Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: Characterization of an AAV6.2FF-hACE2 transduction model for SARS-CoV-2 infection of wild-type mice (A) Diagram of AAV genome expressing hACE2 from the CASI promoter. (B) Western blot of HEK293 cells transduced with AAV6.2FF-hACE and probed with an anti-hACE2 antibody. (C) BALB/c mice were administered 1 x 10 11 vg of AAV-Luc intranasally and imaged 10 days later using an IVIS imager. (D–F) (D) IFA images of lungs harvested from BALB/c mice infected intranasally with 1 x 10 11 vg of AAV-hACE2 or AAV-Luc and euthanized 10 days later. Lungs were stained with a rabbit anit-hACE2 antibody and imaged at 20 X (scale bar, 50mM). Viral RNA (E), and virus TCID50 titers (F) were determined in respiratory tissues on days 2 and 4 post-infection. n = 6 (3M, 3F). Statistical significance determined by Mann-Whitney test. ∗ = p < 0.05, ∗∗ = p < 0.01.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Transduction, Infection, Expressing, Western Blot, Staining, MANN-WHITNEY

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: SARS-CoV-2 spike DNA antigens protect from viral replication in vivo (A) Mice were immunized once or twice separated by four weeks with 10ug of pS via electroporation. Serum was collected at day 18 post-final immunization. At 35 days post-final immunization mice were infected intranasally with adeno-associated virus expressing human ACE2 (white). 17 days following AAV6-ACE2 transduction, animals were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 VIDO-01 P2. Four days post infection, animals were sacrificed to quantify viral replication. SARS-CoV-2 specific serum IgG endpoint titers (B) and pseudoviral neutralization titers (C) at day 18 post-final immunization. Replication competent virus (D), and viral RNA (E) in the lungs four-days post-infection. Pearson correlations between virus titer and serum IgG endpoints (F) and neutralization titers (G). Pearson correlations between viral RNA copies and serum IgG endpoints (H) and neutralization titers (I). Each point represents the average of duplicate samples from an individual animal, bars represent the mean, lines represent the median, and error bars represent the SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by student's t-test (A and B), or Kruskall-Wallis ANOVA (D and E). Spearman correlations were used to determine relationships (F-I). Data are representative of one experiment with n = 5 males (squares) and 5 females (circles) per group.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: In Vivo, Electroporation, Infection, Expressing, Transduction, Neutralization

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet:

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunospot, Software

Journal: bioRxiv

Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

doi: 10.1101/2020.10.15.340604

Figure Lengend Snippet: ( A ) Generation of pseudotyped lentiviral vectors. Second generation LVs pseudotyped with S protein were generated by transfection of HEK-293T cells with a packaging plasmid encoding HIV-1 gag/pol, a transfer vector plasmid with a lacZ reporter gene and one of two envelope plasmids encoding codon-optimized SARS-CoV-2 S with or without (SΔ19) the 19 C-terminal amino acids. The C-terminal endoplasmic reticulum retention signal (purple) and the receptor binding domain (RBD, orange) are indicated. ( B ) Incorporation of S protein into LVs determined by Western blotting. S-LV and SΔ19-LV particles (V) and lysates of their producer cells (C) were stained for the presence of S protein (top) and p24 as particle loading control. Top blot was exposed for 30 s, bottom blot for 5 s. ( C ) Gene transfer activities on the indicated cell lines. The indicated dilutions of 5 μl vector stock of SΔ19-LV or VSV-LV were added to the cells. Cell lysates were prepared three days after vector addition and lacZ reporter activity was quantified as a luminescence readout. Symbols represent means of technical triplicates. Grey shaded area indicates 95% CI of signals from untransduced cells (blanks). ( D ) Effect of ACE2-overexpression on reporter transfer. 293T cells transfected with ACE2 expression plasmid or mock plasmid were incubated with 0.2 μL of SΔ19-LV or VSV-LV. Cell lysates were prepared three days after vector addition and reporter activity was quantified as a luminescence readout. Bars represent geometric means of technical triplicates ±95% CIs.

Article Snippet: The expression plasmid for N-terminally myc-tagged hACE2 (pCMV3-SP-Myc-ACE2) was purchased from Sino Biological (HG10108-NM).

