Journal: bioRxiv

Article Title: Copyback viral genomes trigger stress granule formation independent of the antiviral response to interfere with viral translation in acute and persistent infections

doi: 10.1101/2023.05.17.541157

Figure Lengend Snippet: (A) SG (G3BP1, magenta) and viral protein (RSV NP, yellow) detection 12 and 24 hpi with RSV cbVG-high or cbVG-low at MOI of 1.5 TCID 50 /cell. (B) Percent of SG positive cells within the infected population 12 and 24 hpi with RSV cbVG-high and cbVG-low infections. Approximately 150 infected cells were counted per condition (average of three independent experiments shown). (C) SG (G3BP1, magenta) and viral protein (RSV NP, yellow) detection 24hpi with RSV cbVG-high or cbVG-low at MOIs 0.1, 1.5, 5 and 10 TCID 50 /cell. (D) Percent of SG positive cells within the infected population 24 hpi with RSV cbVG-high and cbVG-low infection at MOIs 0.1, 1.5, 5 and 10 TCID 50 /cell. Approximately 150 infected cells were counted per condition (average of three independent experiments shown). (E) SG detection (TIAR, white) in cells staining via FISH for stVG-high (orange) and cbVG (green) cells 24 hpi with RSV cbVG-high at MOI 1.5 TCID 50 /cell. (F) Percent of SG positive cells within the stVG-high and cbVG-high cell populations during RSV cbVG-high infection (average of three independent experiments shown). All widefield images were acquired with the Apotome 2.0 at 63x magnification and are representative of three independent experiments. Scale bar = 50 μm. Statistical analysis: One way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.00001).

Article Snippet: Slides were imaged using Zeiss Axio observer widefield microscope.

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Copyback viral genomes trigger stress granule formation independent of the antiviral response to interfere with viral translation in acute and persistent infections

doi: 10.1101/2023.05.17.541157

Figure Lengend Snippet: (A) SG (G3BP1, white) and viral protein (SeV NP) detection 24 hpi with SeV cbVG-low and cbVG-high (NP, yellow) at MOI 1.5 TCID 50 /cell. Digital zoomed images for each of the marked cells are shown in the panel on the right. (B) Percent of infected SG positive cells 24 hpi with SeV cbVG-low and cbVG-high at MOI 1.5 TCID 50 /cell. Approximately 150 infected cells were counted per condition (average of three independent experiments shown). (C) SG (G3BP1, white) and viral protein (SeV NP) detection 24 hpi at MOI 1.5 TCID 50 /cell supplemented with either purified cbVG particles or UV-inactivated cbVG particles at increasing hemagglutination units (HAU). (D) Percent of SG positive cells at increasing HAU doses of active/UV inactive cbVG particles. Approximately 200 infected cells were counted per condition (average of three independent experiments shown). All widefield images were acquired with the Apotome 2.0 at 63x magnification and are representative of three independent experiments. Scale bar = 50 μm. Statistical analysis: One way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.00001).

Article Snippet: Slides were imaged using Zeiss Axio observer widefield microscope.

Techniques: Infection, Purification

Journal: bioRxiv

Article Title: Copyback viral genomes trigger stress granule formation independent of the antiviral response to interfere with viral translation in acute and persistent infections

doi: 10.1101/2023.05.17.541157

Figure Lengend Snippet: (A) G3BP1 (red) and TIAR (green) staining for SG in cells treated with sodium arsenite (SA, 0.5mM) for 1 h or infected with RSV cbVG-high (RSV NP, magenta) at MOI 1.5 TCID 50 /cell 23 hpi and treated with DMSO, CHX (10 μg/mL), or ISRIB (200nM) for 1 h. (B) Quantification of SG positive cells after drug treatment in sodium arsenite or RSV cbVG-high infected cells. Approximately 150 cells were counted for each condition. Fold change relative to DMSO-treated cells is shown. (C) RNA granule detection (G3BP1, red; TIAR, green) in control and RNAseL KO cells transfected with poly I:C 10 μg/mL or infected with RSV cbVG-high (RSV NP, magenta) 24hpi at MOI 1.5 TCID 50 /cell. (D) RNA granule detection (G3BP1, red; and TIAR, green) A549 cells transfected with poly I:C or RSV and SeV cbVG derived oligonucleotides RSV 238 and SeV 268. All widefield images were acquired with the Apotome 2.0 at 63x or 40x magnification. Scale bar = 50 μm.

Article Snippet: Slides were imaged using Zeiss Axio observer widefield microscope.

