p62-mediated Selective autophagy endows virus-transformed cells with insusceptibility to DNA damage under oxidative stress

DNA damage response (DDR) and selective autophagy both can be activated by reactive oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in these settings. In this study, we provide evidence that p62 is upregulated through the ROS-Keap1-NRF2 pathway, and considerable levels of p62-mediated selective autophagy are constitutively induced, in virus-transformed cells. Inhibition of autophagy results in p62 accumulation, and promotes ROS-induced DNA damage and cell death, as well as downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but accumulation of the DNA repair proteins CHK1 and RAD51. However, small hairpin RNA (shRNA)-mediated p62 depletion or transient expression of p62 in B cell lines had no apparent effects on the protein levels of CHK1 and RAD51. These results indicate that p62 accumulation resulting from autophagy inhibition is prerequisite for p62-mediated proteasomal degradation of CHK1 and RAD51. Furthermore, shRNA-mediated p62 depletion in EBV-transformed lymphoblastic cell lines results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin ubiquitination in the nucleus, indicative of activation of RNF168-mediated DNA repair mechanisms. Finally, we have evaluated endogenous p62 and autophagy levels and their correlation with endogenous ROS and DNA damage levels in B cell population of AIDS lymphoma patients versus age-matched subjects. Our results have unveiled a pivotal role for p62-mediated selective autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel insight into virus-mediated oncogenesis. Author Summary Reactive oxygen/nitrogen species (ROS/RNS) can induce both DNA damage response (DDR) and selective autophagy, which play crucial roles in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 links their crosstalk. However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). In this study, we provide evidence that p62-mediated selective autophagy is constitutively induced in virus-transformed cells, and that its inhibition exacerbates ROS-induced DNA damage, and promotes proteasomal degradation of CHK1 and RAD51 in a p62-dependent manner. However, rigorous autophagy induction results in accumulation of DNA repair proteins CHK1 and RAD51, and p62 degradation. Furthermore, small hairpin RNA (shRNA)-mediated p62 depletion or transient expression of p62 did not affect CHK1 and RAD51 protein levels; rather, shRNA-mediated p62 depletion activates RNF168-dependent DNA repair mechanisms. Our results have unveiled a pivotal role for p62-mediated selective autophagy in regulation of DDR by overriding traditional DDR mechanisms in the setting of oncogenic virus latent infection, and provide a novel insight into the etiology of viral cancers.