Techniques: Generated, Transfection, Plasmid Preparation, Binding Assay, Western Blot, Staining, Activity Assay, Over Expression, Expressing, Incubation

Journal: bioRxiv

Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

doi: 10.1101/2020.10.15.340604

Figure Lengend Snippet: ( A ) Principle of quantifying cell-cell fusion. Effector cells expressing S protein and the α-fragment contain active β-galactosidase when they fuse with target cells expressing ACE2 and the ω-fragment. ( B-C ) Effector cells transfected with different amounts of S-protein expression plasmid (7500-7.5 ng/T75 flask) were assessed for S protein expression by flow cytometry ( B ) and then applied in the fusion assay ( C ). ( B ) Mean fluorescence intensity (MFI, orange bars) and the percentage of S-positive cells (yellow bars) were determined in flow cytometry. Bars represent means ± 95% CIs, n=3. P-values are from one-way ANOVA. ( C ) Activities of β-galactosidase in cocultures of effector and target cells in absence of ACE2 overexpression. Effector cells were transfected with the indicated amounts of S protein encoding plasmid, detached with trypsin and cultivated for the indicated time periods with target cells which were transfected with the ω-fragment encoding plasmid only. Bars represent means ± 95% CIs, n=3. ( D ) Influence of proteolytic processing and ACE2 overexpression on cell fusion activity. Left panel: Effector cells were detached without trypsin using 5 mM EDTA in PBS. Right panel: Target cells were co-transfected with the ω-fragment and ACE2 encoding plasmids. Bars represent means ± 95% CIs, n=3.

Article Snippet: The expression plasmid for N-terminally myc-tagged hACE2 (pCMV3-SP-Myc-ACE2) was purchased from Sino Biological (HG10108-NM).

Techniques: Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Single Vesicle Fusion Assay, Fluorescence, Over Expression, Activity Assay

Journal: bioRxiv

Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

doi: 10.1101/2020.10.15.340604

Figure Lengend Snippet: The neutralizing activities of an S-protein specific antibody and sera from two convalescent Covid-19 patients were determined against S-protein mediated particle entry ( A ), cell-cell fusion ( B ) and FFWO ( C-D ). Bars represent means, n=3. P-values are from two-way ( A, C ) or one-way ( B ) ANOVA. ( A ) 0.2 μL of SΔ19-LV or VSV-LV were incubated for 30 min with the indicated antibodies or sera before being added to HEK-293T cells transfected with ACE2 expression plasmid. Cell lysates were prepared 3 days after vector addition and reporter activity was quantified as luminescence readout. ( B ) Effector cells coexpressing S protein (7.5 ng S plasmid per T75) and the α-fragment were incubated for 30 min with antibodies or sera before being added to target cells cotransfected with ω-fragment and ACE2 expression plasmids Bars represent means, n=3. ( C ) 5×10 8 SΔ19-VLPs or bald VLPs were incubated for 30 min with antibodies or sera before being added to the cocultures of α/ACE2 and ω/ACE2 cells. Luminescence was quantified after overnight incubation. Select multiplicity-adjusted p-values are reported. ( D ) Neutralizing capacity of the indicated dilutions of antibodies and sera on FFWO. Dilution factors apply to pure serum 80 μg/mL for the antibodies. ( E ) Semi-quantitative Western blots comparing S protein dose applied to the three fusion assays as SΔ19-VLPs, SΔ19-LV particles and effector cells. 2.5×10 5 effector cells transfected with 750 - 7.5 ng/T75 of S protein encoding plasmid were compared with three-fold dilutions of the particles starting with 2.5×10 9 VLPs or 3 μL of LVs. Samples corresponding to the material used in the neutralization experiments are marked with arrowheads.

Article Snippet: The expression plasmid for N-terminally myc-tagged hACE2 (pCMV3-SP-Myc-ACE2) was purchased from Sino Biological (HG10108-NM).

Techniques: Incubation, Transfection, Expressing, Plasmid Preparation, Activity Assay, Western Blot, Neutralization

Journal: bioRxiv

Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

doi: 10.1101/2020.10.15.340604

Figure Lengend Snippet: ( A ) Principle of the assay. HIV-1 derived SΔ19-VLPs were added as effector triggering fusion from without (FFWO) of cocultures of ACE2 overexpressing HEK-293T cells transfected with plasmids encoding the α-fragment or the ω-fragment of β-galactosidase. Cocultures were lysed and galactosidase activity of the lysates determined in luminescence reactions. ( B ) The indicated numbers of SΔ19-VLPs or bald VLP, produced without the S protein, were added to the coculture. Activities were quantified after overnight incubation. Bars represent means ± 95% CIs, n=4. ( C-D ) Effect of proteolytic processing on the induction of FFWO. 5×10 8 SΔ19-VLPs, bald VLPs or no VLPs were incubated in 2 mg/mL trypsin for the indicated time periods before addition to cocultures (10 5 cells). Luminescence was quantified after overnight cultivation. ( C ). Bars represent means of technical triplicates ± 95% CIs. ( D ) Processing of the S protein after trypsin treatment of the VLPs for indicated times by Western blotting for S and p24 proteins.

Article Snippet: The expression plasmid for N-terminally myc-tagged hACE2 (pCMV3-SP-Myc-ACE2) was purchased from Sino Biological (HG10108-NM).

