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SARS-CoV (P SARS-CoV-2 ( 48 h G 1201 Cell- Cell- 100 I Percentage (%) 80 60- 40- 20- SARS-CoV (PV) and B) Schema eudotypes bear H

SARS-CoV (P SARS-CoV-2 ( 48 h G 1201 Cell- Cell- 100 I Percentage (%) 80 60- 40- 20- SARS-CoV (PV) and B) Schema eudotypes bear H

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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A Cell-free D 04 Cell-free infection (RLU x 10") Donor cell Target cell SARS-CoV-2 (PV)____ns____ I 48 h 72 h F 1201 Cell-free (co-culture) G 1201 Cell-free (transwell) SARS-CoV2-S SARS-CoV-S No spike Cell-to-cell Cell-to-cell 100 I 100 I I 80- 80- 60- 60- 1.5- 40- H 40- 20- 1.0- I 20- 0.5- I I 0- 0- 48 h 72 h 10¹ 102 103 104 105 106 Spike 0 SARS-CoV SARS-CoV-2 (PV) (PV) SARS-CoV SARS-CoV-2 (PV) (PV) Fig. 1. The spike protein of SARS-CoV-2 and SARS-CoV mediates cell-to-cell transmission of HIV-1 lentiviral pseudotypes. (A and B) Schematic representa- tions of cell-to-cell and cell-free infection assays (see details in Materials and Methods). Briefly, the inGluc-based lentiviral pseudotypes bearing spike were produced in 293T cells, which were cocultured with the target cells (293T/ACE2) for cell-to-cell transmission; the Gluc activity of cocultured cells was mea- sured over time (A). Cell-free infection was performed by harvesting virus from the same number of producer cells, followed by infecting 293T/ACE2 target cells in the presence of the same number of untransfected 293T cells; alternatively, cell-free infection was carried out in Transwell plates, from which Gluc activity was measured (B). (C) Comparison of cell-to-cell transmission mediated by SARS-CoV-2 or SARS-CoV spike. Results shown were from six independent experiments, with cell-free infection measured at 48 and 72 h after coculture; the portion of cell-free infection was excluded (n = 6). (D) Comparison of cell- free infection mediated by SARS-CoV-2 or SARS-CoV spike. Results were from six independent experiments (n = 6). (E) The expression level of spike proteins on the plasma membrane of donor cells was measured by flow cytometry using a polycolonal antibody T62, which detects both SARS-CoV-2 and SARS-CoV. (F and G) The calculated ratios between cell-to-cell and cell-free infection mediated by SARS-CoV-2 or SARS-CoV-2 spike. Results from cell coculture are shown in F and from Transwell plates shown in G (n = ~3 to 6). PV, pseudotyped virus. *P<0.05, **P<0.01. ns, not significant. Cell-to-cell 20- 15 10- CMV Hey Gagge LTR SARS-CoV-1/2 Spe HV Cappo GLUE B GLM SARS-CoV (PV) SARS-CoV-2 (PV)- E 100- 80- 60- 40 20 Transfected 293T (donor cell) 293T/ACE2 (target cell) Untransfected 293T % of Max Co- culture Cell- free Cell- free 24-72h 24-72 h Cell-to-cell transmission (RLU x 105) 24-48 h 24-48 h Gaussia luciferase 5.0 SARS-CoV (PV) 4.0- 3.0- 2.0- 1.0 Percentage (%) Percentage (%)
Transcribed Image Text:A Cell-free D 04 Cell-free infection (RLU x 10") Donor cell Target cell SARS-CoV-2 (PV)____ns____ I 48 h 72 h F 1201 Cell-free (co-culture) G 1201 Cell-free (transwell) SARS-CoV2-S SARS-CoV-S No spike Cell-to-cell Cell-to-cell 100 I 100 I I 80- 80- 60- 60- 1.5- 40- H 40- 20- 1.0- I 20- 0.5- I I 0- 0- 48 h 72 h 10¹ 102 103 104 105 106 Spike 0 SARS-CoV SARS-CoV-2 (PV) (PV) SARS-CoV SARS-CoV-2 (PV) (PV) Fig. 1. The spike protein of SARS-CoV-2 and SARS-CoV mediates cell-to-cell transmission of HIV-1 lentiviral pseudotypes. (A and B) Schematic representa- tions of cell-to-cell and cell-free infection assays (see details in Materials and Methods). Briefly, the inGluc-based lentiviral pseudotypes bearing spike were produced in 293T cells, which were cocultured with the target cells (293T/ACE2) for cell-to-cell transmission; the Gluc activity of cocultured cells was mea- sured over time (A). Cell-free infection was performed by harvesting virus from the same number of producer cells, followed by infecting 293T/ACE2 target cells in the presence of the same number of untransfected 293T cells; alternatively, cell-free infection was carried out in Transwell plates, from which Gluc activity was measured (B). (C) Comparison of cell-to-cell transmission mediated by SARS-CoV-2 or SARS-CoV spike. Results shown were from six independent experiments, with cell-free infection measured at 48 and 72 h after coculture; the portion of cell-free infection was excluded (n = 6). (D) Comparison of cell- free infection mediated by SARS-CoV-2 or SARS-CoV spike. Results were from six independent experiments (n = 6). (E) The expression level of spike proteins on the plasma membrane of donor cells was measured by flow cytometry using a polycolonal antibody T62, which detects both SARS-CoV-2 and SARS-CoV. (F and G) The calculated ratios between cell-to-cell and cell-free infection mediated by SARS-CoV-2 or SARS-CoV-2 spike. Results from cell coculture are shown in F and from Transwell plates shown in G (n = ~3 to 6). PV, pseudotyped virus. *P<0.05, **P<0.01. ns, not significant. Cell-to-cell 20- 15 10- CMV Hey Gagge LTR SARS-CoV-1/2 Spe HV Cappo GLUE B GLM SARS-CoV (PV) SARS-CoV-2 (PV)- E 100- 80- 60- 40 20 Transfected 293T (donor cell) 293T/ACE2 (target cell) Untransfected 293T % of Max Co- culture Cell- free Cell- free 24-72h 24-72 h Cell-to-cell transmission (RLU x 105) 24-48 h 24-48 h Gaussia luciferase 5.0 SARS-CoV (PV) 4.0- 3.0- 2.0- 1.0 Percentage (%) Percentage (%)
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