Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Full length ACE2 was optimized to fit screening. Synthetic signal sequence and HA tag were fused to mature ACE2 and restriction sites were introduced by optimizing codon optimization for the mutated fragment replacement. ( B ) ACE2 mutant library was expressed in 293T cells and incubated with the RBD of SARS-CoV-2 fused to superfolder GFP (sfGFP). ( C ) Error-prone PCR amplification of ACE2 protease domain induced random mutations. Mutant library-transduced cells were incubated with the RBD-sfGFP. Top 0.05 % population with high level of bound RBD-sfGFP was sorted and underwent DNA extraction, followed by next cycle mutagenesis. ( D ) PD1 mutagenesis and high affinity selection were performed 3 times and followed by PD2 random mutation. Top 0.05% population were harvested and reconstructed into the backbone plasmid to be verified individually. ( E ) Neutralization activity against the RBD was evaluated with sACE2-sfGFP and the RBD-sfGFP. Serial dilution of sACE2-sfGFP was analyzed with the 50-fold diluted RBD-sfGFP in flow cytometry. The experiments were independently performed twice and similar results were obtained. One representative data were shown.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Sequencing, Mutagenesis, Incubation, Amplification, DNA Extraction, Selection, Plasmid Preparation, Neutralization, Activity Assay, Serial Dilution, Flow Cytometry

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Table of all ACE2 variants with mutations. ( B ) Kinetic analysis of sACE2-His binding to RBD-Fc was analyzed by surface plasmon resonance (SPR). ( C ) Neutralization potency of sACE2-Fc against SARS-CoV-2 or SARS-CoV-1 pseudotyped lentivirus was measured in 293T/ACE2 cells. Data are mean ± SD of n = 3 biological replicates. ( D ) Neutralization potency to authentic SARS-CoV-2 was analyzed in Vero6E/TMPRSS2 cells. Data are mean of n = 3 technical replicates.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Binding Assay, SPR Assay, Neutralization

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: An average mass of spike trimer was measured to be ∼560 kDa (top panel). All ACE2 molecules added in excess to the mixture samples gave a peak with an average mass of ∼90 kDa. The spike protein complexed with WT ACE2 showed a broad mass distribution between 550∼850 kDa (the second panel). In contrast, the spike protein complexed with the 3N39 or 3N39v2 showed a monodisperse distribution with an average mass of ∼830 kDa (the third and bottom panels), which corresponds to the mass of the spike trimer bound by three ACE2 molecules.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques:

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: Various versions of ACE2-His proteins were subjected to the differential scanning fluorimetry using SYPRO™ Orange as the probe dye. Denaturation curves were replotted for - d F/ d T and the peak temperature was estimated to be the Tm for each mutant.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: Neutralizing activity of ACE2-Fcs against soluble spike trimer binding to cell-surface ACE2 was evaluated in flow cytometry. Indicated concentration of spike trimer was incubated with 60 µg/ml (∼315 nM) ACE2-Fc proteins for 2h and then the mixture was reacted with ACE2-expressing Expi293F cells. Although WT ACE2-Fc can block binding of soluble spike protein to cells when the concentration of the spike was 0.3 nM, it could not outcompete spike at higher concentration. In contrast, complete (for 3N39v2 and J320v2) or near-complete (for J113v2) inhibition against 30 nM spike protein was achieved with the ACE2 mutants we have isolated, indicating the >100-fold increase in the blocking efficacy from the WT. The experiments were independently performed twice and similar results were obtained. One representative data were shown.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Activity Assay, Binding Assay, Flow Cytometry, Concentration Assay, Incubation, Expressing, Blocking Assay, Inhibition, Isolation

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Overall structure. 3N39 ACE2 and RBD are shown in orange and green, respectively. The mutated residues in 3N39 are shown as cyan stick models. The expanded views of the PD1 region are provided in the inset. ( B ) Structural comparison of the K31N/E35K mutation site in 3N39 (left panel) with its corresponding site in WT (right panel). Hydrogen-bonding interactions (within 3.0 Å) are indicated by dashed lines. ( C ) Structural comparison of the L79F/A25V mutation site in 3N39 (left panel) with its corresponding site in WT (right panel). F486 residue of RBD and hydrophobic residues composing the F486 binding pocket of ACE2 are shown as stick models with transparent sphere models.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Surface representations of the ACE2 structures. All ACE2 molecules are viewed from the same orientation (side view). The RBD-bound 3N39 (left) and the inhibitor-bound WT (center, 1r4l) adopt a closed conformation, while the RBD-bound WT (right, 6m0j) adopts an open conformation. ( B and C ) Superposition of the RBD-bound 3N39 (orange) and the inhibitor-bound WT (cyan, 1r4l) structures. Overall view and the expanded view of the enzymatic active site are provided in (B) and (C), respectively. The active site Zn ions (in both structures) are shown as sphere models, and a sulfate ion (in 3N39) and the inhibitor MLN-4760 (in 1r4l) are shown as stick models. ( D ) Comparison of the distances between C β atoms of S128 and V343 residues in the closed and open conformations. ( E ) SDS-PAGE analysis of WT and S128C/V343C mutant ACE2-His samples conducted under non-reducing (NR) and reducing (R) conditions. ( F ) Enzyme activity was assayed in the form of soluble ACE2-sfGFP by measuring Mca fluorescence resulting from hydrolysis of Mca-Ala-Pro-Lys(Dnp)-OH. ( G ) The RBD neutralization assay was performed in S128C/V343C mutant. The experiments were independently performed twice and similar results were obtained. One representative data were shown.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: SDS Page, Mutagenesis, Activity Assay, Fluorescence, Neutralization

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Protocol of generating escape mutation in SARS-CoV-2. At first, 0.1 MOI of SARS-CoV-2 was cultured in Vero6E/TMPRSS2 cells with indicated concentration of ACE2-Fc or H4 antibody, then a total of 3 × 10 5 copies of virus in partially neutralized well was passed in the presence of ACE2-Fc or H4 antibody dilution. Supernatants were collected from each well and analyzed virus RNA copy number by quantitative PCR. ( B ) The copy number of SARS-CoV-2 genome RNA in cultured medium was analyzed in each passage. The virus from indicated well (arrow head) was passed and escape mutant expansion was observed only in H4 antibody at passage 4. The test for 3J113v2 was discontinued due to no growth of virus at passage 2.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Mutagenesis, Cell Culture, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: Western blot of cultured medium from each ACE2-Fc transfected 293T cells. The experiments were independently performed 3 times and similar results were obtained. One representative data were shown.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Western Blot, Cell Culture, Transfection

Journal: medRxiv

Article Title: Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers and convalescent plasma donors: a cohort study using a rapid and sensitive high-throughput neutralization assay

doi: 10.1101/2020.08.02.20166819

Figure Lengend Snippet: 293T cells seeded on 6-well plates were co-transfected with 0.8 μg HIV-1-NL4.3-inGluc vector plus 0.4 μg SARS-CoV-2 spike-coding plasmid; 48 h post-transfection, viral supernatant was harvested and used to infect target cells. Unless otherwise indicated, 293T/ACE2 cells were used for infection. (A) Schematic representation of the pseudoviral production and infection. Note that Gluc activity can only be detected in virus-infected target cells, but not in the virus-producing cells because of the presence of an intron inserted in the sense of the vector that splits the Gluc gene into two parts. (B and C) Titers of HV-1 inGluc pseudotypes bearing the spikes of SARS-CoV, SARS-CoV-2 or VSV-G; absolute luciferase readouts (B, n=6), and relative infectivity compared to the background (C, n=6) were plotted, respectively. (D) Indicated doses of viral supernatant were used to infect 293T/ACE2 cells seeded in 24-well plates, and 20 μL of supernatant of virus-infected cells were used to measure the Gluc activity as shown. The dashed line indicates the background of luciferase activity. (E) Indicated amounts of culture media harvested from virus-infected cells were used to measure Gluc activity (n=3). (F) Relative infectivity of HIV-inGluc pseudotypes bearing S proteins of SARS-CoV, SARS-CoV-2 or VSV-G in indicated target cells, with parental or those overexpressing ACE2.