Techniques: Derivative Assay, Transfection, Activity Assay, Produced, Incubation, Western Blot

Journal: Journal of Virology

Article Title: Porcine Deltacoronavirus Engages the Transmissible Gastroenteritis Virus Functional Receptor Porcine Aminopeptidase N for Infectious Cellular Entry

doi: 10.1128/JVI.00318-18

Figure Lengend Snippet: Flow cytometry analysis of cell surface expression of pAPN. (A) Soluble pAPN-mFc (10 μg/ml) preincubation was able to block TGEV-S1-hFc and PDCoV-S1-hFc binding to permissive LLC-PK1 and ST cells (gray-filled histogram), whereas preincubation with soluble mFc (10 μg/ml) did not block TGEV-S1-hFc or PDCoV-S1-hFc binding (black-filled histogram). Preincubation with pAPN-mFc followed by hFc binding was used as the control (dashed line). Cell surface binding was detected by an FITC-conjugated anti-human IgG Fc. (B) TGEV-S1-hFc and PDCoV-S1-hFc bound to BHK-21 or Vero cells stably expressing pAPN (BHK-pAPN and Vero-pAPN, respectively; red histograms) but not to BHK-21 or Vero (green histograms) or to BHK-21 or Vero cells expressing ACE2 cells (blue histograms). Soluble pAPN-mFc (10 μg/ml) preincubation was able to block TGEV-S1-hFc and PDCoV-S1-hFc binding to BHK-pAPN and Vero-pAPN cells (yellow histogram). Cell surface binding was detected as described in the legend of Fig. 1A and ​andBB.

Article Snippet: The expression plasmid harboring the full-length cDNA of the SARS-CoV cellular receptor ACE2, pCMV3-Flag-ACE2 (HG10108-NF), was purchased from Sino Biological, Inc. (Beijing, China).

Techniques: Flow Cytometry, Expressing, Blocking Assay, Binding Assay, Stable Transfection

Journal: Experimental & Molecular Medicine

Article Title: Generation of a lethal mouse model expressing human ACE2 and TMPRSS2 for SARS-CoV-2 infection and pathogenesis

doi: 10.1038/s12276-024-01197-z

Figure Lengend Snippet: a An experimental graphic created with BioRender.com. A549 cells or C57BL/6 mice were transfected with plasmid DNA as indicated to overexpress hACE2 and hTMPRSS2 in vitro and in vivo, respectively. b Plasmids expressing hACE2-FLAG (2 μg) or hTMPRSS2-HA (0.3 μg) were transfected into A549 cells on a 6-well plate. Overexpressed proteins at 24 h post transfection were analyzed using western blotting. GAPDH served as a loading control. c At 24 h post-transfection of plasmids, the cells were incubated with SARS-CoV-2-PV (1.3 × 10 5 TCID50/ml). After 24 h, the infectivity of the pseudovirus (PV) was assessed by measuring relative luminescence intensity. d At 2 days post-transfection, the cells were infected with SARS-CoV-2, followed by a further 2 days of incubation. The intracellular RNA was extracted and used to detect viral RNA of the SARS-CoV-2 nucleocapsid protein (NP) via RT-qPCR. e – g In vivo-transfected mice were intranasally infected with SARS-CoV-2 (5 × 10 5 PFU). Expression levels of hACE2 ( e ), hTMPRSS2 ( f ), and viral load ( g ) in the lungs were measured at the indicated days post-infection using RT-qPCR. Symbols represent the means ± SEM. Statistically significant differences between the groups were determined using one-way ANOVA ( c , d ) or unpaired two-tailed t -tests ( g ).

Article Snippet: Cells (2 × 10 5 cells per well) were plated into 6-well plates and transfected with 2 μg pCMV3-hACE2-FLAG (NM_021804.1, HG10108-CF; Sino Biological, Beijing, China) and/or 0.3 μg pCMV3-hTMPRSS2-HA (NM_005656.3, HG13070-CY; Sino Biological) using a TransIT-LT1 Transfection Reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer’s recommendations.

Techniques: Transfection, Plasmid Preparation, In Vitro, In Vivo, Expressing, Western Blot, Incubation, Infection, Quantitative RT-PCR, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Generation of a lethal mouse model expressing human ACE2 and TMPRSS2 for SARS-CoV-2 infection and pathogenesis

doi: 10.1038/s12276-024-01197-z

Figure Lengend Snippet: a Experimental graphic for generating the double-Tg mice. Expression levels of hACE2 ( b ) and hTMPRSS2 ( c ) mRNA in the lungs, brain, heart, liver, kidney, and colon of the double-Tg or wild-type C57BL/6 mice (non-Tg) were assessed via RT-qPCR. Symbols represent the means ± SEM.

Article Snippet: Cells (2 × 10 5 cells per well) were plated into 6-well plates and transfected with 2 μg pCMV3-hACE2-FLAG (NM_021804.1, HG10108-CF; Sino Biological, Beijing, China) and/or 0.3 μg pCMV3-hTMPRSS2-HA (NM_005656.3, HG13070-CY; Sino Biological) using a TransIT-LT1 Transfection Reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer’s recommendations.

Techniques: Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: (A) Infectivity quantification for HIV-1 lentivirus particles carrying various spike variants determined on hACE2 expressing 293T cells (293T-ACE2). Infectivity (mean ± s.d.) was measured from three independent experiments in triplicates. RLU, relative light units. (B) Example fluorescence trace (LD555, green; LD655, red) and resulting quantified FRET traces (FRET efficiency, blue; hidden Markov model initialization, red) of a dually labeled ligand-free spike protein on the surface of HIV-1 lentivirus particle. The single-step photobleaching step of dyes at the single-molecule level define the baseline (dashed black). Four distinguishable FRET-populated states are indicated as color-coded dash lines. ( C, D ) FRET histograms (left) and TDPs (right) of ligand-free D614 spike (S D614 , C ) and G614 spike (S G614 , D ) on lentivirus particles. A number ( N m ) of individual active/dynamic molecules - FRET traces were compiled into a conformation-population FRET histogram (gray lines) and fitted into a 4-state Gaussian distribution (solid black) centered at 0.1-FRET (dashed cyan), 0.3-FRET (dashed red), 0.5-FRET (dashed green), and 0.75-FRET (dashed magenta). TDPs show the distributions of initial and final FRET values for every observed transition in FRET traces. TDPs are displayed as initial FRET vs. final FRET with relative frequencies (max red scale = 0.01 transitions/second), originated from the idealization of individual FRET traces in FRET histograms. TDPs trace the locations of state-to-state transitions and their relative frequencies. ( E, F ) Experiments as in (C, D), respectively, conducted in the presence of 200 μg/mL monomeric hACE2. The soluble hACE2 activates spike proteins on the virus by shaping the conformational landscape toward stabilizing the “all-RBD-up” conformation (activated state). FRET histograms represent mean ± SEM, determined from three randomly assigned populations of FRET traces under corresponding experimental conditions. N m , number of individual FRET traces. Evaluated relative state occupancies see .

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques: Infection, Expressing, Fluorescence, Labeling, Virus

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: ( A, B ) FRET histograms (left) and TDPs (right) of S Alpha variant (S Alpha ) on lentivirus particles with ( A ) and without ( B ) 200 μg/mL hACE2 presence. The soluble hACE2 activates the S Alpha by shaping the conformational landscape toward stabilizing the “all-RBD-up” conformation. (C) Representative fluorescence traces (LD555, green; LD655, red) and quantified FRET traces of a single ligand-free E484K carrying S Alpha variant (S Alpha+E484K ) on lentivirus particles. (D) The FRET histogram (left) and TDP (right) of ligand-free S Alpha+E484K on lentivirus particles. ( E, F ) Experiments as in (C, D), conducted in the presence of 200 μg/mL hACE2. FRET histograms represent mean ± SEM, determined from three randomly assigned populations of all FRET traces under corresponding experimental conditions. N m , number of individual FRET traces. ( G ) Quantification of the FRET-indicated state occupancy for different spike variants. The occupancy in each FRET state was presented as mean ± SEM, determined by estimating the area under each Gaussian curve in FRET histograms. Fitting parameters see .

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques: Variant Assay, Fluorescence

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: ( A, B ) Example fluorescence traces (LD555, green; LD655, red) and resulting quantified low-FRET dominated traces (FRET efficiency, blue; hidden Markov model initialization, red) of lentivirus-embedded spikes variants G614 ( A ) and Alpha ( B ) in response to hACE2.

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: ( A, B ) Survival probability plots of four FRET-indicated conformations of lentivirus particles-associated spike proteins S D614 ( A ) and S G614 ( B ) in the absence (ligand-free, top row) and the presence of 200 μg/ml hACE2 (bottom row). The survival probability plot is the time-duration distribution of spikes dwelling on one conformation before directionally transition to the other. Transition rates (summarized in ) were estimated by double exponential-fitting of survival probability plots.

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques:

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: ( A - D ) Free-energy models of parental S D614 ( A ) and variants S G614 ( B ), S Alpha ( C ), and S Alpha+E484K ( D ). The differences in free energies between states were roughly scaled based upon the relative state occupancies of each state. The ligand-free D614G and E484K carrying spikes are dominated by the intermediate-FRET state (“one/two-RBD-up”), which exhibit the lowest relative free energy among all four FRET states. In contrast, all hACE2-bound spikes show the lowest relative free energy at the low-FRET state (“all-RBD-up”).

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques: