Journal: Biotechnology Journal

Article Title: An Ad5‐Based COVID‐19 Vaccine Encoding SARS‐CoV‐2 Spike Glycoprotein Induces Measurable Antibody and Cytokine Responses in Mice

doi: 10.1002/biot.70216

Figure Lengend Snippet: Construction, expression, and characterization of recombinant adenoviral vectors encoding the SARS‐CoV‐2 Spike protein. (A) Schematic representation of the adenoviral expression plasmid construction using Gateway LR recombination. The entry plasmid (pDONR223 SARS‐CoV‐2 S) containing attL1/attL2 recombination sites was recombined with the destination vector (pAd/CMV/V5‐DEST) containing attR1/attR2 sites using LR clonase enzyme mix. The resulting transfer plasmid (pAd5Spike) includes the full‐length Spike gene under a CMV promoter along with adenoviral vector backbone sequences. Simulation images were generated using SnapGene. (B) Agarose gel electrophoresis analysis of pAd5Spike plasmids. The left panel shows the simulated gel image, and the right panel displays actual gel electrophoresis of plasmids isolated from three independent bacterial colonies (lanes 1–3). MW: 1 kb Plus DNA Ladder (Thermo, SM0313). (C) Immunocytochemical analysis of SARS‐CoV‐2 Spike protein expression in HEK293T cells transfected with pAd5Spike. Cells transfected with pAd5Spike show strong brown immunostaining, indicating Spike expression, while control cells (transfected with PEI alone) show no signal. (D) CsCl density gradient ultracentrifugation of recombinant adenoviral particles. Two distinct bands are visible: the lower band represents genome‐containing Ad5Spike particles, and the upper band corresponds to empty viral capsids, indicating successful viral purification. (E) Immunofluorescence analysis of spike protein expression in HT1080 cells transduced with Ad5Spike at increasing multiplicities of infection (MOI: 10 2 –10 5 ). Cells were stained 72 h post‐transduction using an anti‐Spike antibody (green, Alexa Fluor 488). Nuclei were counterstained with DAPI (blue). Scale bars: 100 µm.

Article Snippet: Neutralizing antibody responses were assessed using a pseudovirus neutralization assay based on third‐generation lentiviral particles pseudotyped with SARS‐CoV‐2 Spike (Wuhan‐1 sequence; Addgene, #155130), encoding an RFP reporter gene.

Techniques: Expressing, Recombinant, Plasmid Preparation, Generated, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Isolation, Transfection, Immunostaining, Control, Purification, Immunofluorescence, Transduction, Infection, Staining

Journal: Biotechnology Journal

Article Title: An Ad5‐Based COVID‐19 Vaccine Encoding SARS‐CoV‐2 Spike Glycoprotein Induces Measurable Antibody and Cytokine Responses in Mice

doi: 10.1002/biot.70216

Figure Lengend Snippet: Humoral immune responses induced by Ad5Spike vaccination in BALB/c mice. Female BALB/c mice (6–8 weeks old, n = 5 per group per timepoint) were immunized intraperitoneally with the Ad5Spike vaccine vector at doses of 10 8 , 10 10 , or 10 1 2 viral particles. Control groups received PBS or an AdGFP vector. (A) Serum anti‐Spike IgG antibody titers were quantified by ELISA at days 30 and 90 post‐immunization. Data are presented as mean ± SD, with each data point representing an individual mouse. (B) SARS‐CoV‐2 Wuhan‐1 variant–specific neutralizing antibody titers were assessed in sera collected at days 30 and 90 using a pseudovirus‐based neutralization assay. Titers are expressed as ID 50 values (reciprocal serum dilution). Each dot represents an individual animal; box plots indicate the interquartile range with whiskers representing the 95% confidence interval. Normality was assessed using the Shapiro–Wilk test. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test, with comparisons made against the AdGFP vector–immunized control group. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Neutralizing antibody responses were assessed using a pseudovirus neutralization assay based on third‐generation lentiviral particles pseudotyped with SARS‐CoV‐2 Spike (Wuhan‐1 sequence; Addgene, #155130), encoding an RFP reporter gene.

