Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly. Author Summary Hepatitis C virus (HCV) is a positive strand RNA virus and is a major cause of liver disease worldwide, affecting more than 170 million individuals. localization was decided using anti-core antibodies (red). DNA is usually stained with DAPI (blue). These images provide lower magnification views of similar test samples examined in Physique 1. Scale bars, 5 m. B) Percent colocalization between cytoplasmic Nups and HCV core protein in an average of 10 cells processed as in panel A was decided. Values represent the percent of the cytoplasmic Nup fluorescence signal (±)-Ibipinabant overlapping with the HCV core fluorescence signal calculated using the Manders colocalization coefficient.(TIF) ppat.1003744.s002.tif (9.4M) GUID:?8CB29D49-447D-4147-A52E-C08EA24B9E87 Figure S3: Colocalization between Nups and HCV core proteins. Huh7.5 cells were infected with HCV for 4 days. The localization of Nups and lamin B compared to HCV core was evaluated using indirect immunofluorescence by staining with antibodies specific for Nups and lamin B (green) or HCV core (red). Fluorescence intensity line graphs were plotted using pixel intensity TYP data obtained from red and green fluorescence channels along lines (white) drawn through regions made up of core protein and lipid droplets. DNA was stained with DAPI (blue). Scale bars, 2 m.(TIF) ppat.1003744.s003.tif (5.9M) GUID:?0309CCA9-BB95-4758-9CC0-CF674ADDB9A6 Physique S4: Localization of Nups and tubulin (±)-Ibipinabant in HCV infected cells. ACB) The localization of Nup98 or Nup155 and tubulin was evaluated in uninfected or HCV infected Huh7.5 cells four days after infection. Cells were examined (±)-Ibipinabant by indirect immunofluorescence confocal microscopy using antibodies directed against Nup155 (panel A, green) or Nup98 (panel B, green) and tubulin (red). DNA was stained with DAPI (blue). Scale bars, 5 m.(TIF) ppat.1003744.s004.tif (3.9M) GUID:?17A1B6E9-8D6B-442B-A834-EA8E5D5021E3 Physique S5: Cytoplasmic localization of Nups in dengue and hepatitis A virus infected cells. A) The localization of Nup98 was examined by indirect immunofluorescence microscopy in uninfected Huh7 cells and cells infected with hepatitis A virus (HAV infected) for three weeks using anti-Nup98 antibodies (green). Hepatitis A viral RNA was detected using anti-dsRNA antibodies (red). B) Localization of Nup98 was also examined in dengue virus infected A549 cells (2 days post contamination) as described in panel A (green) and compared to the localization of dengue virus capsid protein using capsid specific antibodies (red). In both panels, DNA was stained with DAPI (blue). Scale bars, 5 m.(TIF) ppat.1003744.s005.tif (3.4M) GUID:?16D04323-A16A-46B0-8BFF-9D14F604A2DA Physique S6: Localization of Nups and HCV proteins in transfected or HCV-infected cells. A) Huh7.5 cells were transfected with constructs encoding for V5-tagged HCV core, NS5A, or NS4A. The localization of the Nup98 and tagged HCV proteins was examined 48 hours later, (±)-Ibipinabant by indirect immunofluorescence confocal microscopy using anti-Nup98 (green) and anti-V5 (red) antibodies. Arrows point to cells expressing the indicated HCV protein and Pearson’s colocalization coefficients are specified in the merge panel. DNA is usually stained with DAPI (blue). Scale bars, 10 m. B) The localization of Nup155 and Nup98 was evaluated in uninfected Huh7.5 cells (Un) or 4 days following contamination with HCV (HCV) by indirect immunofluorescence confocal microscopy using antibodies specific for the indicated Nups (green) and HCV NS5A protein (red). Boxed areas in the middle rows of images are show at higher magnification in the bottom row. DNA is usually stained with DAPI (blue). Scale bars, 5 m. C) Percent colocalization between cytoplasmic Nups and NS5A in an average of 10 cells processed as in panel B was determined. Values represent the percent of the cytoplasmic (±)-Ibipinabant Nup fluorescence signal overlapping with the HCV core fluorescence signal calculated using the Manders colocalization coefficient.(TIF) ppat.1003744.s006.tif (9.5M) GUID:?E7C3EE74-CD14-43D7-A232-43C78C91CAE7 Figure S7: Localization of a subset of Nups.