Journal: The Journal of biological chemistry

Article Title: SARS-CoV-2 hijacks macropinocytosis to facilitate its entry and promote viral spike-mediated cell-to-cell fusion.

doi: 10.1016/j.jbc.2022.102511

Figure Lengend Snippet: Figure 5. Impairment of macropinocytosis signaling blocks SARS-CoV-2 entry. A, knockdown of RhoA, Cdc42, Rac1, and Pak1. HEK293 cells were transfected with siRNA targeting RhoA, Cdc42, Rac1, and Pak1. The expression of the indicated proteins was evaluated by Western blotting. The relative band intensity was determined by ImageJ, and the intensity of the control was set as 1. B, knockdown of RhoA, Cdc42, Rac1, and Pak1 by siRNA inhibited 70 kDa dextran uptake, which was determined by using flow cytometry. Each experiment was performed in triplicate, and the results are presented as the mean ± SD. C, knockdown of Rac1/Pak1 by siRNA blocks SARS-CoV-2 entry into HEK293T–ACE2 cells. HEK293T–ACE2 cells were transfected with siRNA targeting Pak1, RhoA, Cdc42, Rac1, or Pak1 or with scramble siRNA. Cells were then infected with the indicated VSV-ΔG-Luc pseudovirus at 48 h post- transfection. Luciferase values were normalized to scramble siRNA-transfected control cells. D, Vero E6 cells transfected with Cdc42-T17N, Rac1-T17N, Pak1-R299, or RhoA-T19N were incubated with 70 kDa dextran. The uptake of 70 kDa dextran was analyzed by using confocal laser scanning microscopy. E, Vero E6 cells and (F) HEK293T–ACE2 cells transfected with Cdc42-T17N, Rac1-T17N, Pak1-R299, or RhoA-T19N (as a control) were infected with VSV-ΔG-Luc pseudovirus. Luciferase values are normalized to the control. G, quantitative analysis of VSV-ΔG-EGFP pseudovirus infection in dominant negative– expressing Vero E6 cells. The dominant-negative form of the indicated proteins was labeled by an antimyc antibody in Vero E6 cells. VSV-ΔG-EGFP pseudovirus was incubated for 2 h at 37 C, and the infectivity of VSV-ΔG-EGFP pseudovirus was analyzed by microscopy image analysis using confocal laser scanning microscopy. The scale bars represent 10 μm. ACE2, angiotensin-converting enzyme 2; EGFP, enhanced GFP; HEK293, human embryonic kidney 293 cell line; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; VSV, vesicular stomatitis virus.

Article Snippet: The cells were washed three times with PBS and blocked with 2% bovine serum albumin for 1 h. The cells were incubated with a mouse antimyc antibody (Proteintech; catalog number: 60003-2-Ig: 1:1000 dilution) for 2 h at 37 C and then with an Alexa Fluor 568–conjugated goat antimouse immunoglobulin G secondary antibody (Invitrogen).

Techniques: Knockdown, Transfection, Expressing, Western Blot, Control, Cytometry, Infection, Luciferase, Incubation, Confocal Laser Scanning Microscopy, Dominant Negative Mutation, Labeling, Microscopy, Virus