Journal: bioRxiv

Article Title: Cell cycle independent role of cyclin D3 in host restriction of SARS-CoV-2 infection

doi: 10.1101/2022.05.07.491022

Figure Lengend Snippet: (A) Diagram of cell cycle and cyclin expression during cell cycle. Cyclins drive cell cycle changes by interacting with cyclin dependent kinases (CDK). (B-C) VERO AT2 cells were infected with WT and alpha (α) SARS-CoV-2 live virus variants, and heat inactivated α variant at MOI 0.1. (B) Cells were lysed 48h post-infection and viral protein as well as cell cycle associated protein expression was analysed by western blot. N, nucleocapsid. (C) VERO AT2 Cells were fixed 24h post-infection and stained for viral proteins and cyclins. Arrowheads highlight un-infected cells and cyclin D/A nuclear localization. Arrowheads: Nuclear cyclin staining in uninfected cells. (D) VERO AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Statistical analysis was performed using two-sided unpaired Student’s t-tests; ns, non-significant; ****p < 0.0001. (E) A549 AT2 cells were infected with WT, alpha (α), and delta (Δ) SARS-CoV-2 variants. Cells were lysed 48h post-infection and viral, cyclin proteins expression was analysed by western blot. N, nucleocapsid. (F) A549 AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Statistical analysis was performed using two-sided unpaired Student’s t-tests; ns, non-significant; ***p < 0.001; ****p < 0.0001.

Article Snippet: Following cells were a gift from: A549 ACE2/TMPRSS2 Massimo Palmerini, Vero E6 ACE2/TMPRSS2 from Emma Thomson, HeLa-ACE2 from James Voss, 293T (a human embryonic kidney cell line, ATCC CRL-3216).

Techniques: Expressing, Infection, Virus, Variant Assay, Western Blot, Staining

Journal: bioRxiv

Article Title: Cell cycle independent role of cyclin D3 in host restriction of SARS-CoV-2 infection

doi: 10.1101/2022.05.07.491022

Figure Lengend Snippet: (A) A549 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1uM) was added to cells 18h post-infection. Cells were lysed 24h post-addition of inhibitor and cyclins and viral proteins were detected by western blot. (N) nucleocapsid. (B) Densitometry analysis of western blots for D-cyclins (normalized to actin) in A549 AT2 cells. Plots are average of 3 independent experiments. Bars indicate mean with SD. Statistical analysis was performed using ordinary two way ANOVA; ns, non-significant; *p < 0.1. (C) A459 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1uM) was added to cells 8h post-infection. Cells were fixed and stained 24h post-addition of inhibitor. (D) Quantification of D3 cyclin relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells have been counted. Statistical analysis was performed using ordinary two-way ANOVA; ***p < 0.001.

Article Snippet: Following cells were a gift from: A549 ACE2/TMPRSS2 Massimo Palmerini, Vero E6 ACE2/TMPRSS2 from Emma Thomson, HeLa-ACE2 from James Voss, 293T (a human embryonic kidney cell line, ATCC CRL-3216).

Techniques: Infection, Variant Assay, Western Blot, Staining

Journal: bioRxiv

Article Title: Cell cycle independent role of cyclin D3 in host restriction of SARS-CoV-2 infection

doi: 10.1101/2022.05.07.491022

Figure Lengend Snippet: (A,B) D and A-cyclins were depleted using siRNA in A549 AT2 cells. Cells were infected 18h later with Delta (Δ), Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.001, 0.1, 0.1 respectively. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for western blots (A) and TCID50 (B). (C,D) D and A-cyclins were depleted using siRNA in VERO AT2 cells. Cells were infected 18h later with Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.1. Cells were washed 4h post-infection and new media added. Supernatants and cells were collected at 24h and 48h later. (A,C) Representative example of western blot from cell lysates collected at 48h post-infection. (B,D) Virus titres in cell culture supernatants were determined as TCID50 in VERO AT2 cells. Graphs represent average of n=3. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. NT, non-targeting control. ns, non-significant; *p < 0.1; **p < 0.01. Bars indicate mean with SD.

