plenti ace2 vector  (New England Biolabs)

Journal: Nature Communications

Article Title: Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2

doi: 10.1038/s41467-021-24013-y

Figure Lengend Snippet: a ACE2 mutant library was expressed in 293T cells and incubated with the RBD of SARS-CoV-2 fused to superfolder GFP (sfGFP). b 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. Cell sorting was conducted by gating on forward scatter (FSC)-H and FSC-A to exclude doublets, followed by gating on Alexa 647 for HA-ACE2 expression and sfGFP for RBD-binding. c The value of K D and IC 50 against pseudovirus of SARS-CoV-2 and SARS-CoV-1. 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 BamHI or MfeI-SacII (NEB) for ACE2 residues 18–102 or 272–409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 h and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Mutagenesis, Incubation, Amplification, DNA Extraction, FACS, Expressing, Binding Assay, Neutralization

Journal: Nature Communications

Article Title: Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2

doi: 10.1038/s41467-021-24013-y

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. d Comparison of the distances between Cβ atoms of S128 and V343 residues in the closed and open conformations. e Stabilizing effect of S138C/V343C mutation. Various versions of ACE2-His proteins, with (solid lines) or without (dotted lines) the S138C/V343C disulfide mutation (SS), were subjected to the differential scanning fluorimetry using SYPRO™ Orange as the probe dye. The Tm value for each mutant is estimated by the peak temperature of the -dF/dT plot, and the Tm shift caused by the SS mutation is shown at the bottom. The experiments were independently performed three times and similar results were obtained. One representative data were shown.

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

Techniques: Mutagenesis, Binding Assay

Journal: Nature Communications

Article Title: Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2

doi: 10.1038/s41467-021-24013-y

Figure Lengend Snippet: Data collection and refinement statistics.

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

Techniques:

Journal: Nature Communications

Article Title: Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2

doi: 10.1038/s41467-021-24013-y

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. c Viruses at passage 15 were neutralized similarly with the passaged control in ACE2-Fcs.

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

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

Journal: Nature Communications

Article Title: Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2

doi: 10.1038/s41467-021-24013-y

Figure Lengend Snippet: a ACE2-Fcs and monoclonal antibody, REGN10933 neutralized pseudotyped variants recently emerged in the United Kingdom (B.1.1.7), South Africa (B.1.351), and Brazil (P.1) in 293T/ACE2 cells. n = 4 technical replicates. b Neutralization potency of 3N39v2-Fc and monoclonal antibody, REGN10933 against UK-B.1.1.7 and BR-P.1 was analyzed in Vero6E/TMPRSS2 cells. Data are mean ± SEM of n = 3 technical replicates.

