mouse monoclonal anti dsrna  (Novus Biologicals)

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Proof of concept: targeted protein degradation of the stress granules component G3BP1 as an antiviral strategy against norovirus infection

doi: 10.1128/aac.01118-25

Figure Lengend Snippet: Degradation of G3BP1 using HaloPROTAC3 shows antiviral activity against norovirus. BV2 ΔG3BP1 + HaloG3 BP1 cells were treated for 48 h with the shown concentrations of HaloPROTA3 and infected with MNV1. After 1 h of virus adsorption, the virus inoculum was removed, and HaloPROTAC3 was re-added to the cell culture medium for the remaining infection time. ( A ) Scheme of the experimental setup of the antiviral assays. ( B–D ) Cells infected with MOI 1 TCID 50 /cell for 16 h were incubated with fluorescent ligand Halotag Janelia 647 for 2 h, harvested, and stained with anti-dsRNA antibody to assess the percentage of infection. Cells were then subjected to flow cytometry analysis. ( B ) Gating strategy showing first the duplet exclusion for selection of single cells, followed by dead cell exclusion within the single-cell population. Halotag + cells and dsRNA + cells were gated exclusively from the live cell population. ( C ) Quantification of the percentage of Halotag + and ( D ) dsRNA + (infected) cells across HaloPROTAC3 concentrations. ( E ) Total RNA was isolated from cells infected with MOI 1 TCID 50 /cell for 10 h and used to quantify the levels of viral RNA (expressed as vRNA copies/µg RNA, normalized to the DMSO control), by RT-qPCR. ( F ) Extracellular infectious virus yield in supernatants from cells infected with MOI 1 TCID 50 /cell for 16 h and treated with the indicated concentrations of HaloPROTAC3 was quantified by TCID 50 in BV2 cells. ( G, H ) Infection-induced cell death was detected using the NIR dye measured in the live-cell imaging IncuCyte system. ( G ) Representative pictures after 24 h.p.i.; NIR + cells are shown in blue. ( H ) Quantification of the total number of NIR + cells for the shown HaloPROTAC3 concentrations. Data is expressed as mean ± standard error of the mean with ∗ , ∗∗ , ∗∗∗ , and ∗∗∗∗ indicating P ≤ 0.05, P ≤ 0.01, P ≤ 0.001, and P ≤ 0.0001 respectively.

Article Snippet: In samples where the percentage of infection was also examined, the cells were washed and permeabilized for 10 min on ice with FACS buffer supplemented with 0.3% Triton X-100 and stained for 2 h with Mouse anti-dsRNA mAb (J2) (Cell Signaling, 76651).

Techniques: Activity Assay, Infection, Virus, Adsorption, Cell Culture, Incubation, Staining, Flow Cytometry, Selection, Single Cell, Isolation, Control, Quantitative RT-PCR, Live Cell Imaging

Journal: bioRxiv

Article Title: SARS-CoV-2 and MERS-CoV disrupt host protein synthesis via nsp1 with differential effects on the integrated stress response

doi: 10.64898/2025.12.28.696697

Figure Lengend Snippet: (A) The SARS-CoV-2 genome is shown; the 5’UTR with leader sequence (L), ORFs 1a and 1ab encoded non-structural proteins (nsps) shown in blue and indicated by their number, structural proteins shown in gray: spike (S), envelope (E), membrane (M), and nucleocapsid (N), and accessory proteins shown in white and indicated by their number, and polyA tail (top). Full-length nsp1 of SARS-CoV-2 (middle). Sequence alignment among beta-coronaviruses, from top to bottom: MHV strain A59, MERS-CoV, SARS-CoV, and SARS-CoV-2. N-terminal and C-terminal alanine substitutions are labeled with SARS-CoV-2 amino acid residues boxed in solid lines and sequence numbers under solid lines and MERS-CoV amino acids residues boxed in dashed lines and sequences numbers under dashed lines. (B) Diagram showing the activity of the ISR pathway in response to stress induced by dsRNA or ER stress. Nsp1 is also shown to demonstrate that both p-eIF2α and nsp1 inhibit translation initiation.

Article Snippet: For immunofluorescence assays, cells were incubated with rabbit polyclonal anti-GADD34 antibody (1:1000; proteintech, 10449-1-AP), mouse monoclonal anti-G3BP1 antibody (1:1000; Abcam, ab119236), mouse monoclonal dsRNA antibody (K1) (1:1000; Cell Signaling Technology, 28764), rabbit recombinant monoclonal anti-TIA1 antibody (1:1000, Abcam, ab263945) for 4-8 hours.