Techniques: Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Variant Assay, Neutralization

Journal: International Journal of Molecular Sciences

Article Title: Stalling the Enemy: Targeting Nsp13 for Next-Generation SARS-CoV-2 Antivirals

doi: 10.3390/ijms27062587

Figure Lengend Snippet: Pipeline for the identification and characterization of potential inhibitors of nsp13 helicase activity. ( A ) Schematic representation of FRET-based nsp13 helicase activity assay. Helicase activity liberates the fluorescently labeled RNA (6-FAM) from the RNA strand containing a fluorescence quencher (BHQ), thereby increasing fluorescence intensity. ( B ) This assay was optimized for ultra-high throughput screening to test chemical libraries for molecules that inhibit nsp13 activity. If a compound inhibits nsp13 unwinding activity, little to no fluorescence is expected. ( C ) Several inhibitory molecules were identified with IC 50 values ≤ 10 μM. ( D ) Inhibitors were subjected to thermal shift assays (TSA) and microscale thermophoresis assays (MST) for further characterization of their inhibitory properties. ( E ) Inhibitors of nsp13 helicase enzymatic activity were further tested for antiviral activity in cell-based assays using infectious SARS-CoV-2 and SARS-CoV-2 replicon-based systems. Figure created in BioRender. Goins, S. (2026) https://BioRender.com/08k9d3j .

Article Snippet: COVID-SARS2 NSP13 plasmid was obtained through Addgene (plasmid # 159614) [ ].

Techniques: Activity Assay, Helicase Activity Assay, Labeling, Fluorescence, High Throughput Screening Assay, Microscale Thermophoresis

Journal: International Journal of Molecular Sciences

Article Title: Stalling the Enemy: Targeting Nsp13 for Next-Generation SARS-CoV-2 Antivirals

doi: 10.3390/ijms27062587

Figure Lengend Snippet: Identification of nsp13 helicase activity inhibitors through uHTS. Ten inhibitors of nsp13 helicase activity were selected for further characterization based on potency and diversity of their reported cellular targets. Hits were tested across a range of concentrations up to 20 μM in quadruplicates. Fitting of dose-response curves to calculate IC 50 values was performed in GraphPad Prism 10.0.

Article Snippet: COVID-SARS2 NSP13 plasmid was obtained through Addgene (plasmid # 159614) [ ].

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Stalling the Enemy: Targeting Nsp13 for Next-Generation SARS-CoV-2 Antivirals

doi: 10.3390/ijms27062587

Figure Lengend Snippet: Biochemical and Biophysical characterization of nsp13 helicase activity inhibitors. ( A ) Effect of helicase activity inhibitors on nsp13 ATPase activity. In total, 50 nM nsp13 was exposed to 10 μM of ATP and 20 μM inhibitors (2% DMSO control). Experiments were conducted in triplicate (each data point represents a normalized average against control, per experiment). Error bars reflect the standard deviation of three independent experiments. The statistical analysis includes one-way ANOVA with a p -value < 0.05. * p -value < 0.05, ** p -value < 0.01. Graphical representation performed in GraphPad Prism 10.0. ( B ) Identified compounds bind to nsp13, altering its melting temperature. Nsp13 at 1 μM + 0.2% DMSO as a control, or inhibitors tested at 20 μM. Each sample was tested in quadruplicate (each data point represents a T m ). Error bars reflect the standard deviation of four independent experiments. Welch’s t -test was performed with a p -value < 0.05. * p -value < 0.05, *** p -value < 0.001, and **** p -value < 0.0001. Graphical representation of data performed in GraphPad Prism 10.0. ( C , D ) Examples of nucleic acid binding assessed by Microscale Thermophoresis (MST). ( C ) MST traces of FAM-labeled dsRNA in the absence (black) or presence (green) of CDT, showing a negligible change in diffusion of the dsRNA. ( D ) MST traces of FAM-labeled dsRNA in the absence (black) or presence (green) of DXR, showing a large change in diffusion of the dsRNA, indicating that this inhibitor directly interacts with the nucleic acid. ( E ) Summary of the outcome for each of the inhibitors. Green X represents compounds that interact with nucleic acid, while red checkmark represents compounds that did not. shows individual thermophoretic traces for each compound.

Article Snippet: COVID-SARS2 NSP13 plasmid was obtained through Addgene (plasmid # 159614) [ ].

Techniques: Activity Assay, Control, Standard Deviation, Binding Assay, Microscale Thermophoresis, Labeling, Diffusion-based Assay

Journal: International Journal of Molecular Sciences

Article Title: Stalling the Enemy: Targeting Nsp13 for Next-Generation SARS-CoV-2 Antivirals

doi: 10.3390/ijms27062587

Figure Lengend Snippet: Nsp13 deuterium exchange perturbation plots in the presence of compounds. ( A ) Nsp13 with DMSO (control, representing deuterium uptake). ( B ) Nsp13 with AVM. ( C ) Nsp13 with CDT. ( D ) Nsp13 with PLK. For ( B – D ), the colorimetric scale (−50% to 50%) represents the difference in the rate of deuterium exchange between ligand-treated and apo (unliganded) protein. Datum for each amino acid was superimposed on the nsp13 apo structure (PDB: 7NIO). ZBD: Zinc Binding Domain, 1B: beta-barrel 1B domain, 1A: RecA-like domain 1A, and 2A: RecA-like domain 2A. Dashes in the structure represent disordered loops that were unable to be modeled by X-ray crystallography.

