a Counts Counts b 60 50 40 30 20 10 0 (kDa) 150 10⁰ 60 50 40 30 20 10 0 10⁰ T 100- 75- 10¹ 10¹ gp120-c9 S1-c9 IFNAR2-c9 10² FL1-H 10² FL1-H 103 10³ C PNGase F: (kDa) 150- 100- 75- 104 104 S1-lg + + SIFNAR2-lg + Figure 1 A 110 kDa protein associates with the S1 domain of SARS-CoV S protein. a, A fusion protein of the S1 domain (S1-lg, shaded area), or of the first 327 residues of that domain (S1(327)-lg, dotted line) with the Fc domain of human IgG1, or culture medium alone (thick line) was incubated with 293T (top panel) or Vero E6 (bottom panel) cells. Binding of fusion proteins to cells was measured by flow cytometry using a FITC-labelled anti-human IgG secondary antibody. b, Metabolically labelled Vero E6 cell lysates were immunoprecipitated with HIV-1 gp120, SARS-CoV S1 domain, or the ectodomain of human IFNAR2, each containing a tag (C9) at its C terminus, and an anti-tag antibody. Immunoprecipitates were analysed by SDS-PAGE. c, Labelled Vero E6 cell lysates were immunoprecipitated with S1-lg or soluble IFNAR2-lg. Immunoprecipitates were treated or not, as indicated, with PNGase F, and analysed by SDS-PAGE. found that viral genome copies in ACE2-transfected cells increased by more than 100,000-fold in the first 48 h (Fig. 4a, bottom panel). a Counts 100 80 60 40 20 C 10⁰ PNGase F: (kDa) 150- 100- 75- 10¹ S1(327)-lg 10² 10³ FL1-H S1-lg b Counts 104 100 80 60 40 20 0 Anti-ACE2 10⁰ I Mock I SACE2 I Mock + Mock I SACE2 SACE2 + I Mock I SACE2 10¹ 10² 10³ 104 FL1-H Figure 2 A high-affinity association between ACE2 and the S1 domain. a, 293T cells transfected with plasmid encoding ACE2 (shaded area and dotted line), or with vector alone (thick solid line), were analysed by flow cytometry with S1-Ig (shaded area and thick solid line) or S1 (327)-Ig (dotted line). b, Vero E6 cells were incubated with S1-lg (shaded area, thin solid line and thick solid line), or with medium alone (dotted line), in the presence of soluble ACE1 (thin solid line) or ACE2 (thick solid line), or without either protein (shaded area and dotted line). c, Supernatants of radiolabelled 293T cells transfected with plasmid encoding soluble ACE2 or with vector alone (mock) were immunoprecipitated with S1 (327)-lg, S1-lg, or an anti-ACE2 antibody. Immunoprecipitates were treated or not, as indicated, with PNGase F, and analysed by SDS-PAGE. a HIV-1 gp160 + CD4/CCR5 S protein + CD4/CCR5 b S protein + ACE2 + control Ab Figure 3 Syncytia formation between S-protein- and ACE2-expressing cells. a, 293T cells transfected with plasmids encoding the HIV-1 envelope glycoprotein gp160 (top row) or SARS-CoV S protein (bottom row) were mixed at a 1:1 ratio with 293T cells transfected with plasmids encoding HIV-1 receptors CD4 and CCR5 (left column) or ACE2 (right HIV-1 gp160 + ACE2 S protein + ACE2 S protein + ACE2 + anti-ACE2 Ab column). b, 293T cells transfected with ACE2 plasmid were mixed at a 1:1 ratio with 293T cells transfected with plasmid encoding S protein, in the presence of 10 μg ml-1 goat polyclonal control antibody (left) or goat polyclonal anti-ACE2 antibody (right). Ab, antibody.

