Journal: Nature Communications

Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

doi: 10.1038/s41467-021-21213-4

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 (two or more experiments; n = 6 except for Calu-3 in which n = 8; one-way ANOVA with Dunnett’s test; mean ± s.d.). c Evaluation of entry efficiency in different cell lines infected with pseudoviruses bearing spike protein Sfull, Sdel, or S mutant (R682S, R685S). Data are normalized to the Sfull of individual experiments (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). d Effect of TMPRSS2 serine protease inhibitor Camostat and cysteine protease inhibitor E-64d on Sfull or Sdel infection in different cell lines (two experiments; n = 4 or 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the untreated group of individual experiments. **** P < 0.0001; n.s. not significant.

Article Snippet: The human TMPRSS2 (Sino Biological #HG13070-CM) and DPP4 (kindly provided by Yaowei Huang, Zhejiang University) genes were cloned by the similar strategy.

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

Journal: Nature Communications

Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

doi: 10.1038/s41467-021-21213-4

Figure Lengend Snippet: a Viral load in the tissues of nasal turbinate, trachea, and lung. Tissues were harvested at days 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 days 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 3 days. Hamsters were sacrificed and the indicated tissues were harvested for titration. The dashed lines represent the limit of detection by focus-forming assay. Median viral titers ( a – c ): two-tailed Mann–Whitney test; * P < 0.05; ** P < 0.01; n.s. not significant. d H&E staining of lung sections of contact hamsters. Representative images are shown from n = 6 hamsters. Scale bar, 100 μm. e , f RNA ISH of lung and nasal turbinate sections of contact hamsters. Representative images are shown from n = 6 hamsters. Scale bar, 100 μm. g Model of the role of S1/S2 boundary and host factors in regulating cell entry, pathogenicity, and transmissibility of SARS-CoV-2. SARS-CoV-2 enters cells via two pathways. The virus (Sfull) with intact spike protein preferentially enters cells at the plasma membrane (early entry pathway) in airway epithelial cells (Calu-3) or respiratory tract tissues expressing the proteases (e.g., TMPRSS2) to activate the membrane fusion. The deletion at S1/S2 junction site in spike (Sdel), however, propels the virus to enter cells through the endosomal pathway (late entry pathway), which is less efficient than the fusion pathway at the plasma membrane. Host factors such as CTSL, NCP1/2, WDR81/91, and TFE3 are critical for the endosomal entry. Both entry pathways are initiated with virion binding to receptor ACE2 at the cell surface 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 (Sino Biological #HG13070-CM) and DPP4 (kindly provided by Yaowei Huang, Zhejiang University) genes were 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