Journal: bioRxiv

Article Title: The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

doi: 10.1101/2020.08.25.266775

Figure Lengend Snippet: A. Sequence alignment of spike protein encompassing the cleavage site between S1 and S2 subunits. The spike proteins of SARS-CoV-2 without (Sfull strain) and with (Sdel strain) deletion were used to compare with that of SARS-CoV. The insertion of multi-basic amino acids in spike protein of SARS-CoV-2 was shown in red. B. Comparison of the replication property between Sfull and Sdel strains in different cell lines. The percentage of nucleocapsid (N) protein positive cells was analyzed by imaging-based analysis following virus infection. Data shown are an average of two independent experiments performed in triplicate. C. The immunofluorescence staining of N protein in A549-ACE2 and Calu-3 cells infected with Sfull or Sdel virus. A representative of two independent experiments was shown. D. Assessment of live virus production in different cell lines infected with Sfull or Sdel strain. Data are pooled from two independent experiments conducted in triplicate. E. Evaluation of entry efficiency in different cell lines infected with pseudoviruses bearing spike protein Sfull, Sdel, or S mutant (R682S, R685S). Data shown are an average of two independent experiments performed in triplicate and are normalized to the Sfull of individual experiments. F. Effect of TMPRSS2 serine protease inhibitor Camostat and cysteine protease inhibitor E-64d on Sfull or Sdel infection in different cell lines. Data shown are an average of two independent experiments performed in duplicate or triplicate and are normalized to the untreated group of individual experiments. One-way ANOVA with Dunnett’s test (A, B, C); two-way ANOVA with Sidak’s test (B); ****P < 0.0001; ns, not significant.

Article Snippet: The human TMPRSS2 and DPP4 gene (Sino Biological #HG13070-CM) was cloned by the similar strategy.

Techniques: Sequencing, Imaging, Infection, Immunofluorescence, Staining, Mutagenesis, Protease Inhibitor

Journal: bioRxiv

Article Title: The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

doi: 10.1101/2020.08.25.266775

Figure Lengend Snippet: A. SARS-CoV-2 SH01 strain isolated from a patient sample was purified three times by plaque assay on Vero-E6 (thereafter as Vero) cells in the presence of trypsin, resulting the clone of Sfull virus. The Sdel clone was obtained by passaging the Sfull virus twice and plaque-purified once on Vero cells without trypsin. The trace results of Sanger sequencing were generated by SnapGene Viewer, and the 21 nucleotide (nt) deletion was indicated. B. The Sfull strain was passaged twice on Vero cells in the presence of trypsin. C. The Sfull strain was passaged twice on Vero cells expressing the TMPRSS2 in the absence of trypsin. d. The sequence alignment of SARS-CoV-2 strains. The full-length genome sequences obtained by RT-PCR and Sanger sequencing were aligned and compared to the stain Wuhan-Hu-1. Wuhan-Hu-1, accession No. MN908947 ; SH01, accession No. MT121215 .

Article Snippet: The human TMPRSS2 and DPP4 gene (Sino Biological #HG13070-CM) was cloned by the similar strategy.

Techniques: Isolation, Purification, Plaque Assay, Passaging, Sequencing, Generated, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: bioRxiv

Article Title: The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

doi: 10.1101/2020.08.25.266775

Figure Lengend Snippet: A. Immunofluoresence staining of the nucleocapsid (N) protein of Sfull or Sdel virus on wild type Vero, Vero with trypsin treatment, and Vero expressing the TMPRSS2 cells. Virus-infected cells were fixed, permeablized, and stained with the house-made mouse anti-nucleocapsid serum. After washing, cells were incubated with goat anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo # A-21424, 2 μg/ml), followed by staining with 4’,6-diamidino-2-phenylindole (DAPI). Images were collected using an Operetta High Content Imaging System (PerkinElmer), and processed using the ImageJ software. B. The effect of compounds Comostat and E-64d on the infection by Sfull or Sdel virus in different cell types. Cells were pretreated with 25 μM compounds Camostat or / and E-64d and infected with Sfull or Sdel virus in the presence of compounds for 24 h. Immunofluoresence assay was conducted as described above. C. Sfull infection on A549-ACE2 cells were resistant to the treatment of 50 μM or 100 μM of Comostat or / and E-64d. Cells were pretreated with the indicated compounds and infected with the Sfull virus in the presence of compounds, followed by Immunofluoresence staining as described above. D-E. The effect of compounds Comostat and E-64d on the infection by SARS-CoV or MERS-CoV pseudovirus in different cell types. Cells were pretreated with 25 μM compounds Camostat or / and E-64d and infected with Sfull or Sdel pseudovirus in the presence of compounds for 48 h. One-way ANOVA with Dunnett’s test *P < 0.05; ***, P < 0.001; ****P < 0.0001; ns, not significant. Immunofluoresence assay was conducted as described above. Data were pooled from two independent experiments performed in triplicate, and are normalized to the controls of individual experiments.

Article Snippet: The human TMPRSS2 and DPP4 gene (Sino Biological #HG13070-CM) was cloned by the similar strategy.

Techniques: Staining, Expressing, Infection, Incubation, Imaging, Software

Journal: bioRxiv

Article Title: The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

doi: 10.1101/2020.08.25.266775

Figure Lengend Snippet: A. Viral load in the tissues of nasal turbinate, trachea, and lung. Tissues were harvested at day 1, 2 and 4 post-challenge of Sfull or Sdel virus (n=6 per day). B. Viral RNA in fecal samples. Fresh fecal samples were collected at day 2 and 4 post-infection of Sfull or Sdel strain (n=6 per day) for qRT-PCR. C. Transmission of Sfull or Sdel strain in hamsters by direct contact exposure. Naïve hamsters (n=6) were each co-housed with one inoculated donor at day 1 for three days. Hamsters were sacrificed and the indicated tissues were harvested for titration. The dashed lines represent the limit of detection by focus-forming assay. Mean fecal sample weight (B): two-tailed unpaired t-test; median viral titers (A, B, C): two-tailed Mann–Whitney test *P < 0.05; **P < 0.01; ns, not significant. D. H&E staining of lung sections of contact hamsters. Representative images are shown from n = 6 hamsters. Scale bar, 100μm. E-F. RNA ISH of lung and nasal turbinate sections of contact hamsters. Representative images are shown from n = 6 hamsters. Scale bar, 100μm. G. Model of the role of S1/S2 boundary in cell entry, pathogenicity, and transmissibility of SARS-CoV-2. SARS-CoV-2 with intact spike protein (Sfull virus) preferentially enters cells at the plasma membrane (early entry pathway) in respiratory tract tissues expressing the proteases ( e.g ., TMPRSS2) to activate the membrane fusion. The virus (Sdel) with deletion at S1/S2 junction site in spike, however, tends to enter via endosomal pathway (late entry pathway). Both entry pathways are initiated with virion binding to cellular receptor ACE2 that is regulated by host factors including retromer, CCC, and WASH complexes, etc. The more efficient early entry pathway in respiratory tract with intact spike protein than the late pathway promotes virus production, pathogenesis, and transmission in a hamster model. The SARS-CoV with spike lacking the insertion of multi-basic amino acids may resemble the Sdel virus and enter cell less efficiently than SARS-CoV-2 resulting in relatively low transmissibility.

Article Snippet: The human TMPRSS2 and DPP4 gene (Sino Biological #HG13070-CM) was cloned by the similar strategy.

Techniques: Infection, Quantitative RT-PCR, Transmission Assay, Titration, Focus Forming Assay, Two Tailed Test, MANN-WHITNEY, Staining, Expressing, Binding Assay