Journal: Cell Host & Microbe

Article Title: A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition

doi: 10.1016/j.chom.2020.06.020

Figure Lengend Snippet: Generation of a Recombinant Vesicular Stomatitis Virus (rVSV) Bearing the SARS-CoV-2 Spike (S) Glycoprotein (A) Schematic representation of the VSV genome in which its native glycoprotein gene has been replaced by that encoding the SARS-CoV-2 S protein. The VSV genome has been further modified to encode an enhanced green fluorescent protein (eGFP) reporter to easily score for infection. (B) Infectious center formation assay on Vero cells at 24 h post-infection showing growth of the rVSV-SARS-CoV-2 S after the indicated number of rounds of serial passage of the passage #1 virus (carrying wild-type [WT] S sequences) on Huh7.5.1 cell line (scale bar, 100 μm). Two representative images for each virus passage, showing infected cells pseudo-colored in green, from one of the two independent experiments are shown here. (C) Incorporation of SARS-CoV-2 S into rVSV particles captured on an ELISA plate was detected using antiserum from a COVID-19 convalescent donor (average ± SD, n = 12 from 3–4 independent experiments). Serum from a COVID-19-negative donor and rVSVs bearing Ebola virus glycoprotein (EBOV GP) were used as negative controls (average ± SD, n = 6 from 2 independent experiments). (D) Representative images showing Vero cells infected with plaque #2, #3, and #6 viruses at 16 h post-infection (scale bar, 100 μm). (E) Production of infectious virions at 48 h post-infection from Vero cells infected with the indicated plaque-purified viruses. Titers were measured on Vero cells overexpressing TMPRSS2 (n = 4, from two independent titrations).

Article Snippet: Human ACE2 and TMPRSS2 coding sequences were PCR-amplified from the hACE2 plasmid Addgene #1786 (a generous gift from Hyeryun Choe) and TMPRSS2 plasmid Addgene #53887 (a generous gift from Roger Reeves), respectively and cloned into a retroviral pBabe-puro vector.

Techniques: Recombinant, Virus, Modification, Infection, Tube Formation Assay, Enzyme-linked Immunosorbent Assay, Purification

Journal: Cell Host & Microbe

Article Title: A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition

doi: 10.1016/j.chom.2020.06.020

Figure Lengend Snippet: rVSV-SARS-CoV-2 S Infection Requires the Activity of Cysteine Cathepsin Proteases (A) Huh7.5.1 cells pre-treated for 1 h at 37°C with the indicated concentrations of NH 4 Cl were infected with pre-titrated amounts of rVSVs bearing SARS-CoV-2 S or EBOV GP. Infection was scored by eGFP expression at 16–18 h post-infection (average ± SD, n = 8 from 2 independent experiments). (B) Vero cells pre-treated for 90 min at 37°C with the indicated concentrations of pan-cysteine cathepsin inhibitor E-64 were infected with pre-titrated amounts of rVSVs bearing SARS-CoV-2 S, EBOV GP, or VSV G and scored for infection as above (average ± SD, n = 6 from 3 independent experiments, except n = 4 from 2 independent experiments for EBOV GP). (C) Vero cells pre-treated for 90 min at 37°C with the indicated concentrations of cathepsin L/B inhibitor FYdmk were infected with pre-titrated amounts of rVSVs bearing SARS-CoV-2 S, EBOV GP, or VSV G. Infection was scored as above (average ± SD, n = 6 from 3 independent experiments). (D) Vero cells and Vero cells overexpressing TMPRSS2 pre-treated for 120min at 37C with the indicated concentrations of camostat ]were infected with pre-titrated amounts of rVSVs bearing SARS-CoV-2 S and subsequently scored for infection. In panels (B)–(D), all comparisons were made between vehicle- and inhibitor-treated samples. ns,not statistically significant. ∗ p < 0.033, ∗∗∗ p < 0.001.

Article Snippet: Human ACE2 and TMPRSS2 coding sequences were PCR-amplified from the hACE2 plasmid Addgene #1786 (a generous gift from Hyeryun Choe) and TMPRSS2 plasmid Addgene #53887 (a generous gift from Roger Reeves), respectively and cloned into a retroviral pBabe-puro vector.

Techniques: Infection, Activity Assay, Expressing

Journal: Cell Host & Microbe

Article Title: A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition

doi: 10.1016/j.chom.2020.06.020

Figure Lengend Snippet: rVSV-SARS-CoV-2 S Infection in Human Airway Epithelial Cells is ACE2-Dependent (A) Infectivity of rVSV-SARS-CoV-2 S was measured in human airway epithelial Calu3 cells and Vero-TMPRSS2 cells by applying serial dilutions of the virus. Infections were scored as described in Figure 3 B (Average ± SD, n = 4 from two independent titrations). (B and C) Monolayers of Calu3 cells pre-incubated for 1 h at 37°C with indicated amounts of anti-human ACE2 antibody or negative control (hIgG) were infected with pre-titrated amounts of rVSV-SARS-CoV-2 S. (B) Representative images from one of the two independent experiments are shown (scale bar, 100 μm). (C) Infection was scored as above and is represented as % relative infection (no antibody = 100%, Average ± SD, n = 4 from two independent experiments, except for n = 2 for hIgG at 100 nM). (D) Infectivity of rVSV-SARS-CoV-2 S in human respiratory epithelial A549 cells transduced with a retrovirus carrying human ACE2 cDNA or empty vector was evaluated by exposing cells to serial dilutions of rVSV-SARS-CoV-2 S. Infections were scored as described in Figure 3 B (Average ± SD, n = 6 from 3 independent titrations). Red dotted line indicates the assay limit of detection (LOD). Means were compared by unpaired t test. (E) A549 cells transduced as in panel 3D were immunostained for hACE2 expression as described in Figure 3 A. using an anti-ACE2 antibody. Cells were imaged by fluorescence microscopy. The hACE2 signal is pseudo-colored green and representative images are shown (scale bar = 20 μm). ∗∗∗ p < 0.001.

Article Snippet: Human ACE2 and TMPRSS2 coding sequences were PCR-amplified from the hACE2 plasmid Addgene #1786 (a generous gift from Hyeryun Choe) and TMPRSS2 plasmid Addgene #53887 (a generous gift from Roger Reeves), respectively and cloned into a retroviral pBabe-puro vector.

Techniques: Infection, Virus, Incubation, Negative Control, Transduction, Plasmid Preparation, Expressing, Fluorescence, Microscopy

Journal: Cell Host & Microbe

Article Title: A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition

doi: 10.1016/j.chom.2020.06.020

Figure Lengend Snippet:

Article Snippet: Human ACE2 and TMPRSS2 coding sequences were PCR-amplified from the hACE2 plasmid Addgene #1786 (a generous gift from Hyeryun Choe) and TMPRSS2 plasmid Addgene #53887 (a generous gift from Roger Reeves), respectively and cloned into a retroviral pBabe-puro vector.

Techniques: Virus, In Vitro, Recombinant, Reverse Transcription, Sequencing, Plasmid Preparation, Expressing, Software, Fluorescence, Microscopy, Imaging