Journal: The Journal of Clinical Investigation

Article Title: SARS-CoV-2 Delta and Omicron variants resist spike cleavage by human airway trypsin-like protease

doi: 10.1172/JCI174304

Figure Lengend Snippet: ( A – C ) Infectivity of WT ( A ), Delta ( B ), and Omicron BA.1 ( C ) pseudoviruses pretreated with nasal wash samples (NW) from young (20–30 years) and old (55–70 years) participants ( n = 10). ( D ) Infectivity of WT pseudovirus with respiratory tract proteases or proteins ( n = 3). ( E ) WT pseudovirus treated with NW, with or without HAT inhibitors aprotinin and soybean trypsin inhibitor (STI) ( n = 10). ( F ) HAT (2 μg/mL) pretreated with spike proteins, incubated with WT pseudovirus, determined infectivity ( n = 3). ( G and H ) Representative images ( G ) and quantification ( H ) of CPE in Vero E6 cells infected with live ancestral viruses, with or without HAT (0.5–5 μg/mL) ( n = 3). ( I and J ) Cell lysates collected 48 hours after infection with live viruses for gRNA ( I ) and sgRNA ( J ) assays ( n = 3). ( K ) The mRNA levels of tmprss11d in trachea (left) and lung (right) tissues in mice infected with SARS-CoV-2 on day 3 after infection ( n = 3 mice each group). ( L and M ) The representative images displayed conjugates (arrow) formed with SARS-CoV-2 spike protein (green) and released trypsin-like protease (red) in bronchial tissues of infected mice ( L ) and primates ( M ). ( N ) The images indicated SARS-CoV-2 particles (green) trapped by filamentous HAT (red) in the sputum of patients with COVID-19. The inset shows magnified conjugates in merged images. Data are representative ( D , F , G – J , and K ) of 2 independent experiments with 3 replicates each. Unpaired, 2-tailed Student’s t test was performed in D and K , and 1-way ANOVA analysis followed by Tukey’s multiple comparison post hoc test was conducted in A – C , E , F , and H – J . Scale bars: 200 μm in G ; 4 μm in L and M ; and 10 μm in N . Data are presented as mean values ± SEM in A – K . * P <0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For protein detection, the following antibodies were used: rabbit anti-SARS-CoV-2 S1 polyclonal antibodies (Sino Biological, 40591-T62), rabbit anti-SARS-CoV-2 S2 polyclonal antibodies (Sino Biological, 40590-T62), rabbit anti-SARS-CoV-2 N polyclonal antibodies (Sino Biological, 40143-R001), and horseradish peroxidase–conjugated (HRP-conjugated) anti-rabbit polyclonal antibodies (Thermo Fisher Scientific, 31460).

Techniques: Infection, Incubation, Comparison

Journal: The Journal of Clinical Investigation

Article Title: SARS-CoV-2 Delta and Omicron variants resist spike cleavage by human airway trypsin-like protease

doi: 10.1172/JCI174304

Figure Lengend Snippet: ( A ) 5 × 10 4 PFU of ancestral SARS-CoV-2, Delta, and Omicron (BA.1) variants were preincubated with PBS or 80 ng HAT in a total volume of 40 μL. After incubation at 37°C for 2 hours, 6–8 week-old female transgenic hACE2 (hACE2-KI/NIFDC) mice were intranasally instilled with the mixtures. The lung tissues were collected on day 3 after infection to determine the histopathological changes and the viral loads ( n = 5 mice each group). ( B and C ) Representative images of histopathological changes ( B ) and pathological score ( C ) in the lung tissues in each group. Scale bars represent 100 μm in B . ( D ) The levels of gRNA in mouse lung tissues on day 3 after infection in each group were detected by RT-qPCR. 1-way ANOVA followed by Tukey’s multiple comparison post hoc test was conducted in C and D . Data are presented as mean values ± SEM in C and D . **** P < 0.0001.

Article Snippet: For protein detection, the following antibodies were used: rabbit anti-SARS-CoV-2 S1 polyclonal antibodies (Sino Biological, 40591-T62), rabbit anti-SARS-CoV-2 S2 polyclonal antibodies (Sino Biological, 40590-T62), rabbit anti-SARS-CoV-2 N polyclonal antibodies (Sino Biological, 40143-R001), and horseradish peroxidase–conjugated (HRP-conjugated) anti-rabbit polyclonal antibodies (Thermo Fisher Scientific, 31460).

Techniques: Incubation, Transgenic Assay, Infection, Quantitative RT-PCR, Comparison

Journal: The Journal of Clinical Investigation

Article Title: SARS-CoV-2 Delta and Omicron variants resist spike cleavage by human airway trypsin-like protease

doi: 10.1172/JCI174304

Figure Lengend Snippet: ( A ) Coomassie staining analysis of spike protein cleavage by HAT. Spike protein alone was used as control. Numbers represent normalized band intensities. ( B and C ) Western blot assessed spike protein cleavage by HAT using anti-S1( B ) and anti-S2 ( C ) antibodies. ( D ) Live viruses produced in Vero E6 cells, preincubated with HAT (2 μg/mL) with or without aprotinin, then assayed by Western blot using S1 (top) and nucleocapsid (bottom) antibodies. ( E ) The calculated spike cleavage rate in D . ( F ) A molecular model of HAT interacting with SARS-CoV-2 S proteins S1/S2 cleavage site. Proteins are shown in ribbon format, with HAT in cyan and the cleavage site in yellow. Important residues, including the catalytic triad H227, D272, and S368 and salt bridge R682–D362, are shown in stick form. ( G ) RMSD time evolution of SARS-CoV-2 S proteins S1/S2 cleavage site. ( H ) Time evolution of HAT-S1/S2 cleavage site contact area for SARS-CoV-2 S proteins. ( I and J ) Cleavage products of spike protein for mass spectrometry: in-gel collection, enzymatic digestion, and analysis. HAT cleaves spike protein at R682 site. ( K ) Diagram of cleavage and surrounding mutation sites in SARS-CoV-2 variants. ( L ) Infectivity of Mut-1 (R681P in Delta), Mut-2 (K679N in BA.1), and Mut-3 (H681P in BA.1) pseudoviruses preincubated with HAT (2 μg/mL) ( n = 3). ( M ) Infectivity of WT pseudovirus carrying P681R mutation pretreated with or without HAT (0.5–2 μg/mL) ( n = 3). ( N ) Time evolution of the RMSD of the S1/S2 cleavage site for BA.1 (N679K) or (P681H) mutation. ( O ) Time evolution of contact interface area between HAT and S1/S2 site for BA.1 (N679K) or (P681H) mutation. 2-way ANOVA followed by Šidák’s multiple comparisons test was conducted in L and M . Data are presented as mean values ± SEM. ** P < 0.01; **** P < 0.0001.

Article Snippet: For protein detection, the following antibodies were used: rabbit anti-SARS-CoV-2 S1 polyclonal antibodies (Sino Biological, 40591-T62), rabbit anti-SARS-CoV-2 S2 polyclonal antibodies (Sino Biological, 40590-T62), rabbit anti-SARS-CoV-2 N polyclonal antibodies (Sino Biological, 40143-R001), and horseradish peroxidase–conjugated (HRP-conjugated) anti-rabbit polyclonal antibodies (Thermo Fisher Scientific, 31460).

Techniques: Staining, Control, Western Blot, Produced, Mass Spectrometry, Mutagenesis, Infection