Fig 1.
p62-mediated selective autophagy is endogenously induced in viral latency and correlates with ROS-Keap1-NRF2 pathway activity.
A. Endogenous ROS production in viral latency was measured by flow cytometry with the CellRox Green reagent (Invitrogen). Three independent repeats were conducted, and representative results are shown. Results are the mean ± standard error (SE) of duplicates for each sample. MFI = mean fluorescence intensity. B. The correlation of endogenous p62 protein levels and autophagy activity with the Keap1-NRF2 pathway activity in viral latency was evaluated by immunoblotting with indicated antibodies. C. The correlation of p62 expression with viral latency was evaluated at the transcription level by qPCR. The mRNA levels in SavI, P3HR1, and CEM were set to 1, and compared with the paired SavIII, JiJoye, and MT4 cell lines, respectively. D. Interaction of endogenous p62 with LC3b in virus-transformed cells was evaluated by IP. Cell lysates (1 mg each) were pre-cleared with mouse IgG (Sigma) before subjected to IP with IgG or anti-p62 clone D-3 (Santa Cruz). immunoprecipitants and inputs (5% of cell lysates) were probed with indicated antibodies.
Fig 2.
ROS correlate with Keap1-NRF2 pathway activity and contribute to p62 expression and activation of p62-mediated autophagy in viral latency.
A. SavI and SavIII cells, which were derived from the same patient, were treated with 2 µM of the topoisomerase II inhibitor doxorubicin HCl (Doxo) (UBPBio) for different time periods. B-C. SavI, P3HR1, and CEM were treated with 20 mM of the H2O2 catalase 3-amino-1,2,4-triazole (3-AT) (Fisher Scientific) or vehicle control for 48 h. Cells were then subjected to IB, qPCR, and flow cytometry analyses. D. IB4, LCL45, and MT4 were treated with 3 mM of the antioxidant N-acetylcysteine amide (NACA) (Sigma) or vehicle control for 30 h. The treated cells were then subjected to analyses for p62, autophagy, Keap1-NRF2 pathway, and ROS production. E. IB4 cells were treated with 20 mM 3-AT (Fisher Scientific) or vehicle control for 48 h, and analyzed for p62-autophagosome colocalization by confocal microscopy. Living cells were first stained with a Cyto-ID Autophagy Detection kit (Enzo), and then fixed for staining with the mouse p62 antibody (D-3) and Alexa 555 coupled anti-mouse antibody (Invitrogen). Bar = 2 µm. F. IB4 cells were treated with 50 µM H2O2 for 30 min and then medium was replaced and continued in culture for 2 days, or treated with 20 mM 3-AT for 48 h, before subjected to analysis of autophagy flux by flow cytometry with the Cyto-ID Autophagy Detection kit. MFI = mean fluorescence intensity.
Fig 3.
Autophagy inhibition sensitizes EBV+ cells to ROS-induced DNA damage that is associated with p62 accumulation.
A. Cell lines with higher endogenous autophagy levels were treated with 0.4 µM of the vacuolar ATPase inhibitor bafilomycin A1 (BafA1) (Sigma) or vehicle control for 24 h, and then DNA damage (γH2AX) was evaluated by immunoblotting. B-C. The LCL lines IB4 and LCL45 were treated with ionomycin (Iono) (Sigma) with indicated concentrations for 48 h plus 0.4 µM BafA1 or vehicle control for 24 h or plus 50 µM of the lysosome inhibitor chloroquine (Chloro) (MP Biomedicals) or vehicle control for 6 h. p62, autophagy, and γH2AX were analyzed by immunoblotting. D. IB4 cells were treated with 5 µg/ml Iono or vehicle control for different time periods, and ROS production was measured by flow cytometry with the CellRox Green reagent (Invitrogen). A representative result from three independent repeats is shown. E. IB4 and LCL45 cells were treated with 5 µg/ml Iono plus 3 mM NACA or vehicle control for different time periods (upper panel) or for 30 h (lower panel). p62, autophagy, and γH2AX were analyzed by immunoblotting.
Fig 4.
Autophagy inhibition promotes cell death in association with p62 accumulation in the nucleus.
IB4 cells were treated with indicated concentrations of H2O2 in medium for 30 min. Then the medium was removed, and 0.4 µM BafA1 (Sigma) or vehicle control was added in freshly replaced medium for 48 h. A. p62, autophagy, and DNA damage were analyzed by immunoblotting. B-C. Cell death was analyzed by flow cytometry for Annexin V and 7-AAD expression. Results from a representative experiment of five independent repeats are shown (B), and statistical analysis results are expressed as mean ± standard error (SE) (C). D. p62 subcellular localization was visualized under confocal microscope. Bar = 10 µm.
