The cellular Notch1 Protein Promotes KSHV reactivation in an Rta-dependent manner

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi’s sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi’s sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. In infected PEL cells, we show that reduction of vDNA synthesis by GSI is associated with gene specific reduction of viral transcription. Specific inhibition of Notch1 by siRNA partially reduces production of infectious KSHV, and ectopic activated Notch (NICD1) enhances virus production induced by Valproic Acid (VPA) treatment. We conclude that constitutive Notch activity is required for robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk complex during viral reactivation. Importance Kaposi’s sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces expression of specific viral genes yet does not affect growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta’s ability to redistribute RBP-Jk DNA binding to the virus during reactivation.