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

Techniques: Neutralization

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

Journal: Cell Reports

Article Title: An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

doi: 10.1016/j.celrep.2020.108528

Figure Lengend Snippet: Δ19 SARS-CoV-2 Pseudotyped Lentiviral Virion Infection of ACE2.293T Cells and ACE2 Expressing Cell Lines (A) The domain structure of the SARS-CoV-2 spike protein is diagrammed above. Yellow shading indicates the amino acids of the cytoplasmic domain retained following truncation of the 19 carboxy-terminal amino acids (Δ19). Vectors encoding full-length (fl) codon-optimized SARS-CoV-2 spike protein or truncated Δ19 spike protein, with or without a carboxy-terminal HA-tag and the dual nanoluciferase/GFP lentiviral vector pLenti.NLuc.GFP used to generate pseudotyped lentiviral particles, are diagrammed (below). (B) SARS-CoV-2 spike proteins on pseudotyped lentiviral virions were analyzed. Transfected producer cell lysates (left) and supernatant virions (right) were analyzed on an immunoblot probed with anti-HA antibody. Cell lysate blots were probed with anti-GAPDH to normalize protein loading, and virion blots were probed for HIV-1 p24 to normalize for virions. (C) 293T cells transfected with SARS-CoV-2 spike protein expression vectors were analyzed by flow cytometry to detect the protein at the plasma membrane. (D) HA-tagged ACE2 expressed in control transfected 293T and clonal ACE2.293T cells were analyzed on an immunoblot probed with anti-HA antibody. (E) ACE2.293T cells were infected with virus pseudotyped by full-length or SARS-CoV-2 Δ19 spike proteins. Two days post-infection, infectivity was measured by luciferase assay. The reverse transcriptase inhibitor nevirapine was added to one sample to control for free luciferase enzyme contamination of the virus stock. (F) A panel of cell lines were infected with VSV-G, SARS-CoV-2 Δ19 spike protein, or no envelope (no Env) pseudotyped lentivirus (MOI = 0.2). Luciferase activity was measured 2 days post-infection. (G) ACE2.293T cells were treated with ACE2 antibody (1:20) for 30 min at room temperature, and SARS-CoV-2 Δ19 virus was added on the cells. After 2 days of incubation, luciferase activity was measured. The data are represented as the mean of triplicates ± standard deviation. Statistical significance was calculated with the Student’s t test. The experiment was done twice with similar results. IC, intracellular domain; LTR, long terminal repeat; NTD, N-terminal domain; RBD, receptor binding domain; RRE, Rev response element; TM, transmembrane domain.

Article Snippet: ACE2 expressing lentiviral vector pLenti.ACE2 was generated by amplifying an ACE2 cDNA (Origene) with a forward primer containing an Xba-I site and reverse primer containing a Sal-I site.

Techniques: Infection, Expressing, Plasmid Preparation, Transfection, Western Blot, Flow Cytometry, Luciferase, Activity Assay, Incubation, Standard Deviation, Binding Assay

Journal: Cell Reports

Article Title: An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

doi: 10.1016/j.celrep.2020.108528

Figure Lengend Snippet: Wild-Type and H345A ACE2 Microbody Proteins Are Disulfide-Bonded Dimers (A) The domains of ACE2 are shown with the structures of the soluble ACE2 (sACE2), ACE2 microbody, and ACE2.H345A microbody proteins below. The soluble ACE2 proteins are deleted for the transmembrane (TM) and cytoplasmic domains. The ACE2 microbody proteins are fused to the human IgG CH3 domain each with a carboxy-terminal 8× His-tag. (B) The diagram above shows the predicted dimeric structure of the ACE2 microbody protein. The 3D structure of the ACE2:spike protein complex was generated using Chimera software ( Pettersen et al., 2004 ) using published coordinates ( Lan et al., 2020 ). The position of the conserved active site H345 in the ACE2 carboxypeptidase domain is shown lying underneath the ACE2 interaction site. (C) 293T cells were transfected with sACE2, ACE2 microbody, and ACE2.H345A microbody expression vectors. The proteins were pulled down on NTA agarose beads and analyzed under reducing and nonreducing conditions on an immunoblot probed with anti-His-tag antibody. (D) The soluble ACE2 proteins were purified by metal chelate chromatography and size exclusion chromatography (SEC). The oligomerization state was determined by SEC multi-angle light scattering. The calculated molecular mass of each is shown. The experiment was done twice with similar results.

Article Snippet: ACE2 expressing lentiviral vector pLenti.ACE2 was generated by amplifying an ACE2 cDNA (Origene) with a forward primer containing an Xba-I site and reverse primer containing a Sal-I site.

