vRNP export-independent SG inhibition by influenza virus coincides with depletion of cytoplasmic poly(A) RNA and nuclear relocalization of PABP1.

<p>(A and B) Immunofluorescent analysis of mock and PR8 virus-infected cells at 18 hpi, treated with sodium arsenite where indicated. (A) At 6 hpi some A549 cells received 10 ng/ml leptomycin B (+LMB) to block nuclear export of viral ribonucleoproteins up to the time of arsenite treatment and subsequent fixation at 18 hpi. SG formation was analysed by staining for TIA-1 (red) and YB-1 (green) markers and viral ribonucleoproteins were detected with antibody to NP (blue). (B) SG formation and sub-cellular distribution of cellular G3BP, eIF3A (red), PABP1 (PABP, green), and influenza virus proteins (IAV, blue) were analysed in U2OS and A549 cells. Note that while in U2OS cells most of the IAV antigens remain nuclear at 18 hpi, virus material is exported from the nucleus by this time point in A549 cells (open arrows). In both cell lines, however, virus causes redistribution of PABP1 to the nucleus and the SG formation is blocked (filled arrows). (C) Sub-cellular distribution of poly(A) RNA in mock and PR8 virus-infected A549 cells was analysed at indicated times post-infection by fluorescence in situ hybridization. At 18 hpi, many PR8-infected cells have decreased cytoplasmic and increased nuclear poly(A) signal. Upon arsenite treatment (+As) these cells fail to form cytoplasmic poly(A) RNA-containing stress granules (arrows). Scale bars = 20 µm.