Article Snippet: HeLa, A549, HTX and Huh7.5 cells stably expressing ACE2 were generated by transduction of pLenti-GFP vectors expressing ACE2 (OriGene, #RC208442L4), followed by puromycin selection (1 μg/mL) for 6 days.

Techniques: Transfection, Plasmid Preparation, Infection, Activity Assay, Luciferase

Journal: medRxiv

Article Title: Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers and convalescent plasma donors: a cohort study using a rapid and sensitive high-throughput neutralization assay

doi: 10.1101/2020.08.02.20166819

Figure Lengend Snippet: 293T cells were transfected with lentiviral vector (pNL4-3 inGluc or pNL4-3 secNluc) along with the SARS-CoV-2 S-C9 plasmid. Media was 48 hrs after transfection and used to infect 293T/ACE2 cells; luciferase activity was measured at indicated times to determine the viral infectivity. (A) Stability of inGluc and secNanoLuc luciferase signals measured over time. 20 μl of Gaussia luciferase substrate or 20 μl of Nano Luciferase substrate were added simultaneously, and luminescence measurements were then read every 2 minutes for 60 min. Plotted are the luminescence reads relative to the 0 min time point, which was set to 100 percent; secNluc exhibited a signal that was more stable than the inGluc virus infected cells. ( B and C ) Infectivity of SARS-CoV-2 S secNluc pseudotypes. (B) The Nano-luciferase activity of culture medium harvested from virus-infected cells at indicated times. (C) Relative viral infectivity was plotted by setting the mock infection to 1.0. (D) Indicated amounts of viral supernatant were used to infect 293T/ACE2 cells seeded in 24-well plates, and 20 μl of supernatant from virus-infected cells was used to measure the secNuc activity as shown. The dashed line indicates the background luminescence. (E) Experiment was performed as described in the legends of and , except secNluc lentiviral pseudotypes were used for infection.

Article Snippet: HeLa, A549, HTX and Huh7.5 cells stably expressing ACE2 were generated by transduction of pLenti-GFP vectors expressing ACE2 (OriGene, #RC208442L4), followed by puromycin selection (1 μg/mL) for 6 days.

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Infection

Journal: Nucleic Acids Research

Article Title: Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex

doi: 10.1093/nar/gkad483

Figure Lengend Snippet: Production of m7GTP in Nsp14-expressing cells. ( A ) Detection of m7GTP in extracts prepared from cells cultured under the indicated conditions using CE-MS. Each analysis was performed in triplicate. Peak migration times are shown in parentheses. ( B ) 293F_Nsp14_wt2 cells were transiently transfected with the FLAG-tagged DcpS expression vector. Twenty-four hours after transfection, 2.5 μg/ml DOX was added to the culture medium, and the cells were cultured for an additional 48 h. The cells were fixed and subjected to IFA using anti-FLAG M2 mAb followed by FISH using a Cy3-labeled oligo-dT 50 probe. The cells were observed by confocal microscopy. Maximum intensity projections of a single stack (10 consecutive slices, 0.35 μm z -distance) of images are shown. In the merged picture, the fluorescent signals of FLAG-DcpS and poly(A) + RNAs were pseudocolored blue and red, respectively. ( C , D ) 293F_hACE2_DcpS_29 cells were left untreated (–) or induced by DOX for 24 h (DcpS). The cells were mock transfected (mock) or transfected with the GFP-Nsp14 expression vector (Nsp14) and cultured for an additional 48 h. (C) Whole-cell extracts were subjected to Western blotting using the indicated antibodies. (D) Total RNA was subjected to qRT-PCR analysis as in Figure . The amount of downstream RNA was normalized to that of CDS RNA. The data are presented as the means ± SDs of three biological replicates. * means P value < 0.05.