Techniques: Sequencing, Membrane, Labeling, Activity Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 and MERS-CoV disrupt host protein synthesis via nsp1 with differential effects on the integrated stress response

doi: 10.64898/2025.12.28.696697

Figure Lengend Snippet: (A) IFA analyses for stress granule markers, TIA-1 and G3BP1, and FISH for poly(A)+RNA 48 hours post-infection with WT or nsp1 mutant SARS-CoV-2 viruses in A549 ACE2 RNase L-KO (RL-KO) cells. Images representative of 4 or more fields of view. (B) The percentage of FISH poly(A)+RNA granules smaller or larger than 1 μm 2 in cells infected with WT or nsp1 mutants. Minimum of 24 cells were quantified per virus. (C) Histogram displaying the percentage of cells with poly(A)+RNA granules equal to or larger than 1 μm 2 . Minimum of 24 cells quantified per virus. (D) smRNA-FISH for SARS-CoV-2 genome RNA (ORF1a) and poly(A)+RNA. The graph displays the normalized fluorescence intensity (% of maximum intensity) of poly(A)+RNA and SARS-CoV-2 genome RNA along the line in the merged inset image panel. Images representative of 4 or more fields of view. (E) Similar to (D) but IFA for TIA1 and dsRNA. Scale bars represent 10μm.

Article Snippet: For immunofluorescence assays, cells were incubated with rabbit polyclonal anti-GADD34 antibody (1:1000; proteintech, 10449-1-AP), mouse monoclonal anti-G3BP1 antibody (1:1000; Abcam, ab119236), mouse monoclonal dsRNA antibody (K1) (1:1000; Cell Signaling Technology, 28764), rabbit recombinant monoclonal anti-TIA1 antibody (1:1000, Abcam, ab263945) for 4-8 hours.

Techniques: Infection, Mutagenesis, Virus, Fluorescence

Journal: Virology Journal

Article Title: Translation of nervous necrosis virus involves eIF4E but not RPS6 phosphorylation and viral particle assembly in remodeled microtubule-organizing center

doi: 10.1186/s12985-025-02799-3

Figure Lengend Snippet: Subcellular location of newly synthesized RNAs, dsRNA and NNV mRNAs in GGNNV-infected GB cells. A Schematic illustration of bromouridine incorporation experiment. B DMSO-treated or ACD-treated cells were incubated with bromouridine for 1 h prior to the collection at indicated times after GGNNV infection (MOI = 100). The cells were fixed, and newly synthesized RNA was detected with anti-bromodeoxyuridine antibody (green). White arrowhead indicates the scattered mitochondrial pattern with newly synthesized RNA. White arrow indicates the newly synthesized RNA that has moved away from the mitochondria and become concentrated at a particular perinuclear area. C Cellular localization of NNV RNA2 (green) and dsRNA (violet) was detected by RNA FISH and immunocytochemistry in GGNNV-infected GB cells. White arrowhead indicates crescent-shaped clustered mitochondria with RNA2 and dsRNA. White arrow indicates RNA2 but not dsRNA that has moved out of mitochondria and become concentrated at a particular perinuclear area. D Detection of NNV RNA1 (green) and RNA2 (violet) using RNA FISH at indicated time points. White arrowhead indicates the crescent-shaped clustered mitochondria with RNA1 and RNA2. White arrow indicates that RNA1 and RNA2 moved out of mitochondria and concentrated at a particular perinuclear area. Scale bar = 20 μm. The nuclei (blue) were stained with DAPI and mitochondria (red) was labelled with Mito tracker. E Quantification of colocalization between mitochondria and newly synthesized RNAs at 0, 6, 12, 18 and 24 hpi by Pearson’s correlation coefficient. F Quantification of colocalization between Mito-RNA2, Mito-dsRNA and RNA2-dsRNA at 0, 12 and 24 hpi by Pearson’s correlation coefficient. G Quantification of colocalization between Mito-RNA1, Mito-RNA2 and RNA1-RNA2 at 0, 12 and 24 hpi by Pearson’s correlation coefficient. ACD, actinomycin D; BrU, bromouridine; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; dsRNA, double-stranded RNA; GB, grouper brain; GGNNV, giant grouper nervous necrosis virus; hpi, hour post infection; Mito, mitochondria; MOI, multiplicity of infection; RNA FISH, RNA fluorescence in situ hybridization

Article Snippet: Mouse anti-puromycin Alexa Fluor 488 monoclonal antibody (clone 12D10) (MABE343) and mouse anti-dsRNA monoclonal antibody (clone rJ2) (MABE1134) were purchased from Merck.

Techniques: Synthesized, Infection, Incubation, Immunocytochemistry, Staining, Virus, Fluorescence, In Situ Hybridization