Article Snippet: Following cells were a gift from: A549 ACE2/TMPRSS2 Massimo Palmerini, Vero E6 ACE2/TMPRSS2 from Emma Thomson, HeLa-ACE2 from James Voss, 293T (a human embryonic kidney cell line, ATCC CRL-3216).

Techniques: Infection, Western Blot, Virus, Cell Culture, Control

Journal: bioRxiv

Article Title: Cell cycle independent role of cyclin D3 in host restriction of SARS-CoV-2 infection

doi: 10.1101/2022.05.07.491022

Figure Lengend Snippet: Cells were transduced with Fucci containing lentiviral particles for 18h and infected with SARS-CoV-2 variants for additional 24h. (A) VERO AT2 or (B) A549 AT2 cells. Example of gating strategy for cell cycle analysis. The population of cells exposed to SARS-CoV-2 was stained for nucleocapsid (N) protein and gated on N+ve and N-ve population. Cells were further analysed for expression of cdt1 and Geminin. See also Supplemental Figure S5. (C) VERO AT2 or (D) A549 AT2 cells infected with WT SARS-CoV-2. Quantification of Cdt1 +ve cells (G1), Cdt1/Geminin +ve cells (early S phase) and Geminin +ve cells (S,G2,M phase) cells. n = 5; one-way ANOVA with Dunnett’s multiple comparisons test: ns, non-significant; ****p < 0.0001. Bars indicate mean with SD. (E) VERO AT2 or (F) A549 AT2 cells. Quantification of cell cycle arrest after exposure of cells to SARS-CoV-2 variants. α, alpha (MOI 0.5); Δ, delta (MOI 0.1); WT, Wuhan (MOI 0.5). n = 4; one-way ANOVA with Dunnett’s multiple comparisons test: ns, non-significant; ****p < 0.0001; ***p < 0.001. Bars indicate mean with SD.

Article Snippet: Following cells were a gift from: A549 ACE2/TMPRSS2 Massimo Palmerini, Vero E6 ACE2/TMPRSS2 from Emma Thomson, HeLa-ACE2 from James Voss, 293T (a human embryonic kidney cell line, ATCC CRL-3216).

Techniques: Transduction, Infection, Cell Cycle Assay, Staining, Expressing

Journal: bioRxiv

Article Title: Cell cycle independent role of cyclin D3 in host restriction of SARS-CoV-2 infection

doi: 10.1101/2022.05.07.491022

Figure Lengend Snippet: (A-D) A549 AT2 cells were depleted for D and A2 cyclins and 18h later infected with Delta variant SARS-CoV-2 for 24h. Cells were fixed, stained for SARS-CoV-2 nucleocapsid and analysed for infection and Fucci cell cycle sensor. (A) A representative western blot from lysates of uninfected knock-down cells. (B) Example of gating strategy for flow cytometry analysis. (C) Percentage of infected cells in cells depleted for cyclins. n=3; Ordinary two-way ANOVA with Sidak’s multiple comparisons test: ns, non-significant; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.1. Bars indicate mean with SD. (D) Flow cytometry analysis of early S and S/G2/M cell cycle phases comparing cyclin D1, D3, and A2 knockdown to NT (non-target siRNA). n = 3. Statistical analysis was performed using two-sided unpaired Student’s t-tests; ns, non-significant; ***p < 0.001; **p < 0.01; *p < 0.1. Bars indicate mean with SD. (E-G) VERO AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and 18h later infected with Delta variant SARS-CoV-2. Cells were fixed and stained for D-cyclins 24h later. (E) Example of acquisition using automated microscopic platform. Cells are identified for infection, expression of cyclin D3, and cell cycle (Red/arrow=G1phase; Green/arrowhead=S/G2/M; Red+Green/arrowhead=early S). (F-G) Quantification of D-cyclins re-localization from nucleus to cytoplasm and correlation with cell cycle phases using ImageJ and Harmony (PerkinElmer). (F) Cyclin D3. (G) Cyclin D1. At least 50-200 cells were analysed in each condition. Statistical analysis was performed using two-sided unpaired Student’s t-tests; ***p < 0.001; *p < 0.1.

