 replication generates double-stranded RNA (dsRNA), which leads to RNase L activation. RNase L-mediated mRNA decay reduces SARS-CoV-2 full-length mRNA genome and sub-genomic mRNAs. In addition, SARS-CoV-2 expresses the viral Nsp1 protein. Both RNase L activation and Nsp1 expression result in rapid and widespread decay of host basal mRNAs. While RNase L directly cleaves mRNAs, the mechanism of Nsp1-mediated mRNA decay is unclear. The degradation of host mRNAs results in release of RNA-binding proteins (RBPs), and this perturbs late stages of nuclear-cytoplasmic RNA transport. The sequestration of antiviral mRNAs, such as IFNB1 mRNA, in the nucleus prevents their association with ribosomes in the cytoplasm, reducing their translation for protein production. In addition, SARS-CoV-2 inhibits the transcription, an aspect of mRNA processing, or association with early mRNA export factors, and/or rapidly degrades dsRNA-induced antiviral mRNAs, such as IFNB1 mRNA. The result of this is the inability of IFNB1 mRNAs to exit the site of IFNB1 transcription, preventing their transport to the cytoplasm and reducing their translation.

Article Snippet: SARS-CoV-2/WA/20/01 (GenBank MT020880 ) was acquired from BEI Resources (NR-52881) and used for all infections.

Techniques: Infection, Activation Assay, Expressing, RNA Binding Assay