Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 Activation in Latently Infected Endothelial Cells through SQSTM1 Phosphorylation and Interaction with Polyubiquitinated Keap1

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2), the cellular master regulator of the antioxidant response, dissociates from its inhibitor Keap1 when activated by stress signals and participates in the pathogenesis of viral infections and tumorigenesis. Early during de novo infection of endothelial cells, KSHV induces Nrf2 through an intricate mechanism involving reactive oxygen species (ROS) and prostaglandin E2 (PGE2). When we investigated the Nrf2 activity during latent KSHV infection, we observed increased nuclear serine-40-phosphorylated Nrf2 in human KS lesions compared to that in healthy tissues. Using KSHV long-term-infected endothelial cells (LTC) as a cellular model for KS, we demonstrated that KSHV infection induces Nrf2 constitutively by extending its half-life, increasing its phosphorylation by protein kinase C (PKC) via the infection-induced cyclooxygenase-2 (COX-2)/PGE2 axis and inducing its nuclear localization. Nrf2 knockdown in LTC decreased expression of antioxidant genes and genes involved in KS pathogenesis such as the NAD(P)H quinone oxidase 1 (NQO1), gamma glutamylcysteine synthase heavy unit (␥GCSH), the cysteine transporter (xCT), interleukin 6 (IL-6), and vascular endothelial growth factor A (VEGF-A) genes. Nrf2 activation was independent of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1; p62). SQSTM1 levels were elevated in LTC, a consequence of protein accumulation due to decreased autophagy and Nrf2-mediated transcriptional activation. SQSTM1 was phosphorylated on serine-351 and -403, while Keap1 was polyubiquitinated with lysine-63-ubiquitin chains, modifications known to increase their mutual affinity and interaction, leading to Keap1 degradation and Nrf2 activation. The latent KSHV protein Fas-associated death domain-like interleukin-1␤-converting enzyme-inhibitory protein (vFLIP) increased SQSTM1 expression and activated Nrf2. Collectively, these results demonstrate that KSHV induces SQSTM1 to constitutively activate Nrf2, which is involved in the regulation of genes participating in KSHV oncogenesis. IMPORTANCEThe transcription factor Nrf2 is activated by stress signals, including viral infection, and responds by activating the transcription of cytoprotective genes. Recently, Nrf2 has been implicated in oncogenesis and was shown to be activated during de novo KSHV infection of endothelial cells through ROS-dependent pathways. The present study was undertaken to determine the mechanism of Nrf2 activation during prolonged latent infection of endothelial cells, using an endothelial cell line latently infected with KSHV. We show that Nrf2 activation was elevated in KSHV latently infected endothelial cells independently of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1), which was involved in the degradation of the Nrf2 inhibitor Keap1. Furthermore, our results indicated that the KSHV latent protein vFLIP participates in Nrf2 activation. This study suggests that KSHV hijacks the host's autophagic protein SQS...

Nuclear Innate Immune DNA Sensor IFI16 Is Degraded during Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus (KSHV): Role of IFI16 in Maintenance of KSHV Latency

“…To assess the effect of IFI16 KD on TPA-mediated KSHV lytic reactivation, BCBL-1 cells were transduced with a lentivirus containing ShIFI16 or a control lentivirus. Forty-eight hours later, cells were induced with TPA (20 ng/ml), and total RNA was isolated after 0, 3,6,9,12,24,36,48,72, and 96 h, as indicated (Fig. 3A).…”

“…At 72 h postinfection (p.i. ), when latency is established (47), cells were treated with TPA or a vehicle control for 2, 3, and 4 days (48,72, and 96 h), and IFI16 levels were determined by WB analysis. As a control, mock-infected cells were also treated with TPA to rule out any off-target effects of TPA on IFI16 levels (Fig.…”

“…Five TRC (the RNAi Consortium) lentiviral human IFI16 short hairpin RNA (shRNA) constructs (Dharmacon; GE Healthcare, Lafayette, CO) (clones TRCN0000019079, TRCN0000019080, TRCN0000019081, TRCN0000019082, and TRCN0000019083) were tested for their ability to efficiently knock down IFI16. Lentiviral particles were produced by using a four-vector system in HEK293T cells as described previously (48). HEK293T cells were transfected by using the CalPhos mammalian transfection kit (TaKaRa Clontech, Mountain View, CA) according to the manufacturer's instructions.…”

CROSSTALK BETWEEN Nrf2 AND NOTCH SIGNALING

“…This means that both EBV and KSHV might be able to express the cell survival factor NRF2 in infected cells at least at the RNA level. In the case of KSHV, it was demonstrated and seemed to be important that the latent KSHV protein, vFLIP (Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein) increased SQSTM1 expression and led to stabilization of NRF2 [107]. Thus, KSHV could dually activate the NRF2 pathway during oncogenesis by hijacking canonical NOTCH signaling transcriptionally and manipulating the autophagic pathway of KEAP1 degradation through induction of SQSTM1 post-translationally ( Figure 5B).…”

Activated Nrf2 Interacts with Kaposi's Sarcoma-Associated Herpesvirus Latency Protein LANA-1 and Host Protein KAP1 To Mediate Global Lytic Gene Repression

“…In a set of recent studies, we demonstrated that de novo KSHV infection of endothelial cells induced Nrf2 through multiple mechanisms to create a microenvironment conducive to infection (30,32). In the current study, we further investigated the role of Nrf2 in KSHV gene expression, focusing mainly on latently infected, PEL-derived cell lines.…”