Article Snippet: Following cells were a gift from: A549 ACE2/TMPRSS2 Massimo Palmerini, Vero E6 ACE2/TMPRSS2 from Emma Thomson, HeLa-ACE2 from James Voss, 293T (a human embryonic kidney cell line, ATCC CRL-3216).

Techniques: Infection, Variant Assay, Staining, Western Blot, Knockdown, Flow Cytometry, Transduction, Expressing

Journal: Cells

Article Title: NSP4 and ORF9b of SARS-CoV-2 Induce Pro-Inflammatory Mitochondrial DNA Release in Inner Membrane-Derived Vesicles

doi: 10.3390/cells11192969

Figure Lengend Snippet: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection promotes mitochondrial damage and extrusion of mitochondrial DNA (mtDNA) from the mitochondria. ( a ) Schematic representation of the SARS-CoV-2 infection model. Analysis of mitochondrial morphology and function in SARS-CoV-2 (multiplicity of infection = 0.2)-infected A549 cells stably expressing ACE2 and TMPRSS2 (modified A549). ( b ) Representative immunofluorescence images of SARS-CoV-2-infected A549 cells probed with the anti-spike (red) antibodies. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (blue). ( c ) Representative images of cells probed with the anti-TOM20 (green) antibodies to examine mitochondrial morphology. ( d ) Quantitative analysis of different mitochondrial shapes (n = 10 images). The following three mitochondrial morphologies were predominantly observed: blob/puncta, network, and tubular. Healthy cells exhibited increased proportions of tubular-shaped mitochondria, whereas SARS-CoV-2-infected cells exhibited increased proportions of blob/puncta-shaped mitochondria. Data are plotted as the percentage distribution of mitochondrial morphology. ( e ) Representative high-resolution LIGHTNING microscopy images of cells probed with the anti-TOM20 antibodies (green). Mitochondrial morphology was markedly different between the uninfected and infected groups. The insets show healthy tubular mitochondria ( blue arrowheads ) in the control cells and puncta/blob-shaped mitochondria with disrupted inner membranes ( yellow arrowheads ) (damaged mitochondria) in the SARS-CoV-2-infected cells. ( f ) Quantification of the number of damaged mitochondria (mito) per cell (n = 20) in the images shown in ( e ). ( g ) Quantitative analysis of mitochondrial mass (mitomass) in the control and infected groups using the anti-VDAC antibodies (n = 19). Data are plotted as integrated density. ( h ) Representative immunofluorescence images showing the localization of Cyt c (red) with the mitochondria (stained with the anti-TOM20 antibodies; green). ( i ) Quantification of the downregulated Cyt c signal (red) associated with the anti-TOM20 antibody-stained mitochondria (green) in SARS-CoV-2-infected cells from the images (n = 16). Data are plotted as integrated density. ( j ) Representative LIGHTNING microscopy images showing the colocalization of anti-TFAM antibody-stained mtDNA (magenta) within the anti-TOM20 antibody-stained mitochondria (green). Insets show that in the control group, mtDNA appears within the mitochondria. In the SARS-CoV-2-infected group, most mitochondria are devoid of mtDNA ( red arrowheads ) and in some instances, the mtDNA is localized outside the mitochondria ( yellow arrowheads ). ( k ) Quantitative analysis of the downregulation of mtDNA associated with the mitochondria in SARS-CoV-2-infected cells from images shown in ( j ) (n = 13). Data are plotted as integrated density. ( l ) Schematic representation of SARS-CoV-2 infection-induced mitochondrial damage and mtDNA release. All data are represented as mean ± standard error of mean from three independent experiments. Statistical analyses (unpaired t -tests) were performed using Graphpad Prism software. * p < 0.05; *** p < 0.001; **** p < 0.0001; ns: not significant. Scale bars: 50 ( b ), 10 ( c , h , j ), or 5 μm ( e ).

Article Snippet: A549 cells were transduced with lentiviral particles derived from the ACE2/TMPRSS2-expressing vector (Addgene #154987), and cells stably expressing ACE2/TMPRSS2 were selected using puromycin selection.

Techniques: Infection, Stable Transfection, Expressing, Modification, Immunofluorescence, Labeling, Microscopy, Staining, Software