Techniques: Generated, Software, Transfection, Expressing, Western Blot, Purification, Chromatography, Size-exclusion Chromatography

Journal: Cell Reports

Article Title: An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

doi: 10.1016/j.celrep.2020.108528

Figure Lengend Snippet: ACE2 Microbody Proteins Bind with High Affinity to SARS-CoV-2 S Pseudotyped Virions Nickel agarose beads were coated for 1 h with 10 μg of soluble ACE2 proteins. Unbound protein was removed, and SARS-CoV-2 Δ19 S pseudotyped virions or virions lacking spike protein were incubated with the beads. After 1 h, unbound virions were removed, and the bound virions were analyzed on an immunoblot probed with anti-p24 antibody. (A) Input virions and bead-bound virions were analyzed on an immunoblot probed with anti-p24 antibody. (B) Soluble ACE2 proteins bound to the nickel agarose beads were analyzed on an immunoblot probed with anti-His-tag antibody. (C) Soluble ACE2, wild-type ACE2 microbody, and ACE2.H345A microbody proteins were serially diluted and bound to nickel agarose beads. The amount of bound virions was determined by immunoblot analysis with anti-p24 antibody. Quantification of the band intensities from the immunoblot is graphed below for soluble ACE2 (sACE2), wild-type ACE2 microbody (ACE2-mb), and ACE2.H345A microbody (H345A-mb). The experiment was done twice with similar results.

Article Snippet: ACE2 expressing lentiviral vector pLenti.ACE2 was generated by amplifying an ACE2 cDNA (Origene) with a forward primer containing an Xba-I site and reverse primer containing a Sal-I site.

Techniques: Incubation, Western Blot

Journal: Cell Reports

Article Title: An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

doi: 10.1016/j.celrep.2020.108528

Figure Lengend Snippet: ACE2 and ACE2.H345A Microbodies Potently Block Virus Entry and Are Active on Different Cell Lines (A) Serially diluted soluble ACE2, ACE2, and ACE2.H345A microbody proteins were incubated for 30 min with SARS-CoV-2 Δ19.S pseudotyped virus and then added to ACE2.293T cells. Luciferase activity was measured 2 days post-infection. (B) The number of cells infected was determined by flow cytometry to quantify the GFP + cells. The data are displayed as the percent of GFP + cells. Representative fluorescence microscopy images of the infected cells are shown below. Scale bar, 50 μm. (C) VSV-G pseudotyped lentiviral virions were incubated for 30 min with 10 μg/mL soluble ACE2 proteins and then added to ACE2.293T cells. Luciferase activity was measured 2 days post-infection. (D) Δ19 spike protein pseudotyped virus was incubated with serially diluted soluble ACE2 proteins for 30 min and then added 293T cells. (E) Serially diluted soluble ACE2 proteins were incubated with mNeonGreen SARS-CoV-2 for 30 min and then added to ACE2.293T cells. After 24 h, the GFP + cells were counted. Fluorescent microscopy images of representative fields from wells treated with 1 μg soluble ACE2, and ACE2 microbody proteins are shown below. Scale bar, 2.1 mm. The data are displayed as the mean ± SD, and significance is determined by Student’s t tests. The experiments in (A)–(D) were repeated four times, and the experiment in (E) was done twice.

Article Snippet: ACE2 expressing lentiviral vector pLenti.ACE2 was generated by amplifying an ACE2 cDNA (Origene) with a forward primer containing an Xba-I site and reverse primer containing a Sal-I site.

Techniques: Blocking Assay, Incubation, Luciferase, Activity Assay, Infection, Flow Cytometry, Fluorescence, Microscopy

Journal: Cell Reports

Article Title: An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

doi: 10.1016/j.celrep.2020.108528

Figure Lengend Snippet: ACE2 Microbody Can Act after Virus/Cell Binding The kinetics of ACE2 microbody inhibition were analyzed in an escape from inhibition assay. (A) The experimental scheme is diagrammed above. SARS-CoV-2 Δ19.S pseudotyped virus was added to ACE2.293T cells. Soluble ACE2 proteins were added immediately or at time points up to 6 h later. Luciferase activity was measured 2 days post-infection. (B) As diagrammed above, virus was bound to target cells for 2 h at 22°C, and unbound virus was then removed. Soluble ACE2 proteins were added as in (A). The data are displayed as the mean ± SD, and statistical significance was determined by Student’s t tests. The experiment was done twice with similar results.

