Abstract：
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. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation.
OBJECTIVE: 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 the expression of specific viral genes yet does not affect the 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.
OBJECTIVE: 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 the expression of specific viral genes yet does not affect the 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.
摘要：
细胞Notch信号转导途径与卡波西肉瘤相关疱疹病毒(KSHV)和其他γ-疱疹病毒感染密切相关。RBP-Jk,典型Notch通路的细胞DNA结合成分，是病毒感染和未感染动物细胞中的关键Notch下游效应蛋白。从潜伏期重新激活KSHV需要病毒裂解开关蛋白，Rta,在病毒DNA内的许多位点上与RBP-Jk形成复合物。在卡波西肉瘤（KS）和原发性积液淋巴瘤（PEL）模型中，本构Notch活性对于KSHV病理生理学至关重要，我们证明Notch1在受感染的Vero细胞中也具有组成活性。尽管KSHV基因组包含>100个RBP-JkDNA基序，我们表明，在高度定量的反式互补报告病毒系统中，激活的Notch的四种同工型都不能有效地重新激活病毒的潜伏期。然而，Notch对再激活有积极贡献，因为用γ-分泌酶抑制剂（GSI）广泛抑制Notch1-4或显性阴性策划者样1（dnMAML1）共激活剂的表达严重减少了Vero细胞感染性KSHV的产生。KSHV产生的减少与Vero和PEL细胞中病毒转录的基因特异性减少有关。siRNA对Notch1的特异性抑制部分减少了感染性KSHV的产生，和NICD1在再激活过程中与病毒DNA形成启动子特异性复合物。我们得出的结论是，组成型Notch活性是感染性KSHV的强大生产所必需的，我们的结果表明，在病毒再激活过程中，激活的Notch1是MAML1/RBP-Jk/DNA复合物的前病毒成员。
目的：卡波西肉瘤相关疱疹病毒（KSHV）操纵宿主细胞致癌Notch信号通路，从潜伏期和细胞发病机制中重新激活病毒。KSHV再激活需要病毒蛋白Rta在功能上与RBP-Jk相互作用，Notch通路的DNA结合成分，并与启动子DNA驱动生产周期基因的转录。我们表明，Notch途径在KSHV再激活期间具有组成性活性，并且对于感染性病毒后代的强大生产至关重要。在再激活过程中抑制Notch会降低特定病毒基因的表达，但不会影响宿主细胞的生长。虽然Notch不能单独重新激活KSHV,Rta的必要表达揭示了Notch在重新激活中的先前未被重视的作用。我们建议激活的Notch以启动子特异性方式与Rta合作，该方式部分由Rta在再激活过程中重新分配与病毒结合的RBP-JkDNA的能力编程。
目的：卡波西肉瘤相关疱疹病毒（KSHV）操纵宿主细胞致癌Notch信号通路，从潜伏期和细胞发病机制中重新激活病毒。KSHV再激活需要病毒蛋白Rta在功能上与RBP-Jk相互作用，Notch通路的DNA结合成分，并与启动子DNA驱动生产周期基因的转录。我们表明，Notch途径在KSHV再激活期间具有组成性活性，并且对于感染性病毒后代的强大生产至关重要。在再激活过程中抑制Notch会降低特定病毒基因的表达，但不会影响宿主细胞的生长。虽然Notch不能单独重新激活KSHV,Rta的必要表达揭示了Notch在重新激活中的先前未被重视的作用。我们建议激活的Notch以启动子特异性方式与Rta合作，该方式部分由Rta在再激活过程中重新分配与病毒结合的RBP-JkDNA的能力编程。