Article Snippet: COVID-SARS2 NSP13 plasmid was obtained through Addgene (plasmid # 159614) [ ].

Techniques: Control, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Stalling the Enemy: Targeting Nsp13 for Next-Generation SARS-CoV-2 Antivirals

doi: 10.3390/ijms27062587

Figure Lengend Snippet: Nsp13 helicase inhibitors tested in a transient transfection or a stable cell line system expressing SARS-CoV-2 subgenomic replicon (SARS-2R). ( A ) HEK293 cells were transfected with SARS-2R expressing NanoLuc Luciferase as a reporter. Transfected cells were treated with inhibitors at 10 or 50 μM. After a 2-day incubation, NanoLuc substrate was added, and luminescence was measured using a Promega luminometer. ( B ) BHK-21 cells stably expressing SARS-2R with a NanoLuc Luciferase reporter were seeded into a plate with inhibitor at 10 or 50 μM ( B ). After a 2-day incubation, NanoLuc luciferase activity was measured as above. For measuring % cell viability, the media was removed, and PrestoBlue was added. The data represent the mean of two independent experiments with two technical replicates each. Remdesivir (RDV) was used as a positive control. For both experiments, graphical representation and statistical analysis were performed in GraphPad Prism 10.0.

Article Snippet: COVID-SARS2 NSP13 plasmid was obtained through Addgene (plasmid # 159614) [ ].

Techniques: Transfection, Stable Transfection, Expressing, Luciferase, Incubation, Activity Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Motif V is an allosteric couple between the SARS-CoV-2 nsp13 nucleotide triphosphatase and helicase active sites

doi: 10.1016/j.jbc.2026.111198

Figure Lengend Snippet: Structure and conservation of Motif V in the SARS-CoV-2 nsp13 helicase . A , structure of the SARS-CoV-2 nsp13 helicase based on PDB: 7NNO. Motif V in Domain 2A is colored in red , proximal to bound AMPPNP between Domains 2A and 1A, and the RNA binding domain is highlighted in yellow between Domains 1 A/2A and 1B. RBD = RNA Binding Domain, ZBD = Zinc Binding Domain. B , close-up view of the SARS-CoV-2 nsp13 Motif V region ( colored salmon ). Positions of residues ST532, D534 and S535 are noted. C , conservation of Motif V across the beta-coronavirus genus. Residues 532, 534, and 535 are noted.

Article Snippet: All point mutations were made on a plasmid containing the gene encoding N-terminally 6xHis-ZBasic-tagged SARS-CoV-2 helicase, nsp13 (Addgene plasmid # 159614).

Techniques: RNA Binding Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Motif V is an allosteric couple between the SARS-CoV-2 nsp13 nucleotide triphosphatase and helicase active sites

doi: 10.1016/j.jbc.2026.111198

Figure Lengend Snippet: Interaction of D534 with R560 is dependent on phosphate binding in the NTPase binding site . Motif V and adjacent interacting residues are shown from nsp13 in the apo form (PDB:7NIO), nsp13 bound to ADPPNP and Mg 2+ modeling the pre-ATP hydrolysis step (PDB: 7NN0), and phosphates modeling the post-ATP hydrolysis step (PDB: 6ZSL).

Article Snippet: All point mutations were made on a plasmid containing the gene encoding N-terminally 6xHis-ZBasic-tagged SARS-CoV-2 helicase, nsp13 (Addgene plasmid # 159614).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Motif V is an allosteric couple between the SARS-CoV-2 nsp13 nucleotide triphosphatase and helicase active sites

doi: 10.1016/j.jbc.2026.111198

Figure Lengend Snippet: Fluorescence polarization analysis of nsp13 mutant DNA-binding affinity . Fluorescence polarization was used to determine binding affinity for each mutant. Nsp13 WT and designated mutants were titrated into reactions with 10 nM 5′-labeled AlexaFluor 488-dsDNA. Fluorescence polarization values represent the fraction of labeled dsDNA bound to nsp13. K d values were derived from a four-parameter logistic curve fit using Graphpad Prism 10 and are reported in . n = 3.

Article Snippet: All point mutations were made on a plasmid containing the gene encoding N-terminally 6xHis-ZBasic-tagged SARS-CoV-2 helicase, nsp13 (Addgene plasmid # 159614).