Article Snippet: ACE2 expressing lentiviral vector pLenti.ACE2 was generated by amplifying an ACE2 cDNA (Origene) with a forward primer containing an Xba-I site and reverse primer containing a Sal-I site.

Techniques: Binding Assay, Inhibition, Luciferase, Activity Assay, Infection

Journal: Cell Reports

Article Title: An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

doi: 10.1016/j.celrep.2020.108528

Figure Lengend Snippet: ACE2 Microbody Blocks Entry of the D614G Variant Spike Protein Pseudotyped Virus Infection and ACE2 Using β Coronavirus Spike Proteins (A) The domain structure of the SARS-CoV-2 D614G Δ19 S expression vector is diagrammed above. Red star indicates the D614G mutation in the spike protein. (B) A panel of cell lines was infected with equivalent amounts (MOI = 0.2) of wild-type and D614G Δ19 spike protein pseudotyped virus. (C) Serially diluted soluble ACE2 and ACE2 microbody proteins were mixed with D614G Δ19 spike protein pseudotyped virus and added to target cells. Luciferase activity was measured 2 days post-infection. The data are shown as the mean of triplicates ± SD. The statistical significance of the data was calculated with the Student’s t test. (D) Ni-NTA agarose beads were coated with serially diluted soluble ACE2 and ACE2 microbody proteins. Wild-type, Δ19 spike protein, and no Env pseudotyped virions (28.2 ng p24) were added and allowed to bind. Unbound virions were removed after 30 min, and the bound virions were detected by immunoblot analysis with anti-p24 antibody. The amount (ng) of bead-bound virus p24 was calculated based on band intensities from the immunoblot and indicated in the bottom side. (E) Lentiviral virions pseudotyped by β coronavirus lineage 2 spike proteins were treated with serially diluted soluble ACE2 proteins and then used to infect ACE2.293T cells. The identity of the virus from which the spike protein RBD is derived is indicated above each histogram. Luciferase activity was measured after 2 days. The data are displayed as the mean ± SD, and significance is determined by Student’s t tests. The experiments in (B) and (C) were done three times, and those in (D) and (E) were done twice.

Article Snippet: ACE2 expressing lentiviral vector pLenti.ACE2 was generated by amplifying an ACE2 cDNA (Origene) with a forward primer containing an Xba-I site and reverse primer containing a Sal-I site.

Techniques: Variant Assay, Infection, Expressing, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Western Blot, Derivative Assay

Journal: Cell Reports

Article Title: An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

doi: 10.1016/j.celrep.2020.108528

Figure Lengend Snippet: ACE2.H345A Microbody Protects K18-hACE2 Transgenic Mice from SARS-CoV-2 Infection (A and B) ACE2.H345A microbody (7.5 μg) or control buffer was incubated for 30 min at room temperature with viral inoculum (1 × 10 3 plaque-forming units [PFUs] SARS-CoV-2). The virus was administered intranasally to K18h-ACE2 littermates (control: n = 9; microbody: n = 10), and the mice were then monitored for weight loss (A) and survival (B). Data are pooled from two independent experiments. Weight loss was analyzed by a mixed-effects model. Survival was analyzed by a log rank (Mantel-Cox) test. ∗∗∗∗ p < 0.0001.

Article Snippet: ACE2 expressing lentiviral vector pLenti.ACE2 was generated by amplifying an ACE2 cDNA (Origene) with a forward primer containing an Xba-I site and reverse primer containing a Sal-I site.

Techniques: Transgenic Assay, Infection, Incubation

Journal: Cell Reports

Article Title: An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

doi: 10.1016/j.celrep.2020.108528

Figure Lengend Snippet:

Article Snippet: ACE2 expressing lentiviral vector pLenti.ACE2 was generated by amplifying an ACE2 cDNA (Origene) with a forward primer containing an Xba-I site and reverse primer containing a Sal-I site.

Techniques: Recombinant, Transfection, Luciferase, Software