Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies
Fig 8
KSHV-mediated autophagy utilizes MK2.
A: HUVECs were transduced with recombinant lentiviruses expressing shRNAs targeting MK2 or a non-targeting control (NS) and selected with puromycin (1 μg/mL). After selection, transduced cells were infected with wild-type (WT) or delete KapB (delB) BAC16 KSHV via spinoculation and infection was allowed to proceed for 96 h. Cells were harvested in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for MK2. B: Samples from A were quantified for MK2 protein level and normalized to the total protein in each lane using ImageLab software (BioRad). MK2 levels were then graphed by normalizing to the matched NS for each infection condition in GraphPad, and a one-way repeated measures (RM) ANOVA with a Šidák’s multiple comparison test was performed, ±SEM; n = 3, *** = P<0.001, **** = P<0.0001. C: HUVECs were transduced with shMK2-expressing lentiviruses or NS controls, selected, and infected as in A. Cells were harvested in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for total hsp27 and phospho-hsp27 (S82). D: Samples from C were quantified by normalizing phospho- and total hsp27 protein levels to the total protein in each lane using Image Lab (BioRad) then by dividing phospho-hsp27 over total hsp27 and normalizing to the matched NS for mock, WT KSHV and delB infection, respectively. Results were plotted in GraphPad, a one-way RM ANOVA with a Šidák’s multiple comparison test was performed, ±SEM; n = 3, * = P<0.05. E: HUVECs were transduced, selected, and infected as in C. Cells were treated with Bafilomycin A1 (BafA1, 10 nM) or a vehicle control (DMSO) for the indicated times prior to harvest in 2X Laemmli buffer. Protein lysates were resolved by SDS-PAGE and immunoblot was performed for LC3. F-G: Samples from E were quantified using Image Lab (BioRad) software and then normalized, first to total protein and then to their respective starting time points (0 h). In F, the accumulation of LC3-II in control cells was compared with accumulation in MK2-silenced cells in either uninfected (top) or WT KSHV infected (bottom) cells. Results were plotted in GraphPad and a linear regression statistical test was performed between control or silenced cells. ±SEM; n = 3, * = P<0.05. In G, results were plotted in GraphPad to show accumulation of LC3-II in MK2-silenced cells to compare between the following groups: WT-infected vs uninfected (left), uninfected vs delB infected (middle) and WT infected vs delB infected (right). A linear regression statistical test was performed between infection conditions in each group as indicated. ±SEM; n = 3, * = P<0.05.