The master antioxidant defense is activated during EBV latent infection

Abstract

ABSTRACT Our recent publication shows for the first time that the master antioxidant pathway Keap1-NRF2 is constitutively activated in EBV- or HTLV1-transformed cells, with the mechanism being unclear (L. Wang, M. E. A. Howell, A. Sparks-Wallace, C. Hawkins, et al., PLoS Pathog 15:e1007541, 2019, https://doi.org/10.1371/journal.ppat.1007541 ). In this follow-up investigation, we show that EBV-triggered ROS production activates the Keap1-NRF2 pathway in EBV-transformed cells, and LMP1 plays a major role in this event. Further investigation shows that the mitochondria in cell lines with EBV Type 3 latency produce higher levels of mitochondrial ROS that are responsible for the activation of the Keap1-NRF2 pathway, compared with Type 1 latency. Moreover, using both pharmaceutical inhibitors and CRISPR-mediated gene editing, we show that the stress-related kinase TBK1 is required for NRF2 activation. As to the functional consequences, shRNA-mediated knockdown of NRF2 in EBV-transformed IB4 and HTLV1-transformed MT4 cells elevates endogenous reactive oxygen species levels, promotes DNA damage, downregulates cell proliferation, and promotes EBV reactivation. These findings, together with our previous report, disclose how EBV controls the intricate balance between oxidative stress and antioxidant defense, which greatly improve our understanding of EBV latency and pathogenesis and may be leveraged to opportunities toward the improvement of therapeutic outcomes in EBV-associated diseases. IMPORTANCE To our knowledge, this is the first report delineating the activation of the master antioxidant defense during EBV latency. We show that EBV-triggered reactive oxygen species production activates the Keap1-NRF2 pathway in EBV-transformed cells, and LMP1 plays a major role in this event, and the stress-related kinase TBK1 is required for NRF2 activation. Moreover, we show that the Keap1-NRF2 pathway is important for cell proliferation and EBV latency maintenance. Our findings disclose how EBV controls the balance between oxidative stress and antioxidant defense, which greatly improve our understanding of EBV latency and pathogenesis and may be leveraged to opportunities toward the improvement of therapeutic outcomes in EBV-associated diseases.