Journal: Nature Communications

Article Title: SARS-CoV-2 virulence factor ORF3a blocks lysosome function by modulating TBC1D5-dependent Rab7 GTPase cycle

doi: 10.1038/s41467-024-46417-2

Figure Lengend Snippet: A GST and GST-RILP proteins immobilized on beads were incubated with lysates from uninfected and SARS-CoV-2-infected Vero E6 cells. The precipitates were IB with indicated antibodies, n = 3 . The values in ( A ) and graph ( B ) represent densitometric analysis of the levels of active Rab7 pulldown, n = 3 , p = 0.0116. C Lysates of Vero E6 cells infected with SARS-CoV-2 and transfected with either control or ORF3a-siRNA were IB for the indicated proteins, n = 3 . The values represent densitometric analysis of LC3b-II and p62 levels normalized to β-tubulin. D Relative fold-change in intracellular N-antigen levels in lysates of SARS-CoV-2-infected Vero E6 cells transfected with control or ORF3a-siRNA, n = 3 , p < 0.0001. E Relative expression of SARS-CoV-2 E and orf1ab genes measured in the culture supernatants, n = 3 , p = 0.0003 ( E gene), 0.002 ( orf1ab gene). F Protein levels of N-antigen in the culture supernatants. The values represent a densitometric analysis of the levels of extracellular N-antigen, n = 1 . G Representative confocal images of Vero E6 cells uninfected or infected with SARS-CoV-2 and transfected with either control or ORF3a-siRNA and immunostained for endogenous Rab7 and N-antigen. The insets show the differences in the sizes of the Rab7 puncta, n = 3 . Scale bars: 10 µm (main); 2 µm (inset). H Quantification of Rab7 puncta size in uninfected and SARS-CoV-2-infected Vero E6 cells transfected with control or ORF3a-siRNA, n = 114, 113 and 152 cells examined over three independent experiments, as indicated, p < 0.0001. I GST and GST-RILP proteins immobilized on beads were incubated with lysates from uninfected and SARS-CoV-2-infected Vero E6 cells transfected with control or ORF3a-siRNA. The precipitates were IB with the indicated antibodies, n = 3 . The values in ( I ) and graph ( J ) represent densitometric analysis of the levels of active Rab7 pulldown, n = 3 , p = 0.0002 (uninfected versus control siRNA-infected), 0.0012 (control versus ORF3a siRNA-infected). Quantified results are presented as mean ± S.D. using unpaired two-tailed Student’s t test.

Article Snippet: HeLa, HEK293T, and A549 (from ATCC), Vero E6 (from the NCCS Pune Cell Repository), and HEK293T-hACE2 (HEK293T cells expressing human angiotensin-converting enzyme 2 (ACE2); NR-52511; from BEI Resources) cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) in a humidified cell culture incubator (Thermo Scientific) with 5% CO 2 at 37 °C.

Techniques: Incubation, Infection, Transfection, Control, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: SARS-CoV-2 virulence factor ORF3a blocks lysosome function by modulating TBC1D5-dependent Rab7 GTPase cycle

doi: 10.1038/s41467-024-46417-2

Figure Lengend Snippet: A Representative confocal images of HeLa (wild-type) and ORF3a-GFP-expressing cells immunostained for indicated proteins. The insets 1 and 2 mark untransfected and ORF3a-GFP-transfected cells, respectively. B The colocalization of Rab7 and Arl8b was evaluated using PCC, n = 75 cells examined over three independent experiments, p < 0.0001. C Representative confocal images of uninfected and SARS-CoV-2-infected Vero E6 cells immunostained for Arl8b and Rab7. D The colocalization of Rab7 with Arl8b was measured using PCC, n = 60 cells examined over three independent experiments, p < 0.0001. E Representative micrographs from live-cell imaging experiments performed on untreated and Dox-treated HeLa ORF3a-Strep cells expressing GFP-Rab7 and Arl8b-tomato. The arrows highlight the fusion between GFP-Rab7 and Arl8b-tomato vesicles; see Supplementary Movie . F Quantification of the number of fusion events per ROI (region of interest), n = 27 (-DOX) and n = 22 ( + DOX), p < 0.0001. G Ferrofluid (FF) uptake was performed in untreated and Dox-treated HeLa ORF3a-Strep cells for 20 min at 37 °C (pulse), followed by various periods of time (chase). At the indicated time points, the cells were homogenized, and the FF-containing cellular compartments were purified and immunoblotted for the presence of the indicated proteins, n = 1 . The asterisk (*) denotes a non-specific signal detected using the anti-Rab5 antibody. Representative live-cell imaging micrographs of untreated ( H ) and Dox-treated ( I ) HeLa ORF3a-Strep cells expressing GFP-Rab7 and Arl8b-tomato were captured at the start of time-lapse imaging ( T = 0 s). Single-particle tracking analysis of GFP-Rab7 and Arl8b-tomato was performed until T = 200 s with color coding to show the maximum velocity (V max ; blue, immobile; red, maximum mobility), n = 10 (-DOX) and n = 8 (+DOX) cells. J − M Graphs ( J ) and ( L ) represent the mobile fractions of Arl8b-tomato and GFP-Rab7 vesicles, respectively. Graphs ( K ) and ( M ) represent the maximum speeds of the Arl8b-tomato and GFP-Rab7 vesicles, respectively, n = 10 (-DOX) and n = 8 (+DOX) cells, p = 0.1343 ( J ), 0.0952 ( K ), 0.0001 ( L ), < 0.0001 ( M ). Quantified results are presented as mean ± S.D. using unpaired two-tailed Student’s t test. Scale bars: 10 µm (main); 2 µm (inset).

Article Snippet: HeLa, HEK293T, and A549 (from ATCC), Vero E6 (from the NCCS Pune Cell Repository), and HEK293T-hACE2 (HEK293T cells expressing human angiotensin-converting enzyme 2 (ACE2); NR-52511; from BEI Resources) cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) in a humidified cell culture incubator (Thermo Scientific) with 5% CO 2 at 37 °C.

Techniques: Expressing, Transfection, Infection, Live Cell Imaging, Purification, Imaging, Single-particle Tracking, Two Tailed Test