Fig 5.
p62 accumulation upon autophagy inhibition destabilizes HR DNA repair proteins CHK1 and RAD51 in viral latency.
A. Correlation of p62 with CHK1, RAD51 and 53BP1 protein levels in viral latency were analyzed in paired cell lines. B. Cell lines with higher levels of p62 protein were treated with 0.4 µM BafA1 (Sigma) for 48 h. Indicated proteins were probed by immunoblotting. C. Cell lines with higher levels of p62 protein were treated with 10 µM of the proteasome inhibitor MG132 for 6 h (left panel), or pre-treated with 0.4 µM BafA1 before MG132. Indicated proteins were probed by immunoblotting. D. Cell lines with lower levels of p62 protein were transfected with 5 µg (+) or 10 µg (++) of HA-p62 plasmids, its mutants with Flag tag, or vector control in each electroporation (1X107 cells). Indicated proteins were analyzed by immunoblotting 48 h post-transfection. E-F. IB4 cells stably harboring p62 shRNA or shRNA control in 1 µg/ml puromycin were treated with 0.4 µM BafA1 for 48 h or 10 µM MG132 for 6 h, before subjected to immunoblotting or flow cytometry. shRNA expression was induced by 1 µg/ml doxycycline for 2 days before the drug treatments. MFI = mean fluorescence intensity. G. CHK1 and RAD51 protein stability was evaluated by immunoblotting in virus-transformed IB4 cells stably expressing control shRNA or p62 shRNA that were induced by 1 µg/ml doxycycline for different time points.
Fig 6.
p62 depletion by RNA interference promotes RNF168-mediated chromatin ubiquitination and DNA repair in viral latency.
IB4 cells stably expressing control or p62 shRNA were maintained with 1 µg/ml puromycin, and shRNA expression was induced by 1 µg/ml doxycycline for 3 days before subjected to confocal microscopy analysis for: A. RNF168, γH2AX and their colocalization in the nucleus with a rabbit RNF168 antibody (Millipore) and an Alexa 488 coupled anti-rabbit antibody (Invitrogen), and a mouse γH2AX(S139) antibody (BioLegend) and an Alexa 555 coupled anti-mouse antibody (BioLegend); C. chromatin ubiquitination, γH2AX and their colocalization in the nucleus with the mouse ubiquitin antibody clone FK2 (Millipore) and the Alexa 555 coupled anti-mouse antibody, and a rabbit γH2AX(S139) antibody (Cell Signaling Technol.) and the Alexa 488 coupled anti-rabbit antibody. Bar = 6 µm. B and D. Quantification of RNF168/γH2AX foci or FK2/γH2AX foci in A and C, respectively. E. Interaction between RNF168 and p62 was assessed by immunoprecipitation. The indicated cell lines were treated with 0.4 µM BafA1 for 48h, and then cell lysates (0.5 mg total proteins for each) were subjected to immunoprecipitation with a rabbit RNF168 antibody (Proteintech Group Inc.), and immunoprecipitated proteins were analyzed by immunoblotting with the p62 antibody and the sheep RNF168 antibody (Invitrogen). F. Histone H3 ubiquitination was assessed in p62-depleted IB4 cells treated with 2.5 µg /ml ionomycin for 48 h. Cell lysates (0.5 mg total proteins for each) from IB4 cells stably expressing control or p62 shRNA were subjected to denatured immunoprecipitation with a rabbit H3 antibody (Invitrogen), and immunoprecipitated H3 was probed with the FK2 antibody and the mouse H3 antibody (1G1) (Santa Cruz).
Fig 7.
A diagram showing the interaction of p62-mediated autophagy with DDR in EBV latency.
EBV latent infection produces ROS, which further induce p62 expression through the Keap1-NRF2 pathway and activate p62-mediated selective autophagy. ROS also cause DNA damage, including double strand breaks (DSBs). p62 accumulated in the nucleus due to autophagy inhibition inhibits DSB repair through promoting proteasome-mediated degradation of CHK1 and RAD51 and interacting with RNF168. A moderate level of endogenous p62-mediated autophagy in virus-transformed cells endows them with resistance to DNA damage. Loss of the p62-autophagy balance by exogenic stresses that inhibit autophagy or inducing ROS will exacerbate endogenous DNA damage.