Journal: mBio

Article Title: Caspase-mediated DDX46 cleavage unchains antiviral immunity

doi: 10.1128/mbio.03519-25

Figure Lengend Snippet: Virus infection or treatment with RNA ligands induces reduced DDX46 protein levels. ( A–C ) HeLa cells were mock-infected or infected with VSV ( A ), NDV ( B ), or HSV-1 ( C ) at an MOI of 1 for 6, 12, 18, and 24 h. Protein levels of VSV-G, NDV-NP, or HSV-1-gD were analyzed by WB. β-Actin served as the loading control. ( D–F ) Representative results, with graphs representing the band intensity ratios of DDX46/β-actin normalized to the control conditions for the VSV ( D ), NDV ( E ), and HSV-1 ( F ) infection groups. ( G–J ) HeLa cells were transfected with RNA ligands [poly(I:C) or 3p-hpRNA, panels G and H ] or DNA ligands [poly(G:C) or HSV-60, panels I and J ] for 18 h. Protein levels of DDX46 were analyzed by WB. β-Actin served as the loading control. Representative results, with graphs representing the band intensity ratios of DDX46/β-actin normalized to the control conditions for the RNA ligands ( H ) or DNA ligand treatment groups ( J ). ( K ) Schematic diagram of DDX46 isoform I (full-length; DDX46-I) and isoform II (lacking valine at amino acid 872; DDX46-II). ( L–N ) HeLa cells were transfected with the empty vector p3×Flag, Flag-DDX46-I, or Flag-DDX46-II for 24 h, then mock infected or infected with VSV ( L ), NDV ( M ), or HSV-1 ( N ) at an MOI of 1 for 6, 12, 18, and 24 h. Protein levels of exogenous Flag-DDX46 and viral proteins (VSV-G, NDV-NP, or HSV-1-gD) were analyzed by WB. β-Actin served as the loading control. Data are presented as means from three independent experiments. *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-HSV-1 antibody (NB600-516) was obtained from Novus Biologicals.

Techniques: Virus, Infection, Control, Transfection, Plasmid Preparation

Journal: mBio

Article Title: Caspase-mediated DDX46 cleavage unchains antiviral immunity

doi: 10.1128/mbio.03519-25

Figure Lengend Snippet: Viral infection induces DDX46 cleavage and translocation from the nucleus to the cytoplasm. ( A–D ) HeLa cells were transfected with the Flag-DDX46 for 24 h, then mock-infected or infected with VSV ( A, B ) or HSV-1 ( C, D ) at an MOI of 1 for 6 and 12 h. Cells were fixed and subjected to IF analysis using anti-Flag and anti-viral-protein (VSV-G or HSV-1-gD) antibodies. Nuclei were counterstained with DAPI. Quantification of the relative percentages of cells with nuclear (Nuc) and cytoplasmic (Cyto) Flag signal after VSV ( B ) or HSV-1 ( D ) infection. Six randomly selected fields were analyzed using ImageJ. ( E ) Schematic of DDX46 mutants: point mutant D226A, N-terminal truncation (1–225), and C-terminal truncation (227–1,032). ( F, G ) HeLa cells were transfected with Flag-tagged WT-DDX46 or mutants (D226A, 1–225, and 227–1,032) for 24 h, then mock-infected or infected with VSV (MOI = 1) for 12 h. Cells were fixed and subjected to IF analysis using anti-Flag and anti-VSV-G antibodies. Nuclei were counterstained with DAPI. The two panels on the left show wider fields at 63×, and the two panels on the right show smaller fields at 20× ( F ). Quantification of the relative percentages of cells with nuclear or cytoplasmic Flag signal after VSV infection. Six randomly selected fields were analyzed using ImageJ ( G ). ( H and I ) HeLa cells were transfected with Flag-tagged WT-DDX46 or D226A-DDX46 for 24 h, then mock-infected ( H ) or infected with VSV at an MOI of 1 ( I ) for 12 h. Cells were harvested, and nuclear and cytoplasmic fractions were prepared using a nucleocytoplasmic isolation kit. Protein levels of exogenous Flag-DDX46 and VSV-G were analyzed by WB. β-Tubulin and Lamin B1 served as the loading controls for cytoplasmic and nuclear fractions, respectively. ( J ) Representative results, with graphs showing the band intensity ratios of DDX46/β-actin normalized to the control conditions for the VSV infection group ( I ). Data are presented as means from three independent experiments. *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-HSV-1 antibody (NB600-516) was obtained from Novus Biologicals.

Techniques: Infection, Translocation Assay, Transfection, Mutagenesis, Isolation, Control