Utilization of the Tango beta-arrestin recruitment technology for cell-based EDG receptor assay development and interrogation.

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. These LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. These receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. This complicates examination and comparison of these receptors across the entire family. The Tango technology uses the conserved beta-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. This method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. The authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG(1) and EDG(3) receptors.

Transactivator IE1 is required for baculovirus early replication events that trigger apoptosis in permissive and nonpermissive cells.

Immediate early viral protein IE1 is a potent transcriptional activator encoded by baculoviruses. Although the requirement of IE1 for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is well established, the functional roles of IE1 during infection are unclear. Here, we used RNA interference to ablate IE1, plus its splice variant IE0, and thereby define in vivo activities of these early proteins, including gene-specific regulation and induction of host cell apoptosis. Confirming an essential replicative role, simultaneous ablation of IE1 and IE0 by gene-specific double-stranded RNAs inhibited AcMNPV late gene expression, reduced yields of budded virus by more than 1,000-fold, and blocked production of occluded virus particles. Depletion of IE1 and IE0 had no effect on early expression of the envelope fusion protein gene gp64 but abolished early expression of the caspase inhibitor gene p35, which is required for prevention of virus-induced apoptosis. Thus, IE1 is a positive, gene-specific transactivator. Whereas an AcMNPV p35 deletion mutant caused widespread apoptosis in permissive Spodoptera frugiperda cells, ablation of IE1 and IE0 prevented this apoptosis. Silencing of ie-1 also prevented AcMNPV-induced apoptosis in nonpermissive Drosophila melanogaster cells. Thus, de novo synthesis of IE1 is required for virus-induced apoptosis. We concluded that IE1 causes apoptosis directly or contributes indirectly by promoting virus replication events that subsequently trigger cell death. This study reveals that IE1 is a gene-selective transcriptional activator which is required not only for expedition of virus multiplication but also for blocking of its own proapoptotic activity by upregulation of baculovirus apoptotic suppressors.

The highly conserved basic domain I of baculovirus IE1 is required for hr enhancer DNA binding and hr-dependent transactivation.

The immediate-early protein IE1 is the principal transcriptional regulator of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV). Transactivation by IE1 is dramatically stimulated by cis linkage of the affected promoter to AcMNPV homologous region (hr) elements that contain palindromic 28-bp repeats (28-mers) with enhancer activity. This hr-dependent transcriptional enhancement requires binding of the 28-mer by dimeric IE1. Here, we have defined IE1 domains required for this DNA binding in order to investigate the mechanism of IE1 function. Analysis of a panel of IE1 insertion mutations indicated that disruption of a highly conserved domain (residues 152 to 161) consisting of mostly positive-charged residues (basic domain I) abolished hr-dependent transactivation. Targeted mutagenesis of basic residues within basic domain I caused loss of hr-dependent transactivation but had no effect on IE1 oligomerization, nuclear localization, or hr-independent transactivation of viral promoters. Alanine substitutions of K(152) and K(154) or K(160) and K(161) impaired IE1 binding to 28-mer DNA as a homodimer, indicating that these basic residues are required for enhancer binding. Consistent with a DNA-binding defect, 28-mer interaction was improved by heterodimerization with wild-type IE1 or by increasing mutated IE1 concentrations. DNA binding mediated by basic domain I was also required for IE1 transactivation that occurred through physically separated, unlinked hr elements. We concluded that basic domain I is the enhancer-binding domain for IE1. Our data also suggest that DNA binding activates IE1 for transcriptional enhancement, possibly through a conformational change involving basic domain I.

Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo.

Caspases play a critical role in the execution of metazoan apoptosis and are thus attractive therapeutic targets for apoptosis-associated diseases. Here we report that baculovirus P49, a homolog of pancaspase inhibitor P35, prevents apoptosis in invertebrates by inhibiting an initiator caspase that is P35 insensitive. Consequently P49 blocked proteolytic activation of effector caspases at a unique step upstream from that affected by P35 but downstream from inhibitor of apoptosis Op-IAP. Like P35, P49 was cleaved by and stably associated with its caspase target. Ectopically expressed P49 blocked apoptosis in cultured cells from a phylogenetically distinct organism, Drosophila melanogaster. Furthermore, P49 inhibited human caspase-9, demonstrating its capacity to affect a vertebrate initiator caspase. Thus, P49 is a substrate inhibitor with a novel in vivo specificity for a P35-insensitive initiator caspase that functions at an evolutionarily conserved step in the caspase cascade. These data indicate that activated initiator caspases provide another effective target for apoptotic intervention by substrate inhibitors.

Baculovirus transregulator IE1 requires a dimeric nuclear localization element for nuclear import and promoter activation.

Immediate-early protein IE1 is a principal regulator of viral transcription and a contributor to origin-specific DNA replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Since these viral functions involve interaction of dimeric IE1 with palindromic homologous region (hr) enhancer-origin elements of the AcMNPV genome within the nucleus, it is presumed that proper nuclear transport of IE1 is essential for productive infection. To investigate the mechanisms of IE1 nuclear import, we analyzed the effect of site-directed mutations on IE1 subcellular distribution. As demonstrated by fluorescence microscopy and biochemical fractionation of plasmid-transfected cells, wild-type IE1 localized predominantly to the nucleus. Substitution or deletion of amino acid residues within a positively charged domain (residues 534 to 538) adjacent to IE1's oligomerization motif impaired nuclear import and caused loss of transactivation. Moreover, upon coexpression, these import-defective mutations prevented nuclear entry of wild-type IE1. In contrast, double-mutated IE1 defective for both nuclear import and dimerization failed to block nuclear entry or transactivation by wild-type IE1. Thus, import-defective IE1 dominantly interfered with wild-type IE1 by direct interaction and cytosolic trapping. Collectively, our data indicate that the small basic domain encompassing residues R(537) and R(538) constitutes a novel nuclear localization element that functions only upon IE1 dimerization. These findings support a model wherein IE1 oligomerizes within the cytosol as a prerequisite for nuclear entry and subsequent high-affinity interaction with the symmetrical binding sites comprising AcMNPV hr enhancer-origin elements.