The RING-CH Ligase K5 Antagonizes Restriction of KSHV and HIV-1 Particle Release by Mediating Ubiquitin-Dependent Endosomal Degradation of Tetherin
Figure 5
Effects of proteasomal and endosomal inhibitors on tetherin levels and localization in cells expressing K5 and Vpu.
(A) Western blots of cell lysates from HT/THN-HA cells and isogenic pools stably expressing K5 or Vpu from retroviral vectors. Cells were treated for 16h with BafA1 (100nM), MG132 (1µg/ml) or DMSO as a control, lysed and separated by SDS-PAGE. THN-HA was detected by anti-HA polyclonal antibody, with Hsp90 serving as a loading control and visualized using Licor fluorescently coupled 650 and 800 nm secondary antibodies. Percent mature tetherin levels, normalized to Hsp90 loading are displayed below each lane. (B) Representative examples of HT/THN, HT/THN-HA K5 and HT/THN-HA Vpu cells immunostained for tetherin with a rabbit anti-HA antibody (red) and an antibody for the late endosomal marker CD63 (green). Nuclei were counterstained with DAPI (blue) and cells examined by confocal microscopy. (C) HT/THN-HA Vpu cells stained for THN-HA (green) and the trans-Golgi marker TGN46 (red) were processed as above. (D) Cells from A were surface stained for tetherin levels and analyzed by flow cytometry. Purple histograms represent tetherin levels on DMSO treated cells. Green overlays indicate tetherin levels after treatment with the indicated drug. The pink histogram overlays show the levels of tetherin on untreated HT/THN-HA from the upper row for comparison. (E) Confocal image of a representative HT/THN-HA K5 cell treated with MG132 and stained for THN-Ha (red) and CD63 (green). (F) HT/THN-HA Y6,8A were imaged by confocal microscopy for tetherin (red). The cells were then manipulated to express K5 and analyzed by flow cytometry for surface tetherin and by western blot for total cellular tetherin levels.
doi: https://doi.org/10.1371/journal.ppat.1000843.g005