a Counts Counts b 60 50 40 30 20 10 0 (kDa) 150 10⁰ 60 50 40 30 20 10 0 10⁰ T 100- 75- 10¹ 10¹ gp120-c9 S1-c9 IFNAR2-c9 10² FL1-H 10² FL1-H 103 10³ C PNGase F: (kDa) 150- 100- 75- 104 104 S1-lg + + SIFNAR2-lg + Figure 1 A 110 kDa protein associates with the S1 domain of SARS-CoV S protein. a, A fusion protein of the S1 domain (S1-lg, shaded area), or of the first 327 residues of that domain (S1(327)-lg, dotted line) with the Fc domain of human IgG1, or culture medium alone (thick line) was incubated with 293T (top panel) or Vero E6 (bottom panel) cells. Binding of fusion proteins to cells was measured by flow cytometry using a FITC-labelled anti-human IgG secondary antibody. b, Metabolically labelled Vero E6 cell lysates were immunoprecipitated with HIV-1 gp120, SARS-CoV S1 domain, or the ectodomain of human IFNAR2, each containing a tag (C9) at its C terminus, and an anti-tag antibody. Immunoprecipitates were analysed by SDS-PAGE. c, Labelled Vero E6 cell lysates were immunoprecipitated with S1-lg or soluble IFNAR2-lg. Immunoprecipitates were treated or not, as indicated, with PNGase F, and analysed by SDS-PAGE. found that viral genome copies in ACE2-transfected cells increased by more than 100,000-fold in the first 48 h (Fig. 4a, bottom panel). a Counts 100 80 60 40 20 C 10⁰ PNGase F: (kDa) 150- 100- 75- 10¹ S1(327)-lg 10² 10³ FL1-H S1-lg b Counts 104 100 80 60 40 20 0 Anti-ACE2 10⁰ I Mock I SACE2 I Mock + Mock I SACE2 SACE2 + I Mock I SACE2 10¹ 10² 10³ 104 FL1-H Figure 2 A high-affinity association between ACE2 and the S1 domain. a, 293T cells transfected with plasmid encoding ACE2 (shaded area and dotted line), or with vector alone (thick solid line), were analysed by flow cytometry with S1-Ig (shaded area and thick solid line) or S1 (327)-Ig (dotted line). b, Vero E6 cells were incubated with S1-lg (shaded area, thin solid line and thick solid line), or with medium alone (dotted line), in the presence of soluble ACE1 (thin solid line) or ACE2 (thick solid line), or without either protein (shaded area and dotted line). c, Supernatants of radiolabelled 293T cells transfected with plasmid encoding soluble ACE2 or with vector alone (mock) were immunoprecipitated with S1 (327)-lg, S1-lg, or an anti-ACE2 antibody. Immunoprecipitates were treated or not, as indicated, with PNGase F, and analysed by SDS-PAGE. a HIV-1 gp160 + CD4/CCR5 S protein + CD4/CCR5 b S protein + ACE2 + control Ab Figure 3 Syncytia formation between S-protein- and ACE2-expressing cells. a, 293T cells transfected with plasmids encoding the HIV-1 envelope glycoprotein gp160 (top row) or SARS-CoV S protein (bottom row) were mixed at a 1:1 ratio with 293T cells transfected with plasmids encoding HIV-1 receptors CD4 and CCR5 (left column) or ACE2 (right HIV-1 gp160 + ACE2 S protein + ACE2 S protein + ACE2 + anti-ACE2 Ab column). b, 293T cells transfected with ACE2 plasmid were mixed at a 1:1 ratio with 293T cells transfected with plasmid encoding S protein, in the presence of 10 μg ml-1 goat polyclonal control antibody (left) or goat polyclonal anti-ACE2 antibody (right). Ab, antibody.

Biology 2e

2nd Edition

ISBN:9781947172517

Author:Matthew Douglas, Jung Choi, Mary Ann Clark

Publisher:Matthew Douglas, Jung Choi, Mary Ann Clark

Chapter16: Gene Expression

Section: Chapter Questions

Problem 38CTQ: A scientist discovers a virus encoding a Protein X that degrades a subunit of the elF4F complex....

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