Article Snippet: Mammalian expression vectors of the SARS-CoV-2 proteins , a DOX-inducible mammalian expression vector pInducer20 , and a human ACE2 expression vector pLENTI_hACE2_HygR ( ) were obtained from Addgene (listed in ).

Techniques: Expressing, Cell Culture, Migration, Transfection, Plasmid Preparation, Labeling, Confocal Microscopy, Western Blot, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex

doi: 10.1093/nar/gkad483

Figure Lengend Snippet: Gene expression analysis of SARS-CoV-2-infected cells. ( A ) Distribution of the 9.48 × 10 9 (uninfected) and 8.49 × 10 9 (infected) mapped bases on different gene features was analyzed by CollectRNAseqMetrics from gatk4-4.1.6.0–0 and shown in percentages. CDS: coding sequence, UTR: 5′- and 3′-untranslated regions. ( B ) Intron retention ratios were calculated by IR finder. Overall changes in IR ratios are shown. The centerlines show the medians; the box limits indicate the 25th and 75th percentiles as determined by R software; the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; and the crosses show the means. Orange transparent dots are individual data points. ( C ) Changes in RNA-seq read coverage at replication-dependent (RD) histone genes before (uninfected) and after (infected) SARS-CoV-2 infection. The heatmaps range from 0.5 kb upstream of the transcription start site (TSS) to 2 kb downstream of the transcription termination site (TES) of 69 RD histone genes. ( D – F ) IGV view of the RNA-seq data at the H2BC12 , H3C10 and H4C5 loci. The CPM-normalized RNA-seq read coverage of uninfected and infected samples is shown (top two rows). As a comparison, the read coverage of control (uninduced) and Nsp14-expressing (induced) samples is also shown (bottom two rows). ( G ) Total RNA prepared from 293F_hACE2_21 cells infected with SARS-CoV-2 at the indicated multiplicity of infection (MOI) was subjected to qRT-PCR analysis as in Figure . The amount of the downstream RNA was normalized to that of the CDS RNA. Shown is a representative of two independent experiments. The data are presented as the means ± SDs of three technical replicates. ** and *** indicate P values <0.01 and <0.001, respectively. ( H ) 293F_hACE2_DcpS_29 cells were left untreated (–) or induced GFP-DcpS expression by DOX for 24 h (DcpS). The cells were infected with SARS-CoV-2 at an MOI of 1.0 and cultured for another 24 h. Left : Total RNAs prepared from each culture were subjected to qRT-PCR to analyze the read-through transcription of the H4C5 locus as in Figure . The data are presented as the means ± SDs of three biological replicates. ** and *** mean P values <0.01 and <0.001, respectively. Right : The amount of SARS-CoV-2 N mRNA normalized to that of GAPDH mRNA was also quantitated. The data are presented as the means ± SDs of three biological replicates. *** means P value <0.001. ND means none detected.

Article Snippet: Mammalian expression vectors of the SARS-CoV-2 proteins , a DOX-inducible mammalian expression vector pInducer20 , and a human ACE2 expression vector pLENTI_hACE2_HygR ( ) were obtained from Addgene (listed in ).

