1C). (LMP1) is essential for B lymphocyte transformation into proliferating lymphoblastoid cell line (LCLs) (for review see ref.1). LMP1 has a 24-aa cytoplasmic N-terminus, six hydrophobic transmembrane domains (amino acids 25 to 186) and a 200-aa cytoplasmic C-terminus (amino acids 187 to 386). The transmembrane domains induce LMP1 aggregation in plasma membrane lipid rafts and barges and constitutively activate two C-terminal cytoplasm signaling domains referred to as C-terminal activation regions (CTAR) or transformation effector sites (TES) 1 and 2, which were initially defined as amino acids 187231 and 352386, respectively (Fig. 1A) (29). CTAR1 amino acids 201 to 210 engage BAY 80-6946 (Copanlisib) tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) 1, 3, 2, and 5 and CTAR2 amino acids 376386 engage TNFR-associated death domain proteins TRADD and RIP1 (412). Both signaling domains activate NF-B, p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase, thereby altering cell gene expression, cell survival, and immune responses (1317). == Fig. 1. == LMP1 activates IRF7 through CTAR2. (A) A schematic representation of LMP1WT, LMP1 1231(CTAR1), and LMP1 187351 (CTAR2). TM, transmembrane domain. HEK293 (B) or DG75 (C) cells were cotransfected with pSG5-HA-IRF7 and pSG5 (lane 1), pSG5-FLAG-LMP1 WT (lane 2), pSG5-FLAG-LMP1 1231 (lane 3) or pSG5-FLAG-LMP1 187351 (lane 4) along with an ISRE or NF-B-dependent luciferase and a pGK–galactosidase reporter plasmid. To calculate relative luciferase activity, empty vector and LMP1 induced luciferase activities were set to 1 1 and 100%, respectively. LMP1 also induces and activates IFN regulatory factor 7 (IRF7) (1820), a critical transcription factor for type 1 IFN (IFN)-mediated innate and adaptive immune responses (2124). IRF7 is comprised of DNA binding, constitutive activation, virus activated, inhibitory, and signal response domains and is activated by serine 477 and 479 phosphorylation (25). Although IRF7 is expressed at low levels in nonstimulated cells, IRF7 is critical for type I IFN expression, particularly in plasmacytoid dendritic cells (24,26,27). Toll-like receptor activation, LMP1 expression, viral infection, and other signals that activate IRF3 and Mmp2 induce BAY 80-6946 (Copanlisib) IFN (IFN), can up-regulate IRF7 expression (18,2124,28,29). IRF7 can bind and localize to MyD88 until ligand-activated Toll-like receptors 7, 8, or 9 engage MyD88 and activate IRF7, which enters the nucleus and up-regulates IFN stimulated BAY 80-6946 (Copanlisib) response elements (ISREs) to activate IFN (24,27,29,30). IRF7 accumulation on MyD88 is likely to be important for rapid IFN and activation (2224,30). Because the same MyD88 residues affect IRF7 binding and activation, the importance of IRF7 binding for activation has been difficult to assess (30). LMP1 residues that activate IRF7 have not been identified (20). We therefore investigated the LMP1 residues required for IRF7 activation, assessed whether IRF7 binding to these residues is critical for activation, and evaluated the role BAY 80-6946 (Copanlisib) of RIP1, TRAF2, TRAF3, and TRAF6 in IRF7 activation. == Results == == LMP1 Primarily Activates IRF7 Through CTAR2. == To assess the roles of the two LMP1 C-terminal signaling domains in IRF7 activation, LMP1 mutants deleted for CTAR1 or CTAR2 (Fig. 1A) were tested for IRF7 activation by using an IFN stimulated response element (ISRE)-dependent luciferase reporter. An NF-B dependent luciferase reporter was used as a control for a known CTAR1 or CTAR2 activity. Because HEK293 cells do not express detectable IRF7, LMP1 minimally activated an ISRE-dependent luciferase reporter (data not shown). IRF7 expression in HEK293 cells enhanced ISRE luciferase BAY 80-6946 (Copanlisib) activity 12-fold. LMP1 expression further activated to 200-fold, without altering IRF7 expression levels (Fig. 1Band data not shown)..