Techniques: Staining, Infection, Transfection, Derivative Assay

Journal: bioRxiv

Article Title: Copyback viral genomes trigger stress granule formation independent of the antiviral response to interfere with viral translation in acute and persistent infections

doi: 10.1101/2023.05.17.541157

Figure Lengend Snippet: (A) Phosphorylation of PKR 24 hpi with RSV cbVG-low and cbVG-high infection at MOI 1.5 TCID 50 /cell. (B) Western blot analysis showing efficient KO of PKR and MAVS in A549 cells. (C) Expression of RSV G gene mRNA 24 hpi with RSV cbVG-high infection at MOI 1.5 TCID50/cell in control, MAVS or PKR KO A549 cells (average of three independent experiments are shown). (D) SG (G3BP1, magenta) and viral protein (RSV NP) detection in PKR KO and MAVS KO A549 cells 24 hpi with RSV cbVG-high virus at MOI 1.5 TCID 50 /cell. (E) Quantification of SG positive cells 24 hpi with RSV cbVG-high at MOI 1.5 TCID 50 /cell in PKR or MAVS KO A549 cells. Approximately 300 cells were counted per condition. All widefield images were acquired with the Apotome 2.0 at 63x magnification and are representative of three independent experiments. Scale bar = 50 μm. Statistical analysis: One way ANOVA (*p < 0.05, **p < 0.01)

Article Snippet: Slides were imaged using Zeiss Axio observer widefield microscope.

Techniques: Infection, Western Blot, Expressing

Journal: bioRxiv

Article Title: Copyback viral genomes trigger stress granule formation independent of the antiviral response to interfere with viral translation in acute and persistent infections

doi: 10.1101/2023.05.17.541157

Figure Lengend Snippet: (A) Western blot analysis validating A549 G3BP1 KO, G3BP2 KO and G3BP1/2 dKO (B) Expression of RSV G gene mRNA 24 hpi with RSV cbVG high infection at MOI 1.5 TCID50/cell in control, G3BP1 KO, G3BP2 KO and G3BP1/2 dKO (C) G3BP1 (red) and TIAR (green) staining for SG and viral protein (RSV NP) detection in control and G3BP1 KO cells 24 hpi with RSV cbVG-high at MOI 1.5 TCID 50 /cell and treated with DMSO (upper panel) or CHX (10μg/mL) (lower panel). (D) G3BP1 (red) and TIAR (green) staining for SG and viral protein (RSV NP) detection in control, G3BP1 KO, G3BP2 KO and G3BP1/2 dKO cells infected 24 hpi with RSV cbVG-high at MOI 1.5 TCID 50 /cell. All widefield images were acquired with the Apotome 2.0 at 63x magnification and are representative of three independent experiments. Scale bar = 50 μm. Statistical analysis: One way ANOVA (**p < 0.01).

Article Snippet: Slides were imaged using Zeiss Axio observer widefield microscope.

Techniques: Western Blot, Expressing, Infection, Staining

Journal: bioRxiv

Article Title: Copyback viral genomes trigger stress granule formation independent of the antiviral response to interfere with viral translation in acute and persistent infections

doi: 10.1101/2023.05.17.541157

Figure Lengend Snippet: (A) G3BP1-GFP (green) expressing A549 cells infected with rCantell-miRF670 (magenta) reporter virus at MOI 3 TCID 50 /cell with 20 HAU of supplemented cbVG purified particles, timelapse microscopy 6 - 72hpi, images every 6 h at a 20x magnification. Series show focus of different cells in the population (b) G3BP1-GFP (green) expressing A549 cells infected with rCantell-miRF670 (magenta) reporter virus at MOI 3 TCID 50 /cell with 20 HAU of supplemented cbVG purified particles, timelapse microscopy 8 - 18hpi, images taken every 1 h. (C) SG (G3BP1 white) and viral protein (SeV NP) detection in control and MAVS KO A549 cells 24 hpi with SeV cbVG-high virus at MOI 1.5 TCID 50 /cell 24 hpi. Corrected total cell fluorescence (CTCF) quantification of SeV viral protein NP in control and MAVS KO A549 SG-positive cells. All timelapse images were acquired with a widefield microscope at 20x magnification. Control and MAVS KO widefield images were acquired with the Apotome 2.0 at 63x magnification, scale bar = 50 μm.

Article Snippet: Slides were imaged using Zeiss Axio observer widefield microscope.

Techniques: Expressing, Infection, Purification, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Copyback viral genomes trigger stress granule formation independent of the antiviral response to interfere with viral translation in acute and persistent infections

doi: 10.1101/2023.05.17.541157

Figure Lengend Snippet: (A) timelapse microscopy images of G3BP1-GFP (green) expressing A549 cells infected with rCantell-miRF670 (magenta) reporter virus at MOI 3 TCID 50 /cell with 20 HAU of supplemented cbVG purified particles from day 1 to day 13 (B) SG (G3BP1, white) and SeV viral protein (NP, yellow) detection in persistently infected cultures 44 dpi. (C) Corrected total cell fluorescence (CTCF) quantification of SeV viral protein NP in SG positive cells in two persistently infected cultures (pC1 and pC2). Approximately 30 cells were counted for each persistently infected culture. Widefield images obtained at 40x magnification. scale bar = 50 μm.

Article Snippet: Slides were imaged using Zeiss Axio observer widefield microscope.

Techniques: Microscopy, Expressing, Infection, Purification, Fluorescence