Techniques: Gene Expression, Infection, Sequencing, Software, RNA Sequencing, Comparison, Control, Expressing, Quantitative RT-PCR, Cell Culture

Journal: JCI insight

Article Title: Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers, and convalescent plasma donors.

doi: 10.1172/jci.insight.143213

Figure Lengend Snippet: Figure 1. inGluc-based HIV-1 lentiviral S pseudotypes bearing SARS-CoV-2 spikes. 293T cells seeded on 6-well plates were cotransfected with 0.8 μg HIV-1–NL4.3–inGluc vector plus 0.4 μg SARS-CoV-2 spike-coding plasmid. For- ty-eight hours after transfection, viral supernatant was harvested and used to infect target cells. Unless otherwise indicated, 293T/ACE2 cells were used for infection. (A) Schematic representation of the pseudoviral production and infection. Note that Gluc activity can only be detected in virus-infected target cells — and not in the virus-produc- ing cells — because of the presence of an intron inserted in the sense of the vector that splits the Gluc gene into 2 parts. (B and C) Titers of HIV-1 inGluc pseudotypes bearing the spikes of SARS-CoV (n = 6), SARS-CoV-2 (n = 6), or VSV-G (n = 3); absolute luciferase readouts at 48 hours after infection, and relative infectivity compared with the background, were plotted, respectively. (D) Indicated doses of viral supernatant were used to infect 293T/ACE2 cells seeded in 24-well plates, and 20 μL of supernatant of virus-infected cells were used to measure the Gluc activity as shown. The dashed line indicates the background of luciferase activity; n = 3. (E) Indicated amounts of culture media harvested from virus-infected cells were used to measure Gluc activity; n = 3. (F) Relative infectivity of HIV-inGluc pseudotypes bearing S proteins of SARS-CoV, SARS-CoV-2, or VSV-G in indicated target cells, with parental or those overexpressing ACE2; n = 6. Data were analyzed as mean ± SD.

Article Snippet: HeLa, A549, HTX, and Huh7.5 cells stably expressing ACE2 were generated by transduction of pLenti-GFP vectors expressing ACE2 (OriGene, RC208442L4), followed by puromycin selection (1 μg/mL) for 6 days.

Techniques: Plasmid Preparation, Transfection, Infection, Activity Assay, Virus, Luciferase

Journal: JCI insight

Article Title: Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers, and convalescent plasma donors.

doi: 10.1172/jci.insight.143213

Figure Lengend Snippet: Figure 6. A secreted Nluc–based lentiviral SARS-CoV-2 S neutralization assay with improved stability and sensitivity, and its application in measuring SARS-CoV-2 neutralizing antibody levels in COVID-19 patients. 293T cells were transfected with lentiviral vector (pNL4.3 inGluc or pNL4.3 secNluc) along with the SARS-CoV-2 S-C9 plasmid. Media was 48 hours after transfection and used to infect 293T/ACE2 cells; luciferase activity was measured at indi- cated times to determine the viral infectivity; n = 3 for all experiments. (A) Stability of inGluc and secNluc luciferase signals measured over time. A total of 20 μL of Gaussia luciferase substrate or 20 μL of Nano Luciferase substrate were added simultaneously, and luminescence measurements were then read every 2 minutes for 60 minutes. Plotted are the luminescence reads relative to the 0 minutes time point, which was set to 100%; secNluc exhibited a sig- nal that was more stable than the inGluc virus infected cells. (B and C) Infectivity of SARS-CoV-2 S secNluc pseudotypes. (B) The Nano-luciferase activity of culture medium harvested from virus-infected cells at indicated times. (C) Relative viral infectivity was plotted by setting the mock infection to 1.0. (D) Indicated amounts of viral supernatant were used to infect 293T/ACE2 cells seeded in 24-well plates, and 20 μL of supernatant from virus-infected cells was used to measure the secNluc activity as shown. The dashed line indicates the background luminescence. (E) Experiment was performed as described in the legends of Figure 3A and Figure 4B, except secNluc lentiviral pseudotypes were used for infection. Data were analyzed as mean ± SD.

Article Snippet: HeLa, A549, HTX, and Huh7.5 cells stably expressing ACE2 were generated by transduction of pLenti-GFP vectors expressing ACE2 (OriGene, RC208442L4), followed by puromycin selection (1 μg/mL) for 6 days.

Techniques: Neutralization, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Infection, Virus