We report a new pathway of translation regulation that may operate in interferon-treated or virusinfected mammalian cells. This pathway is activated by P56, a protein whose synthesis is strongly induced by interferons or double-stranded RNA. Using a yeast two-hybrid screen, we identi®ed the P48 subunit of the mammalian translation initiation factor eIF-3 as a protein that interacts with P56. The P56±P48 interaction was con®rmed in human cells by co-immunoprecipitation assays and confocal microscopy. Gel ®ltration assays revealed that P56 binds to the large eIF-3 complex that contains P48. Puri®ed recombinant P56 inhibited in vitro translation of reporter mRNAs in a dose-dependent fashion, and that inhibition was reversed by the addition of puri®ed eIF-3. In vivo, expression of transfected P56 or induction of the endogenous P56 by interferon caused an inhibition of overall cellular protein synthesis and the synthesis of a transfected reporter protein. As expected, a P56 mutant that does not interact with P48 and eIF-3 failed to inhibit protein synthesis in vitro and in vivo. Keywords: double-stranded RNA/eIF-3/interferon/P56/ translational regulation IntroductionAlthough the antiviral effect of interferon (IFN) is the most well known, IFNs have many other effects on cell physiology (Stark et al., 1998). The multiple effects of IFNs are mediated by the numerous cellular proteins whose synthesis is induced by IFNs (Sen and Ransohoff, 1993). However, the speci®c biochemical and cellular functions of most of these proteins are unknown. Among the notable exceptions are the enzymes PKR and 2±5(A) synthetases that are known to affect virus replication and cell growth (Stark et al., 1998). Here, we report the function of another IFN-inducible protein, P56.The cDNA for P56 was cloned in the process of cloning IFN-inducible mRNAs (Chebeth et al., 1983;Kusari and Sen, 1986). We have studied extensively the transcriptional regulation of 561 mRNA that encodes P56 (Bandyopadhyay et al., 1990). The mRNA is undetectable in untreated cells but it is induced rapidly upon IFN treatment (Kusari and Sen, 1986). The 561 mRNA level is quite high in IFN-treated cells and, in a recent gene array analysis, it scored as the most abundant IFN-induced mRNA among >100 such mRNAs (Der et al., 1998). Because the 561 mRNA and the encoded protein, P56, both turn over rapidly, cells are depleted of them quickly after IFN treatment ceases. The 561 gene is transcriptionally induced not only by IFN but also by double-stranded (ds) RNA or virus infection (Tiwari et al., 1987). Our studies using various mutant cells have clearly established that the Jak±STAT pathway used by IFN to induce it is dispensable for its induction by dsRNA or viruses (Bandyopadhyay et al., 1995). Thus, there are alternative ways to induce the 561 gene, and the encoded protein, P56, may have cellular functions beyond the IFN system.Because the cellular level of P56 is drastically enhanced upon treatment of cells with IFN or dsRNA, we suspected that it has important cellular fu...

Double-stranded (ds) RNA, a common component of virus-infected cells, is a potent inducer of the type I interferon and other cellular genes. For identifying the full repertoire of human dsRNA-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from dsRNA-treated GRE cells. Because these cells lack all type I interferon genes, the possibility of gene induction by autocrine actions of interferon was eliminated. Our screen identified 175 dsRNA-stimulated genes (DSG) and 95 dsRNA-repressed genes. A subset of the DSGs was also induced by different inflammatory cytokines and viruses demonstrating interconnections among disparate signaling pathways. Functionally, the DSGs encode proteins involved in signaling, apoptosis, RNA synthesis, protein synthesis and processing, cell metabolism, transport, and structure. Induction of such a diverse family of genes by dsRNA has major implications in host-virus interactions and in the use of RNA i technology for functional ablation of specific genes.

P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm.

For determining cellular functions of the interferon-inducible human cytoplasmic protein P56, we undertook a Saccharomyces cerevisiae two-hybrid screen that identified Int6 as a P56-interacting protein. That the interaction also occurs in human cells was confirmed by coimmunoprecipitation and the observed cytoplasmic displacement of nuclear Int6 upon coexpression of P56. Because Int6 has been claimed to be both a cytoplasmic and a nuclear protein, we investigated the structural basis of this discrepancy. By mutational analyses, we showed that the Int6 protein contains a bipartite nuclear localization signal and a nuclear export signal at the far end of the amino terminus. The 20 amino-terminal residues of Int6, when they were attached to a different nuclear protein, were sufficient to translocate that protein to the cytoplasm. Within this region, replacement of any of the three leucine residues with alanine destroyed the function of the export signal. The specific domain of P56 that is required for its interaction with Int6 was mapped using the yeast two-hybrid assay and a mammalian coimmunoprecipitation assay. Both assays demonstrated that the C-terminal region of P56 containing three specific tetratricopeptide motifs is required for this interaction. In contrast, removal of an internal domain of P56 enhanced the interaction, as quantified by the two-hybrid assay.

The authors have developed a cellular analysis platform, based on encoded microcarriers, that enables the multiplexed analysis of a diverse range of cellular assays. At the core of this technology are classes of microcarriers that have unique, identifiable codes that are deciphered using CCD-based imaging and subsequent image analysis. The platform is compatible with a wide variety of cellular imaging-based assays, including calcium flux, reporter gene activation, cytotoxicity, and proliferation. In addition, the platform is compatible with both colorimetric and fluorescent readouts. Notably, this technology has the unique ability to multiplex different cell lines in a single microplate well, enabling scientists to perform assays and data analysis in novel

Gene prediction by computer algorithms and approaches based on comparative genomics identifies the presence of potential genes in a genomic sequence. By placing these predicted genes onto microarrays and searching for interactions with complementary DNA from different types of tissue or cell, novel genes are discovered.