Interferon establishes an antiviral state in numerous cell types through the induction of a set of immediateearly response genes. Activation of these genes is mediated by phosphorylation of latent transcription factors of the STAT family. We found that infection of primary foreskin fibroblasts with human cytomegalovirus (HCMV) causes selective transcriptional activation of the alpha/beta-interferon-responsive ISG54 gene. However, no activation or nuclear translocation of STAT proteins was detected. Activation of ISG54 occurs independent of protein synthesis but is prevented by protein tyrosine kinase inhibitors. Further analysis revealed that HCMV infection induced the DNA binding of a novel complex, tentatively called cytomegalovirus-induced interferonstimulated response element binding factor (CIF). CIF is composed, at least in part, of the recently identified interferon regulatory factor 3 (IRF3), but it does not contain the STAT1 and STAT2 proteins that participate in the formation of interferon-stimulated gene factor 3. IRF3, which has previously been shown to possess no intrinsic transcriptional activation potential, interacts with the transcriptional coactivator CREB binding protein, but not with p300, to form CIF. Activating interferon-stimulated genes without the need for prior synthesis of interferons might provide the host cell with a potential shortcut in the activation of its antiviral defense.Alpha interferon (IFN-␣) and IFN-␤ are unique among the continuously growing superfamily of cytokines in their ability to confer resistance to viral infection (20,36). The synthesis of IFN-␣ and IFN-␤ is induced at the transcriptional level after a cell encounters virus or double-stranded RNA (dsRNA) (16,46). The subsequent secretion of the newly produced interferons and their binding to a common cell surface receptor results in the induction of a set of immediate-early response genes (12, 21, 24-26, 32, 44, 47). The activation of these interferon-stimulated genes (ISGs) represents the first step towards the development of an antiviral state. Control over ISGs is exerted by an IFN-␣/␤-activated transcription factor complex termed interferon-stimulated gene factor 3 (ISGF3), which binds to a common enhancer element referred to as the interferon-stimulated response element (ISRE) (10,14,23,27,43). ISGF3 is formed through the interaction of the DNA binding subunit ISGF3␥ (p48) and the regulatory component ISGF3␣ (14,28,48), which itself is composed of two members of the STAT (signal transducers and activators of transcription) family of transcription factors, STAT1 and STAT2 (14,15,43). Both STAT proteins become tyrosine phosphorylated in response to IFN-␣/␤ stimulation, which enables their nuclear translocation and DNA binding (8,10,13,41). Transcriptionally active STAT1 has been shown to be a requirement for the antiviral and antiproliferative effects of IFN-␣/␤ (5, 11, 31). The phosphorylation of STAT1 and STAT2 is mediated through the action of two related tyrosine kinases, Jak1 and Tyk2, which are enzymatic...

Protein phosphorylation is one of the major regulatory mechanisms involved in signal-induced cellular events, including cell proliferation, apoptosis, and metabolism. Because many facets of biology are regulated by protein phosphorylation, aberrant kinase and/or phosphatase activity forms the basis for many different types of pathology. The disease relevance of protein kinases and phosphatases has led many pharmaceutical and biotechnology companies to expend significant resources in lead discovery programs for these two target classes. The existence of >500 kinases and phosphatases encoded by the human genome necessitates development of methodologies for the rapid screening for novel and specific compound inhibitors. We describe here a fluorescence-based, molecular assay platform that is compatible with robotic, ultra-high throughput screening systems and can be applied to virtually all tyrosine and serine/threonine protein kinases and phosphatases. The assay has a coupled-enzyme format, utilizing the differential protease sensitivity of phosphorylated versus nonphosphorylated peptide substrates. In addition to screening individual kinases, the assay can be formatted such that kinase pathways are re-created in vitro to identify compounds that specifically interact with inactive kinases. Miniaturization of this assay format to the 1-microl scale allows for the rapid and accurate compound screening of a host of kinase and phosphatase targets, thereby facilitating the hunt for new leads for these target classes.

Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner. A key determinant of the intracellular concentration of a given protein is its stability or "half-life." We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells. The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (UbG76V) that cannot be cleaved by ubiquitin hydrolases. We show that increasing the number of UbG76V moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins. Cells expressing a destabilized beta-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.

Autographa californica nuclear polyhedrosis virus (AcMNPV) contains five homologous regions (hrl through hr5) interspersed throughout its genome. Analysis of plasmid transfections indicates that the hrs function as transcriptional enhancers and possible origins of viral DNA replication. The role of these repetitive elements in regulating expression from the AcMNPV genome was examined by constructing a series of recombinant viruses that tested the effect of hr5 on expression of the adjacent p35 gene (p35). When embedded within the viral genome, hr5 stimulated transcription from the early p35 promoter in a position-and orientationindependent manner. Moreover, hr5 and the upstream activating region ofp35 were functionally interchangeable. A 28-bp imperfect palindrome, repeated six times within hr5, was the minimal sequence required forp35 promoter activation. hr5 also stimulated another early AcMNPV promoter but not a late promoter or a host-derived promoter, suggesting that enhancement is promoter specific during infection. To investigate its role during AcMNPV replication, hr5 was deleted from its normal position within the viral genome. The resulting hr5 mutants exhibited no apparent defects in replication, as judged by production of budded virus and levels of very late gene expression, even though steady-state levels of p35 RNA were reduced. These results indicated for the first time that hr5 functions as a transcriptional enhancer within the viral genome. However, the element is not required for AcMNPV replication in cultured cells. Thus, loss of one of five possible origins of DNA replication is not deleterious to viral growth. Since p26 was removed from the hr5 deletion mutants, this gene is also nonessential for viral replication.

No abstract