Article Title: Species difference in ANP32A underlies influenza A virus polymerase host restriction
Figure Lengend Snippet: Knockdown of ANP32 reveals avian influenza polymerase dependence on chANP32A and dependence on huANP32A and B in human cells by human adapted influenza polymerase a, DF-1 cells transduced with VSV-G lentiviral vectors delivering transgenes expressing puromycin and shRNA targeting chANP32A or Negative. Puromycin selected cells transfected with pCOM1-firefly minigenome reporter, avian 50-92 polymerase (627E), and Renilla expression control . b, siRNA (100nM) applied to DF-1 chANP32A shRNA cells. After 48hrs, cells transfected with avian 50-92 polymerase (627E), minigenome reporter and Renilla expression control. Luciferase activity measured 20hrs later. Knockdown in chicken DF-1 cells verified by immunoblotting using antibody against vinculin and chANP32A. c, DF-1 cells depleted of chANP32A by siRNA infected with avian-like influenza virus (PR8 virus bearing H5N1 Ty05 polymerase genes with PB2 627E, MOI 0.01). 24hrs later cell supernatants titrated for infectious virus by plaque assay on MDCK cells. d , 293T cells transduced with lentiviral vectors delivering transgenes expressing puromycin and shRNA targeting huANP32A, huANP32B, both huANP32A and B, or Negative. Puromycin selected cells transfected with, pHOM1-firefly minigenome reporter, human-adapted avian 50-92 polymerase (627K), and Renilla expression control.Luciferase activity measured after 20hrs. Knockdown in 293T cells verified immunoblotting using antibody against vinculin, huANP32A and huANP32B. (a,b d, data are firefly activity normalised to Renilla, plotted as % of Negative or Allstars; error as SEM; One-way ANOVA comparisons to ALLStars or Negative, ns= not significant, **p
Article Snippet: Chicken fibroblast (DF-1) (ATCC) cells were maintained in DF-1 cell culture media (DMEM supplemented with 10% FCS, 5% tryptose phosphate broth (Sigma-Aldrich) and 0.1% Penicillin-streptomycin (Invitrogen)).
Techniques: Transduction, Expressing, shRNA, Transfection, Luciferase, Activity Assay, Infection, Plaque Assay