Techniques: Fluorescence, Mutagenesis, Binding Assay, Labeling, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Motif V is an allosteric couple between the SARS-CoV-2 nsp13 nucleotide triphosphatase and helicase active sites

doi: 10.1016/j.jbc.2026.111198

Figure Lengend Snippet: Michaelis-Menten kinetics enzymatic characterization of nsp13 T532A and S535A mutants . A , reactions were carried out in a 20 mM HEPES, pH 7.50 reaction buffer with 20 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.1% bovine serum albumin, and 1 nM of WT ( black circle ), T532 A ( red square ) or S535 A ( blue up triangle ) nsp13. A , Helicase kinetic characterization using DNA as the variable substrate. Reactions were incubated with 40–500 nM dsDNA with a AlexaFluor 488/IowaBlack fluorophore-quencher pair, and initiated with 500 μM ATP. B , Helicase activity characterization using ATP as the variable substrate. Reactions were incubated with100 nM labeled dsDNA and initiated with 20–500 μM ATP. Initial rates were determined as a function of increase fluorescence, RFU values were converted to ssDNA concentration, and product concentration per second rate units were normalized to enzyme concentration. Plotted values are nM ssDNA product per second per nM enzyme. C , ATP activity characterization. Reactions were incubated with100 nM unlabeled dsDNA and initiated with 20 to 500 μM ATP. Initial rates were determined through the colorimetric BioMOL Green assay to detect free phosphate concentration. Absorbance values were converted to [P i ], and rate data was plotted similarly to helicase data, with rate values in μM phosphate product per second per nM enzyme. K cat , K m , and K cat /K m values are reported in , , , respectively. n ≤ 3.

Article Snippet: All point mutations were made on a plasmid containing the gene encoding N-terminally 6xHis-ZBasic-tagged SARS-CoV-2 helicase, nsp13 (Addgene plasmid # 159614).

Techniques: Incubation, Activity Assay, Labeling, Fluorescence, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Motif V is an allosteric couple between the SARS-CoV-2 nsp13 nucleotide triphosphatase and helicase active sites

doi: 10.1016/j.jbc.2026.111198

Figure Lengend Snippet: Michaelis-Menten Kinetics Enzymatic Characterization of nsp13 L405D and D534 A Mutants . Reactions were carried out in a 20 mM HEPES, pH 7.50 reaction buffer with 20 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.1% bovine serum albumin, and 1 nM of WT ( black circle ), D534 A ( green down triangle ) or L405D (magenta diamond) nsp13. A , Helicase kinetic characterization using DNA as the variable substrate. Reactions were incubated with 40–500 nM dsDNA with a AlexaFluor 488/IowaBlack fluorophore-quencher pair and initiated with 500 μM ATP. B , Helicase activity characterization using ATP as the variable substrate. Reactions were incubated with100 nM labeled dsDNA and initiated with 20–500 μM ATP. Given the lack of helicase activity for D534 A in panel A, helicase activity for D534 A was not determined using ATP as the variable substrate. Initial rates were determined as a function of increase fluorescence, RFU values were converted to ssDNA concentration, and product concentration per second rate units were normalized to enzyme concentration. Plotted values are nM ssDNA product per second per nM enzyme. C , ATP activity characterization. Reactions were incubated with100 nM unlabeled dsDNA and initiated with 20 to 500 μM ATP. Initial rates were determined through the colorimetric BioMOL Green assay to detect free phosphate concentration. Absorbance values were converted to [P i ], and rate data was plotted similarly to helicase data, with rate values in μM phosphate product per second per nM enzyme.K cat , K m , and K cat /K m values are reported in , , , respectively. n ≤ 3.

Article Snippet: All point mutations were made on a plasmid containing the gene encoding N-terminally 6xHis-ZBasic-tagged SARS-CoV-2 helicase, nsp13 (Addgene plasmid # 159614).

Techniques: Incubation, Activity Assay, Labeling, Fluorescence, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Motif V is an allosteric couple between the SARS-CoV-2 nsp13 nucleotide triphosphatase and helicase active sites

doi: 10.1016/j.jbc.2026.111198

Figure Lengend Snippet: Model for the nsp13 enzymatic mechanism . Proposed mechanism of the kinetically-coupled active sites in nsp13 through a single turnover of ATP and the procession of one nucleotide step. Kinetic steps occurring in the ATPase active site are in red, and steps occurring in the helicase domain are in blue .

Article Snippet: All point mutations were made on a plasmid containing the gene encoding N-terminally 6xHis-ZBasic-tagged SARS-CoV-2 helicase, nsp13 (Addgene plasmid # 159614).

Techniques: