Journal: bioRxiv

Article Title: The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

doi: 10.1101/2023.03.03.530798

Figure Lengend Snippet: ( A ) Diagrammatic illustration of the experimental design. ( B ) Representative immunofluorescence images and ( C, D ) the quantification of the relative infection (the number of N-positive cells related to the number of nuclei in C, and number of dsRNA-positive cells/ number of nuclei in D) in A549-AT cells treated with indicated concentration of IMP-1088 or DMSO vehicle control, 1.5 hours before-infection (hbi) or post-infection (hpi), and infected with SARS-CoV-2 Wuh for 24 hours. Immunostainings show viral markers N (magenta) and dsRNA (yellow). Actin and nuclei are shown in green and blue, respectively. ( E ) Representative immunofluorescence images and ( F ) quantification of the relative infection in A549-AT cells treated with indicated concentration of IMP-1088 or DMSO vehicle control 1.5 hbi, and infected SARS-CoV-2 Wuh for 48 hours. Immunostainings show viral markers N (magenta). Actin and nuclei are shown in green and blue, respectively. The boxed areas i (multi-nucleated syncytium of cells), ii and iii (infected single cells) are shown magnified at the bottom of each image. ( G ) The quantification of cell viability of A549-AT cells treated with the indicated concentrations of the IMP-1088 or UCN-01 [9 μM], an inducer of apoptosis, for 24 h. All the results are normalised to DMSO vehicle controls and shown as mean ± SEM of 3 independent experiments. Statistical testing was performed with one-way ANOVA with Tukey’s correction for multiple comparisons, **P<0.01.

Article Snippet: The cells were treated with indicated concentrations of IMP-1088 (MedChemExpress, Cat. no. HY-112258), UCN-01 (Merck, Cat. no. 539644), or 4OH-Dynasore (Merck, Cat. no. D7693), at the indicated times before or after infection.

Techniques: Immunofluorescence, Infection, Concentration Assay, Control

Journal: bioRxiv

Article Title: The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

doi: 10.1101/2023.03.03.530798

Figure Lengend Snippet: ( A, B ) Quantification of the mean fluorescence intensity of N- and dsRNA in IMP-1088 pre-treated A549-AT cells infected with SARS-CoV-2 for 24 h ( A, B ) or 8 h ( C, D ). Cells were treated with indicated concentration of IMP-1088 or DMSO vehicle control, 1.5 hours before-infection (hbi) and fixed at 24 h (A,B) or 8 h (C,D) post infection (hpi). The values represent the mean fluorescence intensity per cell of each immunostained viral component in the cells that were classified as infected by image analysis (see and ). All the results are expressed as mean ± SEM of 3 independent experiments. Statistical analysis was performed with one-way ANOVA with Tukey’s correction for multiple comparisons, ns= non significant.

Article Snippet: The cells were treated with indicated concentrations of IMP-1088 (MedChemExpress, Cat. no. HY-112258), UCN-01 (Merck, Cat. no. 539644), or 4OH-Dynasore (Merck, Cat. no. D7693), at the indicated times before or after infection.

Techniques: Fluorescence, Infection, Concentration Assay, Control

Journal: bioRxiv

Article Title: The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

doi: 10.1101/2023.03.03.530798

Figure Lengend Snippet: ( A ) Diagrammatic illustration of the experimental design. ( B, D ) Representative immunofluorescence images and ( C, E ) the quantification of the relative infection (the number of N-positive cells related to the number of nuclei in (B,C), and number of dsRNA-positive cells/ number of nuclei in (D,E) in A549-AT cells treated with indicated concentration of IMP-1088 or DMSO vehicle control 1.5 hours before-infection (hbi) and infected SARS-CoV-2 Wuh for 8 hours. ( F ) Representative immunofluorescence images and ( G ) the quantification of the relative infection (the number of dsRNA-positive cells related to the number of nuclei in F) in A549-AT cells treated with 4OH-dynasore (30 μM) or DMSO vehicle control, 1.5 hours before-infection (hbi) and infected SARS-CoV-2 Wuh for 8 hours. Immunostaining shows viral marker N (magenta) and dsRNA (magenta). Actin and nuclei are shown in green and blue, respectively. The results are normalised to DMSO vehicle control and shown as mean ± SEM of 3 independent experiments. Statistical testing was performed with ordinary one-way ANOVA with Tukey’s correction for multiple comparisons, ****p<0.0001; ns= non significant.

Article Snippet: The cells were treated with indicated concentrations of IMP-1088 (MedChemExpress, Cat. no. HY-112258), UCN-01 (Merck, Cat. no. 539644), or 4OH-Dynasore (Merck, Cat. no. D7693), at the indicated times before or after infection.

Techniques: Immunofluorescence, Infection, Concentration Assay, Control, Immunostaining, Marker

Journal: bioRxiv

Article Title: The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

doi: 10.1101/2023.03.03.530798

Figure Lengend Snippet: ( A ) Diagrammatic illustration of the experimental design. ( B and C ) Representative immunofluorescence images of A549-AT cells infected with SARS-CoV-2 Omicron for 24 h (B) or 48 h (C) and treated with the indicated doses of IMP-1088 1.5 hours before-infection (hbi). ( D ) The quantification of the relative infection of SARS-CoV-2 Omicron at 24 h and 48 h post infection in A549-AT cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) at 1.5 hbi. ( E and F ) Representative immunofluorescence images of A549-AT cells infected with SARS-CoV-2 Delta for 24 h (E) or 48 h (F) and treated with the indicated doses of IMP-1088 1.5 hours hbi. ( G ) The quantification of the relative infection of SARS-CoV-2 Delta at 24 h and 48 h post infection in A549-AT cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) at 1.5 hbi. All the results are normalized to DMSO vehicle controls and shown as mean ± SEM of 3 independent experiments.

Article Snippet: The cells were treated with indicated concentrations of IMP-1088 (MedChemExpress, Cat. no. HY-112258), UCN-01 (Merck, Cat. no. 539644), or 4OH-Dynasore (Merck, Cat. no. D7693), at the indicated times before or after infection.

Techniques: Immunofluorescence, Infection, Control

Journal: bioRxiv

Article Title: The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

doi: 10.1101/2023.03.03.530798

Figure Lengend Snippet: ( A ) Diagrammatic illustration of the experimental design. ( B ) Representative immunofluorescence images and ( C ) the quantification of the relative RSV-GFP (green) infection (the number of GFP positive cells related to the number of nuclei) at 48 hours post infection (hpi) in A549 cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) 2 hours before infection (hbi), or ( D and E ) 1 hpi. ( F ) Representative immunofluorescence images and ( G ) the quantification of the relative SFV-GFP (green) infection (the number of GFP positive cells related to the number of nuclei) at 12 hpi in A549 cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) 2 hbi, or ( H and I ) 1 hpi. ( J ) Representative immunofluorescence images and ( K ) the quantification of the relative VSV-GFP (green) infection (the number of GFP positive cells related to the number of nuclei) at 12 hpi in A549 cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) 2 hbi, or ( L and M ) 1 hpi. All the results are normalised to DMSO vehicle controls and shown as mean ± SEM of 3 independent experiments. Nuclei are indicated in blue. Statical analysis was performed with one-way ANOVA with Tukey’s correction for multiple comparisons, ***P<0.001, and ****P<0.0001.

Article Snippet: The cells were treated with indicated concentrations of IMP-1088 (MedChemExpress, Cat. no. HY-112258), UCN-01 (Merck, Cat. no. 539644), or 4OH-Dynasore (Merck, Cat. no. D7693), at the indicated times before or after infection.

Techniques: Immunofluorescence, Infection, Control

Journal: bioRxiv

Article Title: The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

doi: 10.1101/2023.03.03.530798

Figure Lengend Snippet: ( A ) Representative immunofluorescence images and ( B ) the quantification of the relative RSV-GFP (green) infection (the number of GFP positive cells related to the number of nuclei) at 17 hours post infection (hpi) in A549 cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) 2 hours before infection (hbi). ( C ) Representative immunofluorescence images and ( D ) the quantification of the relative SFV-GFP (green) infection (the number of GFP positive cells related to the number of nuclei) at 7 hpi in A549 cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) 2 hbi. ( E ) Representative immunofluorescence images and ( F ) the quantification of the relative VSV-GFP (green) infection (the number of GFP positive cells related to the number of nuclei) at 7 hpi in A549 cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) 2 hbi. All the results are normalised to DMSO vehicle controls and shown as mean ± SEM of 3 independent experiments. Nuclei are indicated in blue. Statical analysis was performed with one-way ANOVA with Tukey’s correction for multiple comparisons.

Article Snippet: The cells were treated with indicated concentrations of IMP-1088 (MedChemExpress, Cat. no. HY-112258), UCN-01 (Merck, Cat. no. 539644), or 4OH-Dynasore (Merck, Cat. no. D7693), at the indicated times before or after infection.

Techniques: Immunofluorescence, Infection, Control

Journal: bioRxiv

Article Title: The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

doi: 10.1101/2023.03.03.530798

Figure Lengend Snippet: ( A ) Diagrammatic illustration of the experimental design. ( B and C ) Representative immunofluorescence images and ( D ) the quantification of the relative SARS-CoV-2 Wuh (green) infection (the number of N-positive cells related to the number of nuclei) at 24 hpi in A549-AT cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) 2 hours before infection (hbi). The drug was then washed off (B, and “Wash out” in D) or left on the cell culture dish (C, and “No wash” in D) and the cells were infected with SARS-CoV-2 Wuh for 24 h, fixed and immunostained for N protein (magenta). Actin and nuclei are shown in green and blue, respectively. ( E and F ) Representative immunofluorescence images and ( G ) the quantification of the relative SARS-CoV-2 Wuh (green) infection (the number of N-positive cells related to the number of nuclei) at 48 hpi in A549-AT cells treated with the indicated doses of IMP-1088 or vehicle control (DMSO) 2 hours before infection (hbi). The drug was then washed off (E, and “Wash out”in G) or left on the cell culture dish (F, and “No wash” in G) and the cells were infected with SARS-CoV-2 Wuh for 48 h, fixed and immunostained for N protein (magenta). Actin and nuclei are shown in green and blue, respectively. All the results are normalised to DMSO vehicle controls and shown as mean ± SEM of 3 independent experiments. Statistical analysis was performed with one-way ANOVA with Tukey’s correction for multiple comparisons, ***p<0.001, ****P<0.0001.

Article Snippet: The cells were treated with indicated concentrations of IMP-1088 (MedChemExpress, Cat. no. HY-112258), UCN-01 (Merck, Cat. no. 539644), or 4OH-Dynasore (Merck, Cat. no. D7693), at the indicated times before or after infection.

Techniques: Immunofluorescence, Infection, Control, Cell Culture

Journal: bioRxiv

Article Title: The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

doi: 10.1101/2023.03.03.530798

Figure Lengend Snippet: ( A ) Diagrammatic illustration of the experimental design. A549-AT cells were treated with indicated concentrations of IMP-1088 or DMSO vehicle control at 2 hours before infection (hbi), and the drug was then either washed out ( B and C ) or left in the culture dish ( D and E ) and the cells were then infected with SARS-CoV-2 Wuh. The culture medium was collected at indicated time points (0 h to 48 h) for qRT-PCR detection of viral RNA. Note that C and E are shown from the same experiments shown in B and D, respectively. Statistical analysis was done using ordinary one-way ANOVA with Tukey’s correction for multiple comparisons, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. N = 4 technical replicates per concentration/time point.

Article Snippet: The cells were treated with indicated concentrations of IMP-1088 (MedChemExpress, Cat. no. HY-112258), UCN-01 (Merck, Cat. no. 539644), or 4OH-Dynasore (Merck, Cat. no. D7693), at the indicated times before or after infection.

Techniques: Control, Infection, Quantitative RT-PCR, Concentration Assay

Journal: bioRxiv

Article Title: The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

doi: 10.1101/2023.03.03.530798

Figure Lengend Snippet: ( A ) Diagrammatic illustration of the experimental design. ( B, D ) Representative immunofluorescence images and ( C, E ) the quantification of the relative SARS-CoV-2 Wuh (green) infection (the number of N-positive cells related to the number of nuclei) at 24 hours post infection (hpi) in VeroE6 cells. The viruses where isolated from A549-AT cells pre-treated with indicated IMP-1088 concentrations or DMSO, starting 2 hours before infection (hbi). Drugs were either washed out (B and C) or kept on cells for the duration of the experiment (D and E). VeroE6 cells were infected with inocula containing the same number of viral RNA genomes as measured by qRT-PCR. After infection for 20 h, VeroE6 cells were fixed and immunostained for N protein (magenta). Actin and nuclei are shown in green and blue, respectively. All the results are normalised to DMSO vehicle controls and shown as mean ± SEM of 3 independent experiments. Statistical analysis was performed with ordinary one-way ANOVA with Tukey’s correction for multiple comparisons, ****P<0.0001.

Article Snippet: The cells were treated with indicated concentrations of IMP-1088 (MedChemExpress, Cat. no. HY-112258), UCN-01 (Merck, Cat. no. 539644), or 4OH-Dynasore (Merck, Cat. no. D7693), at the indicated times before or after infection.

Techniques: Immunofluorescence, Infection, Isolation, Quantitative RT-PCR